508 resource limit is reached resource limit is reached the website is temporarily unable to service your request as it exceeded resource limit. please try again later. 508 resource limit is reached resource limit is reached the website is temporarily unable to service your request as it exceeded resource limit. please try again later. 508 resource limit is reached resource limit is reached the website is temporarily unable to service your request as it exceeded resource limit. please try again later. 508 resource limit is reached resource limit is reached the website is temporarily unable to service your request as it exceeded resource limit. please try again later. 141 squalen bull. of mar. and fish. postharvest and biotech. 14 (3) 2019, 141-153 www.bbp4b.litbang.kkp.go.id/squalen-bulletin squalen bulletin of marine and fisheries postharvest and biotechnology issn: 2089-5690 e-issn: 2406-9272 copyright © 2019, squalen bmfpb. accreditation number: 21/e/kpt/2018. doi: http://dx.doi.org/10.15578/squalen.v14i3.399 emerging pollutants from industrial emission and their health hazards in indonesian seafood: a review dwiyitno research and development center for marine and fisheries product processing and biotechnology, jalan ks. tubun petamburan vi, slipi, jakarta pusat 10260, indonesia abstract emerging pollutants (eps) or contaminants of emerging concern (cecs) have become global awareness since few decades ago, including in indonesia. intensive usage of industrial compounds has led to the massive emission to the environment and therefore their potential adverse effects may endanger aquatic organisms and human health. based on the available literatures, organotins and flame retardants are two main groups of e ps from industrial emission identified in indonesian seafood, as well as in those worldwide. however, concentration of both eps group detected in indonesian seafood is relatively low than that in developed countries and the majority of south east asian countries. toxicological studies revealed that eps emitted from industrial activities have to be concern, as most of the eps attributed to endocrine disrupting chemicals. nevertheless, study on the exposure assessment of eps in indonesia is very limited. even though, the concentrations of eps in indonesian seafood produce exposure below tolerable daily intake (tdi) to the local consumer, the long term exposures have to be aware due to their possible continues emission and elevating concentration such as shown by flame retardants. governmental regulation, monitoring programs, and laboratory approach are among issues to be concern in addressing eps in indonesia, especially from industrial emission. keywords: emerging pollutants, industrial emission, organotins, flame retardants, aryl hydrocarbons *corresponding author. e-mail: dwiyitno.brkp@kkp.go.id article history: received: 12 august 2019; revised: 27 september 2019; accepted: 2 december 2019 introduction emerging pollutants (eps) or contaminants of emerging concern (cecs) have become global awareness since few decades ago. the concern was highlighted by a 1962’s book of silent spring that criti cizes the i mpact of widespread usage of dichlorodiphenyltrichloroethane (ddt) for eradicating mosquitoes and other pests on bird’s species (carson, 2002). due to their toxicological harmful to the environment and human health, ddt was finally banned in 1970s followed by other chlorinated pesticides, later on known as persistence organic pollutants (pops). through stockholm convention, those chemicals are currently replaced by more env i ron m ent al l y f ri e ndl y c om pou nds such organophosphate and carbamate pesticides (unep, 2014). united states environmental protection agency/ us-epa (2019) defined cecs as naturally occurring, manufactured or man-made chemicals or materials which have now been discovered or are suspected present in various environmental compartments and whose toxicity or persistence are likely to significantly alter the metabolism of any living organism. similarly, european commission uses eps to define substances that have the potential to enter the environment and causes adverse ecological and human health effects, but are still largely unregulated and whose fate and potential effects are poorly understood (ec, 2018). list of abbreviations: bciphipp: bis(1-chloro-2-propyl)-1-hydroxy-2-propyl phosphate; bde: bromo diphenyl ethane; bfrs: brominated flame retardants; bha: butylated hydroxyl anisole; bht: buylated hydroxyl toluene; bmdl: benchmark dose confidence limit; btbpe: bis-tri-bromo phenoxy ethane; bts: butyltins; cecs: contaminants of emerging concern; crm: certified reference material; dbdpe: decabromo diphenyl ethane; dbt: dibutyltin; ddt: dichloro diphenyl trichloroethane; dipns: di-isoprophyl naphthalenes; dot: dioctyltin; dphp: diphenyl phosphate; eps: emerging pollutants; hbcd: hexabromocyclododecane; imo: international maritime organization; loel: lowest observed effect level; mbt: monobutyltin; moe: margin of exposure; mrl: maximum residue limit; noec: no observed effect concentration; ots: organotins; pbde: polybrominated diphenyl ether; pcbs: polychlorinated biphenyls; pfrs: organophosphate flame retardants; pmn: phenylmethoxy naphthalene; pnts: phenyltins; pops: persistence organic pollutants; ppcps: pharmaceutical and personal care products; tarl: tolerable average residue level; tbbpa: tetrabromobisphenyl a; tbt: tributyltin; tdi: tolerable daily intake; toc:total organic carbon; tpht: triphenyltin; txib: trimethyl pentanediol diisobutirate http://www.bbp4b.litbang.kkp.go.id/squalen-bulletin http://dx.doi.org/10.15578/squalen.v14i3.399 mailto:dwiyitno.brkp@kkp.go.id 142 however, scientists pointed different categories of emerging contaminants, i.e. contaminants which are very recently produced and contaminants which have been established in the environment but much concern is only recently (cantwell & katz, 2017; sauvé & desrosiers, 2014). eps consists of wide range anthropogenic contaminants, including pesticides, cosmetics, pharmaceuticals, and personal care products (ppcps), and other compounds which are associated with modern daily use (thomaidis, asimakopoulos, & bletsou, 2012). some indonesian waters have been known to receive high pollution exposure derived either from upland regions or direct from coastal activities. those contaminants are delivered from various sources, such as domestic disposal, industrial waste, transportation, oil spills, etc. (takarina & adiwibowo, 2010; arifin, puspitasari, & miyazaki, 2012; dwiyitno et al, 2015 & 2016; dzikowitsky et al, 2016). terrestrial pollution load has been shown in jakarta bay, as 39% of river water from jakarta city was categorized as highly polluted and 44% was moderately polluted (bplhd jakarta, 2018). as the main river discard to jakarta bay, 54% of citarum water ini west java was also highly polluted (bplhd jakarta, 2018). hence, the burden of the contaminants potentially increases significantly in accordance with the population growth and the development of industrial activities. studies on pops and other priority contaminants from indonesi an waters hav e been reported. nevertheless, studies on the eps such as emission of pharmaceutical and ppcps, pesticides, hormones, biological metabolites, perfluoroalkylated surfactants, nanoparticles, and other technical or industrial products in indonesia waters are v ery limited (dsikowitzky et al., 2014; 2016; dwiyitno et al., 2016; suzuki, matsumura, moriguchi, & nakano, 2007; syakti et al., 2013). through bio-magnification pathway via aquatic food web, the contaminants potentially accumulate in the aquatic organisms including seafood. the present article would be the first comprehensive review concerning present studies on eps, especially from industrial emission in indonesian seafood and their potential impact to food safety issues compared to that of worldwide. data of contaminant concentration in seafood from certain area in indonesia are then used to estimate the exposure levels in concerned area. emerging pollutants in seafood studies on the occurrence of eps in indonesia was dominated from around java island waters, as the most populated and concentrated island in the country. additionally, jakarta bay is the most investigated ecosystem for anthropogenic pollutants since is located in the most populated industrilized city of indonesia, jakarta. a number of studies on eps have been conducted in jakarta bay, including eps emitted from industrial activities in seafood species. based on earlier literatures, industrial eps could be classified as organotins (ots), flame retardants, and aryl hydrocarbons (table 1). other locations become concern of eps in seafood sample are medan, jambi, lampung, ci rebon, surabaya, and makassar (dwiyitno et al., 2016; ilyas et al., 2013; isobe et al., 2004; isobe, ogawa, ramu, sudaryanto, & tanabe, 2012; monirith et al., 2003; nakata et al., 2012; sudaryanto et al., 2007; sudaryanto, 2002; tsutsumi et al., 2002) as illustrated in figure 1. noteworthy, industrial contaminants associated with pahs, pharmaceuticals, and pesticides are excluded from this review since they are commonly categorized in different pollutant groups such as domestic waste, pharmaceuti cal, and personal care products (ppcps). figure 1. distribution of studies on eps from industrial emission in indonesian seafood (illustrated based on literatures presented in table 1) dwiyitno/squalen bull. of mar. and fish. postharvest and biotech. 14 (3) 2019, 141-153 143 table 1. emerging pollutants from industrial emission in seafood from indonesian water and worldwide dwiyitno/squalen bull. of mar. and fish. postharvest and biotech. 14 (3) 2019, 141-153 group and com m on us e compound location species (year) concentration reference belaw an, medan green mussel (1998) 5.0 ng/g w w kl tungkal, jambi 3.7 ng/g w w t. harun, lampung 9.9 ng/g w w t. lada, banten 6.8 ng/g w w kamal, jakarta 27 ng/g w w ancol, jakarta 58 ng/g w w bondet, cirebon 7.2 ng/g w w genjeran, surabaya 45 ng/g w w maros, makassar 6.1 ng/g w w kamal, jakarta fish (1998) 21-84 ng/g w w t. lada, banten 4.2-18 ng/g w w bondet, cirebon 3.3-25 ng/g w w jakarta bay (tbt) green mussel (2003) 14 ng/g w w midorikaw a et al. (2004) green mussel (1998) 3.5-730 ng/g w w sudaryanto et al. (2002) fish (1997-1998) 17-390 ng/g dw sudaryanto takahashi, iw ata, tanabe, ismail (2004) green mussel (1998) <1-787 ng/g w w fish (1998) 220 ng/g w w thailand green mussel (1998) 3-680 ng/g w w kan-atireklap, sanguansi, tabucanon, & hungspreugs (1997) vietnam green mussel (1998) 2.1-64 ng/g w w cambodia green mussel (1998) 2.4-88 ng/g w w hongkong green mussel (1999) 16-330 ng/g w w india green mussel (1998) <1-570 ng/g w w korea blue mussel (1997-1999) 17-1200 ng/g w w hong et al. (2002) blue mussel (1989) 20-240 ng/g w w fish (1996) 11-182 ng/g w w usa blue mussel (1989-1990) 2-276 ng/g w w short & sharp (1989) taiw an fish (2006) nd-953.7 ng/g w w chen et al. (2008) green mussel (2007) 11+1.4 ng/g lw fish (2007) 9.1-23 ng/g lw cultured snapper (2004) 2.6-6.2 ng/g lw wild snapper (2004) <1-7.3 ng/g lw nile tilapia (2008) 6.6-11 ng/g dw ilyas et al. (2013) jakarta bay green mussel (2003) 17-34 ng/g lw isobe et al. (2012) cambodia green mussel (2004) 2.3-5.3 ng/g lw china green mussel (2004) 10-75 ng/g lw hongkong green mussel (2004) 25-130 ng/g lw india green mussel (2004) 56-640 ng/g lw gren mussel (2003-2008) 18 ng/g lw blue mussel (2003-2008) 49-89 ng/g lw korea blue mussel (2005) 6.7-440 ng/g lw malaysia green mussel (2004) 0.5-10 ng/g lw philippines green mussel (2004) 69-140 ng/g lw vietnam green mussel (2004) 0.7-5.4 ng/g lw china fish (2009) 8.6-74.3 ng/g lw yu et al. (2012) usa fish (2000-2003) 38-125 ng/g w w us-epa (2013) jakarta bay green mussel (2003) 1.7-2.4 ng/g lw cambodia green mussel (2004) 8.8-9.7 ng/g lw china green mussel (2004) 2-21 ng/g lw hongkong green mussel (2004) 51 ng/g lw india green mussel (2004) 0.3-16 ng/g lw green mussel (2003-2008) 10-1,400 ng/g lw blue mussel (2003-2008) 16-980 ng/g lw korea blue mussel (2005) 6-500 ng/g lw malaysia green mussel (2004) 2.2-15 ng/g lw philippines green mussel (2004) 6.6-17 ng/g lw vietnam green mussel (2004) <0.01-140 ng/g lw green mussel (2007) 4.1+1.4 ng/g lw fish (2007) <1-2.8 ng/g lw nile tilapia (2008) 1.6-3.3 ng/g dw ilyas et al. (2013) lampung sudaryanto  (2019) japan isobe et al. (2012) japan jakarta bay, indonesia sudaryanto et al. (2012) butyltins /bts sudaryanto (2002) malaysia 1.organotins catalyst, stabilizer, preservatives, biocides sudaryanto, isobe & tanabe (2012) polymer additive of electronics, building materials and textile products 2.flam e retar dants philippines prudente (2008) sudaryanto et al. (2002) japan harino et al. (2000) -polymer additive of polystyrene foam, thermal isolation a. polybrominated diphenyl ethers/ pbdes jakarta bay b. hexabromo cyclododecanes/ hbcds benowo, surabaya benowo, surabaya 144 organotins a number of studies have reported organotin chemicals especially butyltins (bts) accumulation in green mussel and fish samples from different locations. based on a study conducted by sudaryanto (2002), the highest concentration of total bts in green mussel was found in jakarta bay (58 ng/g), followed by that from surabaya bay (45 ng/g). similar trend was found in fish species as performed by the most concentrated fish was from jakarta bay (84 ng/g), followed by that from cirebon bay (25 ng/g). this fact is in accordance with extensive use of organotins in the last 40 years, especially in industrial applications either as polyurethane and silicone catalyst, plastic stabilizer, or preservative. other application of bts is as biocide, either agricultural pesticide or antifouling paint in boat vessel and other marine equipment (bennett, 1996; hoch, 2001). commercially, total bts consists of mono-bt, di-bt, and tri-bt with tri-bt pre sents pred om i na ntl y. rel a ti v el y hi g her concentration of bts in seafood from jakarta bay and surabaya, compare to those from other indonesia waters could indicate the accumulation of bts emission from industrial activities and/or leaching from marine vessels in those areas. in comparison to other regions of the world, bts contamination in indonesian seafood is much lower than that from malaysia, thailand, hongkong, taiwan, korea, japan, and usa (chen, huang, ho, chang, & liu, 2008; harino, fukushima, & kawai, 2000; hong, takahashi, min, & tanabe, 2002; prudente, 2008; short & sharp, 1989; sudaryanto, 2002) as presented in table 1. the bts concentration in indonesian seafood is comparable to those from cambodia and vietnam, eventhough the use of organotin has been banned by international maritime organization (imo). since indonesia has not established regulation concerning this compound and therefore, possible emission of this harmful chemical should be taken into account. table 1. emerging pollutants from industrial emission in seafood from indonesian water and worldwide (continued) note: nd: not detected; ww: wet weight; lw: lipid weight; dw: dry weight dwiyitno/squalen bull. of mar. and fish. postharvest and biotech. 14 (3) 2019, 141-153 group and com m on us e jakarta bay (2003) green mussel <0.3-1.2 ng/g lw cambodia (2004) green mussel <0.3 ng/g lw china (2004) green mussel <0.3-2.2 ng/g lw hongkong (2004) green mussel <0.3-2.3 ng/g lw india green mussel <0.3 ng/g lw -green mussel <0.3-5.7 ng/g lw -blue mussel <0.3-22 ng/g lw korea (2005) blue mussel <0.3-6.6 ng/g lw malaysia (2004) green mussel <0.3-1.4 ng/g lw philippines (2004) green mussel <0.3 ng/g lw vietnam (2004) green mussel <0.3-1.1 ng/g lw jakarta bay (2003) green mussel <0.1-0.8 ng/g lw cambodia (2004) green mussel <0.1 ng/g lw china (2004) green mussel <0.1-0.7 ng/g lw hongkong (2004) green mussel <0.1-4.3 ng/g lw india green mussel <0.1 ng/g lw -green mussel <0.1 ng/g lw -blue mussel <0.1 ng/g lw korea (2005) blue mussel <0.1-13 ng/g lw malaysia (2004) green mussel <0.1-2.1 ng/g lw philippines (2004) green mussel <0.1 ng/g lw vietnam (2004) green mussel <0.1-0.6 ng/g lw 3. arom atic s e ns itize rs di-isoprophyl naphthalenes/dipns jakarta bay green mussel 40-231 ng/g w w (2012-2013) fish 2-143 ng/g w w -pcbs substitute shrimp 3-13 ng/g w w -thermal paper crab 2 ng/g w w -solvent for dye japan (2005-2006) fish 9-1,000 ng/g w w terasaki et al (2008) jakarta bay green mussel nd -75 ng/g w w (2012-2013) fish nd-27 ng/g w w shrimp nd-5 ng/g w w crab 13 ng/g w w japan (2005-2006) fish 3-66 ng/g w w terasaki et al. (2008) dw iyitno et al (2016; 2017) phenylmethoxy naphthalene /pmn dw iyitno et al (2016; 2017) c. decabromo diphenyl ethane/ dbdpe isobe et al (2012) japan (2003-2008) d. bis-tri-bromo phenoxyethane/ btbpe isobe et al (2012) japan (2003-2008) species conce ntration re fer ence 2. flam e r etardants -polymer additive of electronic circuits com pound location (year) . . . . . 145 data on bts pollutants reported in the present article are predominantly conducted before 2000. however, few current studies showed that the accumulation of bts in marine ecosystem still occur in indonesian waters. razak (2005), for example, reported elevation of tributyltin (tbt) concentration in jakarta bay sediment based on 2003 sampling campaign (500-600 ng/g ww) compared to that in 1998 (190 ng/g ww). furthermore, undap (2016) reported bts accumulation in bitung bay of 667 ng/g dw, elevated in comparison to that from the earlier study of 425 ng/g ww (undap et al., 2013). sediment bts at concentration of 88-593 ng/g dw was also reported recently from patani, thailand (hajisamoh, nur, siddique, & shakya, 2018). this fact suggests that bts are still used in the present time. in general, bts concentration in mussel is relatively higher than that of fish species, possible associated with their different feeding behavior. flame retardants flame retardants are contaminant typically present from industrial emission. there are three main rec ogni zed g roup s of f l am e ret ardan ts i .e. polybrom inateddiphenyl ether (pbde), hex abro m ocy cl odo deca ne (h bcd) , and tet rabromobisphenyl a (tbbpa). pbde has been widely applied as polymer additive of electronics and building materials and also textile products, while hbcd is the main additive of polystyrene foam and thermal insulation. due to their toxicity and persistence in the environment, pbdes have been banned by the stockholm convention in 2013, while hbcds joined the list of restricted chemicals/pops (ecc, 2016; fayiga & ipinmoroti, 2017; poma, binelli, volta, roscioli, & guzzella, 2014). a number of flame retardants are reported to contaminate seafood species in indonesian waters and other regions, i.e. pbdes, hbcds, bis-tri-bromo phenoxy ethane (btbpe), and decabromo diphenyl ethane (dbdpe) as presented in table 1. based on the investigation in seafood species, pbdes was identified as the most concentrated flame retardants, followed by hbcds, dbdpe, and btbpe, respectively. relatively low concentration of dbdpe and btbpe contaminants in seafood might be associated with the relatively minor and limited use of this compound since they are lately replaced by tbbpa as new flame retardant applied for electronic ci r cui ts. in v esti gati o ns on f l am e re tarda nts contamination in indonesian seafood are relatively less, at which jakarta bay is the most interesting environment for the study (sudaryanto et al., 2012). other studies conducted in surabaya (ilyas et al., 2013) and lampung (sudaryanto, 2019) revealed flame retardants concentration was much lower than that of jakarta bay. in comparison to other regions, concentration of flame retardants in indonesian seafood is comparable to that from cambodia, malaysia, and vietnam, but much lower than that from china, hongkong, korea, japan, india, and usa (table 1). as performed in bts contaminants, concentrations of flame retardant in mussel are also relatively higher than that of fish species. aryl hydrocarbons aryl hydrocarbons are reported as relatively new eps and very limited identified in aquatic ecosystem. these contaminants are first reported by terasaki, fukazawa, tani, & makino, m. (2008) as typical organic pollutants from paper industries in himeji coast and shizouka coast, japan. they include di-isoprophyl naphthalenes (dipns), phenylmethoxy naphthalene (pm n),di m ethy l di ph enyl m etha ne, d i m eth yl phenylethane, bismethyl-phenoxyethane, m-terphenyl, chloromethyl phenoxymethyl phenoxyethane, and benzylbiphenyl. due to global ban of pcbs usage, dipns are mainly produced to replace various use of pcbs in industrial applications, as well as stabilizer of carbonless/thermal-paper, heat carrier and pesticide solvent (suzuki et al., 2007). there is no official data of dipns production globally, but japan has produced them since 1970s with a production rate of approx. 6,000 ton/year (suzuki, matsumura, nakano, & imaishi, 2012). contamination of dipns in seafood samples from indonesian water was so far only reported by dwiyitno et al. (2016). the samples include green mussel, fish, banana shrimp, and blue crab from jakarta bay. based on their study, dipns concentration in fish species are relatively low (2-143 ng/g ww fish tissue) compared to that from himeji coast, japan (9-1,000 ng/g ww) as presented in table 1. the relatively high concentration of dipns in fish from himeji coast could be attributed to the high load of dipns emission in this area since not only paper industries are located, but also paper recyclers. earlier study reported that paper recycling could reduce 50% dipns concentration in thermal paper (jamnicki, lozo, rutar, & barušiæ, 2012). study in jakarta bay also reported that green mussel accumul ates double concentrations of dipns compared to fish species, due to mussel is known as filter feeding species. lower concentration of dipns contaminant was also found shrimp and crab samples (dwiyitno et al., 2016). howev er, no regulation concerning either maximum residue limit (mrl) or tdi dwiyitno/squalen bull. of mar. and fish. postharvest and biotech. 14 (3) 2019, 141-153 146 of dipns is established and therefore the health risk could not be estimated. the only study on the accumulation of pmn contaminant in indonesian seafood was also reported by dwiyitno et al. (2016) from jakarta bay ecosystem. they found that pmn concentration in fish samples was relatively low compared to that from shizouka coast, japan as reported by terasaki et al. (2008). this fact could be attributed to the different pollution loads between those areas as could be seen from the contamination level of pmn in sediment sample in shizouka was much higher (3,700 ng/g ww) than that in jakarta bay (1,600 ng/g toc) as reported by terasaki et al. (2008) and dwiyitno et al. (2016). similar to dipns contaminant, concentration of pmn in green mussel was relatively higher than that in fish, crab, and shrimp. even though m-terphenyl was not detected in seafood from jakarta bay, this compound is present in water and sediment samples as reported by dwiyitno et al. (2015) that indicates contamination occurrence at lower concentration and/or less persistence than dipns and pmn. this could be seen from the lower octanol/water coefficient (log kow) of mterphenyl (5.52) compared to that of dipns (6.08). plasticizers and antioxidants are among other industrial emission frequently contaminating aquatic ecosystem (debonde, cossu-leguille, & hartemann, 2011). dsikowitzky et al. (2016) reported trimethyl pentanediol diisobutirate (txib), bisphenol-a, tri aceti ne, and tri ethyl ci trate are pl asti ci zer compounds frequently identified in surface water of jakarta bay. triethylcitrate is also employed as food additive. txib was detected at relativ ely high concentrations (up to 6000 ng/l). additionally, bisphenol-a is also commonly applied as plasticizer and dsikowitzky et al. (2016) detected in jakarta river water at concentration exceeds the ec50 (13.9 ng/l). antioxidants were not detected, but the derivative of buylated hydroxytoluene/bht, known as 3,5-di-tert.butyl-4-hydroxybenzoic acid and 3,5-di-tert.-butyl-4hydroxybenzaldehyde were detected in nearly all jakarta river water (dsikowitzky et al., 2016). bht is an antioxidant commonly used in packaging materials, adhesives, cosmetics, and ppcps. other antioxidant commonly used in various products is butylated hydroxyanisole (bha). however, neither plasticizers nor antioxidants are detected in seafood species from jakarta bay. they might be used in less amounts or more rapidly degraded in water compartment. health hazard of the priority pollutants organotins bts c om pound m ay conta m i nate aq uati c ecosystem through leaching from the antifouling paints used in boat, ship, and other marine equipment, from the pvc products disposal, as well as from the runoff of farming fields. in the environment, organotins have bioaccumulative potential and the predominantly compound of tbt may degrade into derivative products mbt and dbt with the similar toxicity properties (who-ipcs, 1999). tbt, dbt, and mbt have been detected in different marine species including fish, mollusks, crustaceans, and cephalopods in different regions. organotin compounds could pose harmful effects on a variety of aquatic organisms, even at low concentrations and are also suspected to disrupt human endocrine. their toxicological effect may cause abnormalities in male reproductive systems and disrupt the critical function of human immune cells (atsdr, 2005). recent findings have shown biologically significant effect of organotins in human blood samples from the usa, and finland, as well as in human liver from poland (barbosa, ferrão, & graceli, 2018). several investigations have revealed that seafood is the primary source of human exposure to organotin compounds (de araújo et al., 2018). this could be attributed to tbt leach from antifouling paints from commercial vessels and/or due to the persistence of tbt in sediments. since the majority of fish in indonesia are captured from the sea, it becomes necessary to assess the levels of organotin compounds in seafood at the markets in order to ascertain their potential health risks to the public. through the ingestion of polluted seafood, organotin compounds may enter the human body. sudaryanto (2002) has estimated contamination level of organotin from indonesian seafood based on tolerable average residue level (tarl) approach. based on the bts tolerable daily intake (tdi) value of 250 ng/kg bw/day (efsa, 2004), and to bts concentration in green mussel from sudaryanto (2002) which are 3.7-64 ng/g for green mussel and 3.3-84 ng/g for fish species, the daily intake of bts to individual 60 kg indonesian consumer is estimated between 154 and 2,662 ng/day bts through green mussel consumption and 137-3,494 ng/day bts via fish. this estimated indonesian daily intake of bts through seafood is relatively low in comparison to that of other country such as japan (3,000-100,000 ng/person/day), thailand (228-45,714 ng/person/day), and philippines (2,361-68,312 ng/person/day) as compiled by sudaryanto et al. (2002). as a comparison, if the bts exposure around jakarta bay is calculated based on the more recent consumption data reported by dwiyitno et al. (2017) i.e. 2.5-25 g/person/day for green mussel and 6.6-53 g/person/day for fish species, the bts exposure for consumer in this area is estimated as 9.25-1,600 ng/person/day from green mussel consumption and 21.78-4,460 ng/person/day dwiyitno/squalen bull. of mar. and fish. postharvest and biotech. 14 (3) 2019, 141-153 147 from fish. these number are still below the tdi of 15,000 ng/person/day. tbt concentration in blue mussel (mytilus galloprovincialis) and fish (tapes spp) over the tarl has been reported from other region such as from the lagoon of venice, italy (zanon, rado, centanni, zharova, & pavoni, 2009). flame retardants flame retardants, such as brominated flame retardants (bfrs) may be released into the aquatic environment during their production emission or the disposal of plastics and electronic wastes that contain these compounds. bfrs such as pbdes and hbcds have gained public attention as eps due to their characteristics such as persistency, bioaccumulative properties, and possible harmful effects to wildlife and human. toxicological studies have shown that pbdes and hbcds could disrupt thyroid hormones and endocrine, change the neurobehavioral, affect to fetal development, and possible cause cancer in animal study (gill, chu, ryan, & feeley, 2004; hallgren & darnerud, 2002). the wide use of flame retardants in v ari ous dai l y products has resul ted i n thei r accumulation at all environmental compartments (covaci et al., 2006; hites, 2004). as these compounds are relatively lipophilic and persistent (kow: 5.8-7.0), they can easily accumulate and biomagnify through f ood webs. for that reason deca-and hex abromodiphenyl ether are recently categorized as new pops according to stockholm convention on pops in june 2017 (unep, 2017). theref ore, their bioaccumulation and biomagnification in aquatic ecosystems could generate the risk in higher trophic level organisms including human. the commercial pbde products are predominantly composed of penta-, octa-, and deca-bromodiphenyl ether products. each product may elicit different toxicological properties. generally, the penta-bdes seem to cause toxicity at the comparably lowest dose, while much higher doses are required for effects of the deca-bdes. the critical effects of penta-bdes include neurobehavioral disorder (from 0.6 mg/kg body weight) and at higher dose will alter thyroid hormone levels in rats and mice study. exposure of 2 mg/kg body weight octa-bdes showed fetal toxicity/ teratogenicity in rats and rabbits, while 80 mg/kg body weight of deca-bdes revealed thyroid, liver, and kidney morphology disorder in adult animals (hendriks & westerink, 2015). since the official threshold/tdi for bfr compounds has not been established, data of animal studies are used to estimate their bmdl (benchmark dose confidence limit) for calculating margin of exposure (moe). us-epa (1995) and efsa (2011) have suggested bmdl of bfrs compounds, such as bde47 (309 µg/ kg bw/day), bde99 (12 µg/kg bw/day), bde153 (83 µg/kg bw/day), bde209 (1700 µg/kg bw/day), hbcds (790 µg/kg bw/day), and tbbpa (16000 µg/kg bw/ day ). st udi es show onl y deca -bdes l i nk to carcinogenic effects at very high levels and therefore iarc (1990) revealed deca-bdes not classifiable as carcinogenic to humans. based on the animal studies, a critical effect of hbcd was seen in liver and on thyroid hormones (loel 100 mg/kg bw/day). this value can also be furthermore used to estimate its bmdl. however, behavioral effects of hbcd on infantile were observed at concentration of 0.9 mg/kg body weight and a sensitive behavior effects endpoint may also present as for other bfrs. based on the bfrs concentration in green mussel and fish from jakarta bay reported by sudaryanto et al. (2012), moe of these contaminants to consumer around jakarta bay could be estimated. using dietary record in 4 districts around jakarta bay of other study (dwiyitno et al., 2017), i.e. 25 g/person/day for green mussel and 53 g/person/day for fish species, the moe of bfrs for consumer in this area is estimated as 9.5 x 104 and 6.8 x 104 for pbde99 from green mussel and from fish species, respectively and 3.1x104 and 1.3x104 for hbcd from green mussel and from fish consumption, respectively. these moe values are practically much higher than the moe threshold of pbde (2.5) and hbcds (100), indicating consumer is still safe from bfrs exposure through seafood consumption. moes of bfrs contaminant over the threshold have been reported from the minority seafood consumer in portugal (bde99<2.5) as reported by aznar-alemany et al. (2016). aryl hydrocarbons since the structural properties of aryl hydrocarbons are analog to pahs and biphenyls (table 1), they are considered accumulative and toxic in the environment (terasaki et al., 2008). according to stockholm convention, dipns is classified as slow biodegradable compound with t50 in water of 2 months. further toxicity analysis showed ld50 on yellowtail (seriola quinqueradiata) was approximately 2 ml/kg which corresponds to highly aquatic toxicity according to the globally harmonized system. long-term toxicity test showed the no observed effect concentration (noec) study with daphnia is 13 g/l. further bioassay test on rat indicated an increase of liver weight, disturbance of lipid metabolism in the liver, and serum and disturbance of glucose metabolism. even though earlier studies showed that dipns, for example, exhibit toxicity to aquatic animals (s. dwiyitno/squalen bull. of mar. and fish. postharvest and biotech. 14 (3) 2019, 141-153 148 quinqueradiata and daphnia) with ld50 and noec 2 ml/kg and 13 g/l, respectively, those test were not sufficient to establish the exposure limit. however, subchronic exposure on rat showed a lethal dose of 100 µg/kg bw when directly administered into stomach (un 2011; unep 2014). unfortunately, no acceptable exposure limits levels are available (unep, 2014). in contrast with dipn, a bioassay study of pmn in rat has been conducted by nicnas (1999). based on an oral test, pmn showed a lowest observed effect level (loel) concentration for hepatic toxicity of 300 mg/kg bw/day. at this concentration, animal test showed a liver hypertrophy (increase of liver weight). other study revealed that liver hypertrophy is among common response on stress of rat and in long term may induce hepato-carcinogenesis (hall et al., 2012). for this reason, moe for pmn contaminant for consumer around jakarta bay could be estimated based on consumption rate reported by dwiyitno et al. (2017). based on the loel value of pmn 300 mg/ kg bw/day with correction factor of 100 and the estimation of seafood consumption 25 g/person/day for green mussel and 53 g/person/day for fish species, moe of pmn is estimated of 230,760 for green mussel consumption and 142,852 for fish consumption. this value is categorized as fairly safe since the risk threshold for moe of pmn is 10,000 (efsa, 2005). future concern with regard to the adverse effects of eps, global concerns have been undertaken in order to initiate m i ti gati on, especi all y to protect the aquati c environment. different countries have banned the use of tbts since the mid-1980s (konstantinou & albanis, 2004). organotin-based antifouling paints have been prohibited in france for the use on boats less than 25 m long since 1982, followed by north america, uk, australia, new zealand, hong kong, and most european countries a few years later (champ, 2000). a global ban on the application of tbt-based paints was supported by imo since 1 january 2003, and total prohibition by 1 january 2008 (imo, 2001). in europe, deci sion no.2455/2001/ec amended directive 2000/60/ec on water policy to agree 11 hazardous chemicals, including tbts as subject to prohibit the emissions and discharges into aquatic environment. furthermore, decision no. 415/2004/ec prohibits the usage of organotin compounds on ships. in national level, the royal decree 995/2000 of the spain regulated the level of organotins less than 20 ng/l in waste discharges to continental. most developed countries, such as usa, uk, canada, france, new zealand, australia, and the european union have adopted the restrictions on the use of tbt containing paints (birchenough et al., 2002). in 1998, the united states introduced organotin antifouling paint control act to the federal level a leaching rate of 4 g/cm2/day. additionally, the use compounds as food additive has been regulated by the food and drug administration (atsdr, 2005). the use of organotin compounds in taiwan as antifouling agents was restricted to boats under 25 m in length since 2003. however, bt compounds were still identified in water and sediments (zanon et al., 2009). in indonesia, organotins, flame retardants, and aryl hydrocarbons specifically emitted from industrial activities as mentioned in this article have not beenincluded specifically in the regulation of waste management of hazardous and toxic chemicals (indonesian government, 2004). since there is no official regulation, it is likely that organotins still continue to be used as effective biocides and/or used as wood preservatives. since bts are rapidly absorbed by organic materials such as bacteria and plankton, once released to the environment, these compounds are readily accumulated into filter-feeding species and higher organisms such as seafood as well as adsorbed onto suspended particles (harino et al., 2008). it has to be noted that recent findings concerning organotins contaminant is not only limited to bts, but also phenyltins (phts) and dioctyltin (dot). the phts, especially triphenyltin (tpht) has become concern as environment contaminant since it poses harmful effect similar to tbts as endocrine disrupting compounds either to aquatic organism or human. concerning the exposure of organotins, who has regulated a tdi of 0.25 µg/day/kg bw for tbt contaminant, while tdi for tpht is 0.5 µg/day/kg bw (who-ipcs, 1999). in contrast, european set a tdi for total organotins (2 ots) of 0.25 µg/day/kg bw as the sum of bts, tpht and dot (efsa, 2004). therefore regulation concerning the use of tpht needs to be addressed in order to protect the harmful effects to the environment. it has been proved that commercial formulation of flame retardants is dominated by three degrees bromination pbdes i.e., penta-bde, octa-bde, and deca-bde. commercial market of pbdes in 2001 indicated that asian countries consumed as the second (24,650 tons or 37%) after americas (33,100 tons or 49%), while europe was the lowest (8,360 tons or 12%). however, due to toxicological reason, organophosphate flame retardants (pfrs) have recently replaced pbdes. consequently, global concerns about pfr contamination and its impact on aquatic environment and human health have increased. a recent study on human exposure suggested that seven out of eleven target pfr metabolites, including diphenyl phosphate (dphp) dwiyitno/squalen bull. of mar. and fish. postharvest and biotech. 14 (3) 2019, 141-153 149 and bis(1-chloro-2-propyl)-1-hydroxy-2-propyl phosphate/bciphipp (figure 2), were frequently detected in human urine and also the most frequently detected metabolite in both serum and hair (xu et al., 2019). based on limited studies in japan and indonesia, some compounds from aryl hydrocarbons group showed si gn i f i cant concentrati on i n aquati c environment, in particular dipns and pmn. however, global concern to these contaminants seems very minor, hence no regulatory body addresses this compound. specifically to terphenyl, which was also detected in japan coast and jakarta bay (terasaki et al., 2008; dwiyitno et al., 2016), the occurrence to develop complex compound such as polychlorinated terphenyls/pcts (when chlorine is present) must be monitored. this is due to pcts are classified as polyhalogenated compounds previously used in similar applications as pcbs (figure 3), but the industrial production was closed in the 1970s. pcts and pcbs hav e si mi lar chem ical and physi co-chemi cal properties, as well as the toxicological behavior. restrictions of ots and other pollutants will not immediately remove their residue in the environment. tbts and their degradation products for example, are retained in the aquatic compartments where they persist. illegal use of this compound and other prohibited chemicals may also be occured if not supported by law enforcement. for that reason a continued monitoring program needs to be established either in the aquatic ecosystem or potential points of pollution sources in the terrestrial. in indonesia, ministry of env ironment is responsible for the coordination of monitoring environmental pollution, including industrial emission. related ministries and local environmental agencies may contribute for concerning ecosystem, such as ministry of marine affairs and fisheries could be in charge for monitoring aquatic ecosystem in order to assure the safe seafood. however, monitoring management has not been established comprehensively due to some limitati on such as hum an resource ex pertise, supporti ng f acili ti es, budget, and com muni ty awareness (wahjono & satmoko, 2006; yudo, 2016). laboratory approach is another issue concerning eps analysis. since target compounds are present at relatively minor levels (ppb), their quantification requires highly sensitive equipments. the possible interference content from the environment, such as salt content, may cause difficulties in the determination step (brunori, ipolyi, massanisso, & morabito, 2005). an accurate characterization of concentration levels of organotin, particularly tbt and its derivates requires sample preparation and chemical analysis, based-on the analyte (e.g. content of water and lipid). every anal yti cal step (e. g. ex tr acti on, separat i on, derivatization, and detection) potentially can affect the accuracy and precision of the final results (diez, jover, albaiges, & bayona, 2006). in the case of flame retardants, most of the studies reported the concentration of total pbdes. practically, commercial pbdes may present in more than 50 possible congeners and each congener has different physico-chemical properties, accumulation pattern, and toxi cologi cal pathways. for this reason, identification of each pbde congener will be beneficial to estimate the more accurate adverse level on the expose assessment which consequently needs more efforts in the laboratory analysis, such as fractionation/ derivatization step, the availability of concerning certified reference materials (crm) and advanced laboratory equipments. the limited study of the contamination level of aryl hydrocarbons maybe caused by the rarity of supporting toxicological studies of these compounds. additionally, due to limited toxicological data, regulation on tdi threshold of flame retardant is not figure 2. molecular structure of diphenyl phosphate/dphp (left) and bis(1-chloro-2-propyl)-1-hydroxy-2-propyl phosphate/bciphipp (right) figure 3. molecular structure of m-terphenyl (left), polychlorinated terphenyls (middle), and pcbs (right) dwiyitno/squalen bull. of mar. and fish. postharvest and biotech. 14 (3) 2019, 141-153 150 available yet. therefore, it is suggested to conduct bioassay studies either in aquatic organisms or mammalian species to ascertain the safety level of this contaminant group and the possible adverse effects to the aquatic environment and human health. conclusion information of eps in aquatic environment and seafood from indonesia, in particular industrial emission is very limited. based on the literatures, the studies were predominantly focused on organotin compounds, followed by flame retardants. however, the rare studies of organotin contaminants have been conducted in recent years. in general, areas of interest for those studies are situated around java island, followed by sumatera. in comparison to eps in seafood from other countries, eps concentrations from industrial emission detected in indonesian seafood samples are relatively lower than those in developed countries, and the majority of south east asian countries, but comparable to those in vietnam and cambodia. aryl hydrocarbons contamination is the least investigated subject globally, including in indonesia. in general, the concentrations are still below the residual limit to pose adverse effect on human health. toxicological studies suggested that eps emitted from industrial activities has to be concern. all the contaminants basically pose 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(2 009 ). time trend of bu tyland ph enyl-t in contamination in organisms of the lagoon of venice (1 99 9 – 20 0 3 ). e n v iro n me nta l mon ito rin g & assessment. 152, 35–45. doi 10.1007/s10661-0080294-6. dwiyitno/squalen bull. of mar. and fish. postharvest and biotech. 14 (3) 2019, 141-153 https:// https:// http://www.epa.gov/fedfac/emerging-contaminants-andhttp:// http://www.inchem.org/documents/cicads/cicad14.htm. https://doi.org/10.1016/ 79 squalen bull. of mar. and fish. postharvest and biotech. 13 (2) 2018, 79-84 www.bbp4b.litbang.kkp.go.id/squalen-bulletin squalen bulletin of marine and fisheries postharvest and biotechnology issn: 2089-5690 e-issn: 2406-9272 copyright © 2018, squalen bmfpb. accreditation number: 21/e/kpt/2018. doi: http://dx.doi.org/10.15578/squalen.v13i2.297. physicochemical characteristics of sodium alginate extracted from turbinaria sp. and sargassum sp. rinta kusumawati,1 jamal basmal1, and bagus bandol utomo1 1)research and development center for marine and fisheries product processing and biotechnology jl. ks. tubun petamburan vi, jakarta 10260 indonesia abstract characterization of sodium alginate extracted from two species of brown seaweeds (turbinaria sp. and sargassum sp.) harvested from binuangeun beach, banten, has been conducted. the aim of the study was to evaluate physicochemical characteristics (moisture, whiteness, viscosity, and functional groups) of sodium alginates extracted from turbinaria sp. and sargassum sp. extraction was conducted in acid solution with the following steps: washing, acid extraction, bleaching, alginic acid conversion, sodium alginate conversion, dehydration, and drying. each extraction was conducted in duplicate using approximately 1 kg of the dry raw materials (turbinaria sp. and sargassum sp.). results of the analysis showed that the yield of sodium alginate powder extracted from sargassum sp. was 24.56+0.56% (w/w) with moisture content of 12.69+1.24%; whiteness degree of 43.80+1.71%; and viscosity of 143.43+3.25 cps, while the yield of sodium alginate powder extracted from turbinaria sp. was 22.69+2.12% (w/w) with moisture content of 14.77+2.55%; whiteness degree of 23.77+0.68%; and viscosity of 133.67­+4.04 cps. meanwhile, the commercial sodium alginate was identified to have moisture content of 16.07+0.09%, whiteness degree of 29.37+0.55% and viscosity of 102.67+4.04 cps. this indicates that physicochemical characteristics of sodium alginate extracted from sargassum sp. is better than those extracted from turbinaria sp. and commercial alginate since it had lower moisture content as well as higher whiteness degree and viscosity. keywords: acid extraction, sodium alginate, sargassum sp., turbinaria sp. *corresponding author. e-mail: tanjung.1979@gmail.com article history: received: 8 january 2018; revised: 20 july 2018; accepted: 25 august 2018 1. introduction alginate is a hydrocolloid extracted from brown seaweed. according to bps (2018), indonesia imported about 1,650 tons of alginate every year which was entirely supplied from imports. as much as 50% of the imported alginate was used in textile industry, 30% for food, 6% for paper production, 5% for welding rods production, and the other 5% for pharmaceutical purposes. brown seaweed, as a raw material for alginate extraction, can be found in indonesian waters. two types of brown seaweeds which grow predominantly in indonesia are sargassum sp. and turbinaria sp. however, not similar to eucheuma cottonii which has been cultured successfully cultured in many areas in indonesia, turbinaria sp. and sargassum sp. has not been cultured intensively (purnomo et al, 2017). therefore, in order to develop alginate industry in indonesia, improving the method for alginate for alginate extraction to produce good quality product is very important, besides providing the high capital investment for the raw material sustainability. there had been several research reports on alginate extraction using acid followed by additional of formalin solution (reyes-tisnado, r.,hernandezcarmona, g., montesinos, y. e. r., higuera, d. l. a., & gutierrez, f.l. 2005, jelynne, p., tamayo, & del rosario, e.j., 2014, viswanathan & nallamuthu, 2014). other methods were reported using acid solution for extraction and dehydrated using suitable solvent to form fibrous material (murdinah et al, 2005; husni, a., subaryono, pranoto, y., tazwir, & ustadi. 2012, latifi, a.m., nejad, e.s., & babavalian, h., 2015). research for alginate production commonly used sargassum sp. as a raw material, however, http://www.bbp4b.litbang.kkp.go.id/squalen-bulletin http://dx.doi.org/10.15578/squalen.v13i2.297. mailto:tanjung.1979@gmail.com 80 r. kusumawati, j. basmal, and b.s.b. utomo/squalen bull. of mar. and fish. postharvest and biotech. 13 (2) 2018, 79-84 alginate can also actually be extracted from turbinaria sp. although the resource of turbinaria sp. is still abundance in indonesia, the experiments on alginate extraction in indonesia from this species is still limited. extraction using acid added with isopropyl alcohol (ipa) was chosen in in this study to avoid the use of formalin which is not recommended to be used in food processing. in addition, the use of ipa is able to speed up the drying process as well. jothisaraswathi, s., babu, b., and rengasamy, r. (2006) reported that alginate extracted from ‘the leafy parts’ of the seaweed has the highest yield than that extracted from stem or entire thallus of turbinaria conoides (j.ag.) kutz. however, it had the lowest viscosity, and molecular weight than alginate from s. binderi and s. baccularia (chee, s., wong, p., & wong, c. , 2010). viscosity and molecular weight are specific characteristics of alginate that can indicating the functional properties of alginate in commercial products. beside the ratio of manuronate and guluronate, other properties such as moisture content, viscosity, whiteness, and functional groups are also important characteristics to determine the quality of alginate. the ai m of th e stu dy wa s to i dent i f y phy si co chem i cal chara cteri sti c s (m o i stu re, whiteness, viscosity, and functional groups) of sodium al gi nates ex tracted f rom turbinaria sp. and sargassum sp harvested from binuangen beach, banten, indonesia. 2. materials and method 2.1. materials the study used sargassum sp. and turbinaria sp. collected from binuangen beach, banten, indonesia, which were harvested during low tide condition. other materials used were commercial sodium alginate (4mular irving ca 92614), sodium alginate powder (aldrich chemistry 9005-38-3), hcl, fresh water, na2co3, naocl, naoh, c3h7oh, and chemicals for fourier-transform infrared (ftir) spectroscopy analysis. 2.2. method 2.2.1.samples preparation the brown seaweeds (sargassum sp. and turbinaria sp.) were washed with fresh water followed by cleaning the samples. the samples were sundried and packed hermetically in plastic bag, then transported to laboratory and stored at room temperature for further experiment. 2.2.2.sodium alginate extraction sodium alginate extraction was conducted based on murdinah et al (2005). kg of dry seaweed, with having moisture content 11.96+0.03%, were soaked in 1% (v/v) hcl with ratio of dry seaweed to hcl solution was 1:30 (w/v), and suspended for 1 hour then neutralized by flowing fresh water. sodium alginate extraction using neutral raw material was conducted in 2% (w/v) na2co3 at 60–70°c for 2 hour, with proportion of dry seaweed to na2co3 solution was 1:30 (w/v).the suspended material from previous extraction step was re-extracted, filtered and the supernatant was mixed with the first extract then bleached by adding 4% (v/v) naocl slowly for 30 minutes. the alginate solution obtained from extraction process was then acidified by adding 10% (v/v) hcl until the acidity of the solution reached a ph of 2 – 3 and then filtered by using vibrator screen. the solution was converted into sodium alginate by adding 10% (w/v) naoh solution until the ph of the solution was 7 – 8. the sodium alginate was then dehydrated using c3h7oh with the proportion of sodium alginate to c3h7oh was 1:2 (v/v) followed by smooth stirring for 30 minutes until solid state of sodium alginate was formed. the solid state of the sodium alginate was dried at 50°c for 10 hours and then milled into powder using 100 mesh of sieve plate. this extraction procedure was applied for both sargassum sp. and turbinaria sp. 2.2.3. mosture content analysis mosture content was analyzed following sni 25341:2015 analysis procedure (bsn, 2015). as much as +2 g sodium alginate powder, were dried for 18-24 hours in the oven at 1050c, then cooled in a desiccator for 30 minutes and weighed after temperature of the samples reach 250c. the value of moisture content is the percentage of the weight ratio of the water evaporated to the initial sample weight. 2.2.4. viscosity analysis viscosity was measured based on brookfiled viscometer manual book using brookfiled lvt model viscometer. 7.5 grams of sodium alginate powder were dissolved in 500 ml of distilled water then stirred slowly at 800c for 30 minutes. analysis was conducted using a spindle number 2 with 60 rpm speed rotation and keeping the temperature of the solution stable. the stable number indicated by the tool was then multiplied by the conversion factor to obtain a viscosity value. 81 2.2.5. whiteness degree analysis w hiteness degree was measured based on whiteness meter manual procedure using kett whiteness meter. measurement using white paper as a standard and powder samples. after measuring absorption of the standard, absorption of the sample measured by filling the sample into whiteness meter chamber until full and compact. the whiteness value indicated by the tool is the percentage of the trend toward the white color of the sample. 2.2.6. functional groups analysis the ftir (perkin-elmer 577) was used to analyze of functional groups of the sample using procedure manual for kbr pellet method and m-80 spedcord. one milligram of sample was crushed with 200 mg kbr until it was homogeneous. the powder was pressed into a thin tablet and placed in a sample pan. infrared uptake was measured at a waveleth of 4004,000 cm-1. 2.2.7. data analysis the experimental design used in this study was com pl ete l y r andom i zed desi g n wi th th ree replications. data obtained were analyzed using msexcel program. 3. result and discussion the yield of sodium alginate extracted from sargassum sp. was 24.56%, higher than turbinaria sp. which was only 14.77%. it was probably due to the difference in the thallus hardness. the thallus of sargassum sp. was softer than that of turbinaria sp. stiger, deslandes, and payr (2004) found that morphological point of view affected the yield of sodium alginate extract. due to their thallus texture difference, extracting alginate from sargassum sp is easier than from turbinaria sp, thus the yield of alginate which can be extracted from sargassum sp was also higher than that from turbinaria sp, stiger et al (2004) also found that morphologically, sargassum sp has softer and more flexible thallus than turbinaria sp. which was strongly embeded to corral reef. extracting alginate from turbinaria probably needs additional treatment to produce an optimal or even maximum yield. it could be done by using higher concentration of extracting solution or temperature. however, further study related to this needs to be conducted. for commercial production, sargassum sp. as raw material has a better opportunity to be developed because this seaweed is more easily found in indonesian coastal area. however, this type of seaweed has not been cultivated commercialy, even though the cul ti v ati on ex peri ment conducted i n 2000 at bi n uang eun b each was qui t e suc cessf ul (kusumawati, 2011). according to soegiarto, a., soelistijo, atmadja, w.s. &mubarak, h. (1978), yield of alginate is depent on the type of seaweed, the environmental condition and season, whereas budiyanto and djazuliu (1997) mentioned that the yield o alginate depended on light intensity, water current, nutritional condition, the age of the seaweed, method of handling as well as extraction techniques. yulianto (1997) stated that pre-treatment which was conducted 24.56+0.56 12.69+1.24 43.80+1.71 143.43+3.25 22.69+2.12 14.77+ 2.55 23.77+0.68 133.67+4.04 16.07+0.09 29.37+0.55 102.67+4.04 0.00 20.00 40.00 60.00 80.00 10 0.00 12 0.00 14 0.00 16 0.00 yield (%) w ater content (%) w hiteness (%) viscosity (cps) sargassum sp. tu rbinaria sp. alginate comme rcial figure 1. yield and characteristics of alginate extracted from sargassum sp. and turbinaria sp. r. kusumawati, j. basmal, and b.s.b. utomo/squalen bull. of mar. and fish. postharvest and biotech. 13 (2) 2018, 79-84 82 before extraction was v ery important factor in determining the quality of the extracted alginate. water content affected the shelf life of the product. nasir et.al (2013) reported that the shelf life of powder are depending very much on the water content which affects especially on microbiological activity and insect infestation. the higher the moisture content the faster is the growth of fungi and also insects infestation, means that the lower the moisture content of alginate powder the longer is its shelf life. sodium alginate powder extracted from sargassum sp. had moisture content of 12.69+1.24%, lower than that from turbinaria sp., which was 14.77+2.55%. since the structure of sodium alginate extracted from both brown seaweed might has some differences, the position of water trapped inside the polymer of alginate may also be different. it needs more research regarding the type and characteristics of the polymer in relation to water trapped in the products. comparing to commercial sodium alginate powder which had moisture content of 16.07+0.09%, the al ginate extracted from sargassum sp. and turbinaria sp. have lower moisture content, meaning that those products might have longer shelf life due to lower microbial activity and growth of fungi. whiteness degree of alginate powder is correlated with the use of alginate in food and pharmaceutical industry. the high degree of alginate powder ’s brightness or whiteness, would give browning effect to the natural color of the main material in formulation. in this study, the whiteness degree of sodium alginate from sargassum sp. was higher than alginate from turbinaria sp. (43.80+1.71 and 23.77+0.68%)as well as sodium alginate commercial (29.37+0.55%). this phenomenon showed similar contend to the moisture content. similarly, viscosity of alginate powder is associated with its function as emulsifier or binder in product formulation in food and pharmaceutical industry. until recently, indonesia is still importing alginate for this purpose because it requires a specific viscosity of alginate. production of alginate powder that meeting the specification needed can support the industrial demand and reduce alginate imports. the viscosity of sodium alginate from sargassum sp (143.43+3.25 cps) was higher than alginate from turbinaria sp. (133.67+4.04 cps). both of sodium alginates have hi gher v iscosi ty v alues than sodium al ginate commercial (102.67+4.04 cps). viscosity is important phy si cal cha racte ri sti c f o r al g i nate , si nce concentrations of alginate in water causing thickening effect during stirring process (mchugh, 1987). in order to get a thickening effect in a product a small amount of high viscosity alginate can simply be added into formulation. quantitative information to describe the functional groups and composition of sodium alginate is very important. this prarameter was analyzed using ft-ir spectroscopy (figure 2). a band at around 3,400 cm1 was attributed to o-h stretching vibration, a band at around 2,900 and 1,600 cm-1 was attributed to c-h stretching to carboxylate group, while a band at around 1,400 and 1,600 cm-1 indicated the presence of carbonyl group (daemi & barikani (2012), gholamipoor et al. (2013), and liu et al. (2016)). the absorbance values of sodium alginate extracted from sargassum sp. and turbinaria sp. were not at the same band, indicating that they had different ability in absorbing figure 2. ft-ir spectrum of sodium alginic standard, and sodium alginic extracted from sargassum sp. and turbinaria sp. r. kusumawati, j. basmal, and b.s.b. utomo/squalen bull. of mar. and fish. postharvest and biotech. 13 (2) 2018, 79-84 83 infrared light. table 1 is the summary of the absorbance band characteristics around 3400 – 1000 cm1. based on ftir analysis, alginate extracted from sargassum sp. and turbinaria sp. had a characteristic of high absorbance intensity at specific frequency. these specific frequency was very similar to those of the alginate standard used in the experiment, ie. 3,446.38; 2,928.41; 1,628.41; 1.628; 1,420.78, and 1.033.18 cm-1. some frequency were not exactly the same with the standard, they were slightly different (a little bid higher or lower) due to atomic bounding interaction. pereira, l, gheda, s.f., & ribeiro-claro, j.a. (2013) reported that manuronate and guluronate ratio (m/g) of alginate can be tentatively estimated at band value around 1,030 to 1,080 cm-1 in infrared spectra. the result showed that intense broad band of sodium alginate extracted from turbinaria sp. centered at around 1,030 cm-1 of band absorbance, might indicate that the sample contain a lot of guluronate acid (pereira et al, 2013). the band shifting seems to happen on sodium alginate extracted from sargassum sp. which may be caused due to the existence of impurities. to determine which type and quantity of impurities affected the band shifting, further study need to be conducted. 4. conclusion sodium alginate extracted from sargassum sp. has yield of 24.56+0.56%, moisture content of sodium alginate sodium alginate extracted sodium alginate extracted assignments standard from sargassum sp. from sargassum sp. (fertah et al, 2017) (cm-1) (cm-1) (cm-1) 1 3,446.38 3,446.08 3,446.70 hydrogen bonded o-h stretching vibration 2 2,928.41 2,921.50 2,928.81 c-h stretching vibration 3 1,628.00 1,636.21 1,630.35 asymmetric stretching of carboxylate o-c-o vibration 4 1,420.78 1,417.22 1,404.81 symmetric stretching vibration of the carboxylate group 5 1,033.18 1,034.44 1,115.37 due to c-o stretching vibrations no table 1. absorbance band of sodium alginate standard, sodium alginate extracted from sargassum sp. and turbinaria sp. 12.69+1.24%, whiteness degree of 43.80+1.71% and viscosity of 143.43+3.25 cps; while sodium alginate ex t racte d f ro m tur binari a sp. has yi el d of 22.69+2.12%, water content of 14.77+2.55%, whiteness degree of 23.77+0.68% and viscosity of 133.67+4.04 cps. meanwhile, the commercial sodium alginate was identified to have moisture content of 16.07+0.09%, whiteness meter of 29.37+0.55% and viscosity of 102.67+4.04 cps the physicochemical characteristics of sodium alginate extracted from sargassum sp. is better than those extracted from turbinaria sp. and commercial alginate since it had lower moisture content as well as higher whiteness degree and viscosity. references badan pusat statistik (2018). expor dan impor: tabel impor menurut komiditi, tahun 2017. badan standardisasi nasional. 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(2017). extraction and characterization of sodium alginate from moroccan laminaria digitata brown seaweed. arabian journal of chemistry, 10(2), s3711-s3712. gholamipoor, s., nikpour-ghanavati, y., oromiehie, a. r ., & m o ham m adi, m . (2 0 13 ). extractio n an d c h arac terizatio n o f a lg in ate from s arg a ss u m angustifolium collected from northern coasts of persian gulf, bushehr. international symposium on advanced in science and technology, 1-5. husni, a., subaryono, pranoto, y., tazwir, &ustadi. (2012). p eng em b an g an m eto de e k strak si a lg in at dari r u mp u t l aut s a rg as s u m sp . seb ag ai b ah an pengental. agritech, 32(1), 2-3. jelynn e, p.; tam ayo, & del ro sario, e . j. (20 14 ). chemical analysis and utilization of sargassum sp. as substrate for ethanol production. iranica journal of energy & environment, 5(2), 203. jothisaraswathi, s., babu, b., & rengasamy, r. (2006). seasonal studies on alginate and its composition ii: tu rb in a ria c on o id es (j .a g .) ku tz. (f u c ales, phaeophyceae). journal application phycology . springer science business media. 18(2), 161-166. doi.org/10.1007/s10811-006-9089-8. kett. (2011). instant whiteness tester rice and rice powder. california: author. kusu mawati, r. (2011 ). peny ediaan sumber daya hayati kelautan sebagai bahan baku bioenergi dalam perspektif ekosistem. tesis. institut teknologi bandung. p.98-100. latifi, a. m., nejad, e. s., & babavalian, h. (2015). c om pariso n of e xtractio n d ifferent m etho ds o f sodium alginate from brown alga sargassum sp. localized in the southern of iran. journal of applied biotechnology reports, 2(2), 51-255. liu, x., liu, b., w ei, x., sun, z., & w ang, c. (2016). e xtrac tio n , f rac tio n atio n , an d c h em ic al c harac terizatio n o f f u c oid an s from th e b row n seaweed sargassum pallidum. czech. journal of food science, 34(5), 410. mchugh , d.j. (19 87) produ ction and utilization of products from commercial seaweeds. fao fisheries technical paper no. 288, 1-189. murdinah, peranginangin, r., irianto, h.e., amini, s., subaryono, darmawan, m., sinurat, e., dan fransiska, d. (2005). riset optimasi pemanfaatan makro dan mikro alga. laporan teknis. pusat riset pengolahan produk dan sosial ekonomi. jakarta. p.106. nasir, m.; butt, m. s., anum, f. m.;, sharif, k., & minhas, r. (2003). efect of moisture on the self life of wheat flour. international journal of agriculture and biolog, 5(4), 458-459. pereira, l, gheda, s.f., & ribeiro-claro, j.a. (2013). analysis by vibrational spectroscopy of seaweed p o lysacc h arid es with p o tential u se in foo d , p h arm ac eutic al, an d c o sm etic ind u stries. international journal of carbohydrate chemstry , 2013, 1-7. dx.doi.org/10.1155/2013/537202. perkin elmer. (2004). spectrum one user’s guide. connecticut: author. purnomo, a. h., utomo, b. s. b.; wibowo, s.; basmal, j.; aji, n., ....... and octavini, h. (2017). improving seaweed production and processing opportunities in indonesia. research report. p. 6–10. r afsan jan i, h . (20 1 7 ). b ersin erg i men g g arap bioteknologi kelautan indonesia. newsantara. https:/ /newswantara.com/. accessed on august 8th, 2018. r eyes-tisn ad o, r .; h ernan d ez-c armo n a, g.; montesinos, y. e. r.; higuera, d. l. a.; &gutierrez, f. l. (2005). food grade alginates extracted from the giant kelp macrocystis pyrifera at pilot-plan scale. rev. invest. mar, 26(3), 186. soegiarto, a., soelistijo, atmadja, w.s. &mubarak, h. (1978). rumput laut (alga). manfaat, potensi dan usaha budidaya. lon-lipi, jakarta. p 5-15. stiger, v., deslandes, e., &payri, c. e. (2004). phenolic contents of two brown algae, turbinaria ornata and s a rg a s s u m ma n ga re v e ns e o n tah iti (f ren c h po lynesia): in terspecific, o ntog en ic and spatiotemporal variations. botanica marina, 47, 402-409. viswanathan, s. & nallamuthu, t. (2014). extraction of sodium alginate from selected seaweeds and their p h ysio c hem ic al an d b io c hem ic al p rop erties . international journal of innovative research in science, engineering, and technology, 3(4), 10999. yulianto, (1997). ekstraksi lginat dari turbinaria ornate (tu rner) j. gardh (phaeophyceae): s uatu stu di p en d ahu lu an .s emin a r n a s ion a l b io lo g i x v. balitbang sumberdaya laut, p3o-lipi, poka ambon. r. kusumawati, j. basmal, and b.s.b. utomo/squalen bull. of mar. and fish. postharvest and biotech. 13 (2) 2018, 79-84 117 squalen bulletin of marine & fisheries postharvest & biotechnology, 8 (3), 2013, 117-127 copyright © 2013, squalen bmfpb, issn 2089-5690 the quality of alkali treated cottonii (atc) made from eucheuma cottonii collected from different regions in indonesia kualitas alkali treated cottonii (atc) yang dibuat dari rumput laut eucheuma cottonii yang berasal dari beberapa daerah di indonesia muhamad darmawan1*, bagus sediadi bandol utomo1 and raekal amral yuda mulia2 1research and development center for marine and fisheries product processing and biotechnology, jl. k.s. tubun petamburan vi, jakarta pusat 10260, indonesia 2 swiss german university, jalan raya bumi serpong damai sektor 1, tangerang selatan 15321 *correspondence author: m.darmawan22@gmail.com abstract the presented study has been carried out in order to study the quality of alkali treated cottonii (atc) made from eucheuma cottonii which being collected from different regions in indonesia (belitun g, nu sa ten ggara barat and lamp ung). the q uality variables analyzed were the characteristics of raw materials (clean anhydrous weed and impurities) and the characteristics of atc produced (moisture content, ash content, acid insoluble ash content, yield, gel strength, sulphate content, gelling -melting point). the analysis was done in 3 replicates and the data were statistically analyzed using spss 15 package software. results indicated that the raw material from lampung had a better quality than those from nusa tenggara barat and belitung. in addition, the characteristics of atc produced from these three raw materials showed that seaweed from lampung produced better quality atc than those from nusa tenggara barat and belitung in terms of their gel strength, sulphate content and yield. keywords: alkali treated cottonii (atc), eucheuma cottonii, quality abstrak penelitian mengenai pembuatan alkali tretated cottonii (atc) dari rumput laut eucheuma cottoniii dari beberapa wilayah di indonesia (belitung, nusa tenggara barat, dan lampung) telah dilaksanakan dalam rangka mengetahui kualitas atc yang dibuat. pengamatan dilakukan terhadap karakteristik bahan baku rumput laut (clean anhydrous weed dan impurities) serta karakteristik atc yang dihasilkan (kadar air, kadar abu, kadar abu tak larut asam, rendemen, kekuatan gel, kadar sulfat dan titik jendal dan titik leleh). analisa dilakukan dengan 3 kali ulangan dan datanya dianalisa menggunakan paket program (software) spss 15. hasil penelitian menunjukkan bahwa bahan baku yang berasal dari lampung memiliki kualitas yang lebih baik dibandingkan dengan bahan baku yang berasal dari nusa tenggara barat dan belitung. demikian pula dengan hasil karakterissasi atc yang dihasilkan. atc yang dihasilkan dari rumput laut yang berasal dari lampung memiliki mutu paling baik terutama dari segi kekuatan gel, kadar sulfat, dan rendemen yang dihasilkan. kata kunci: atc, eucheuma cottonii, kualitas article history: received: 16 mei 2013; revised: 7 november 2013; accepted: 8 november 2013 available online at website: www.bbp4b.litbang.kkp.go.id/squalen-bulletin 1. introduction seaweed is one of the six primary commodities which being developed recently at several potential locations in indonesia. the production of seaweed has been increasing rapidly in the past following years. according to international finance corporation (2006), seaweed is a major source of income for tens of thousands of small indonesian farmers, collectors, traders, exporters as well as processors. in terms of natural resources, indonesian waters have many advantages on the climatic condition for seaweed growth having warm waters rich in nutrients and extensive coastline with shallow waters which are suited for seaweed production (anonymous, 2004). as an archipelagic nation, indonesia has 1.2 million hectare of potential area that can be used for planting seaweed and produce 16 ton dry seaweed per hectare. if all of that potential areas are used optimally, then the production of seaweed in indonesia can reach 17.774.400 tons per annum (dkp, 2008). the government has realized about the potential to develop this commodity. the minister of marine affairs and fisheries has made a target that in 2014, indonesia can produce 10 million tons of seaweed. that target permalink/doi: http://dx.doi.org/10.15578/squalen.v8i3.37 118 squalen bulletin of marine & fisheries postharvest & biotechnology, 8 (3), 2013, 117-127 copyright © 2013, squalen bmfpb, issn 2089-5690 was double compared with seaweed production in 2012 which was only 5 million tons a year (anonymous, 2013). the world demand on dry carrageenan producing seaweed increases every year. the world annual production of this extract was about 250.000 tones (ifc, 2006), so that the government intends to increase the production of seaweed in indonesia to fulfill the world demand and become one of the biggest exporters in the world. in the other side, the government also intends to uplift the socio-economic status of coastal population by developing seaweed industry. this seaweed farming is very promising to be developed by the coastal people to improve their economic status. the seaweed farming technology is relatively simple and it requires low initial capital investment. with only 45 days of farming cycles with a very good price, seaweed farming can provide a high return of investment for the coastal population. seaweed farming is developed in several regions in indonesia like south sulawesi, bali, lampung, nusa tenggara barat, and many other regions. nusa tenggara barat province is well known as one of the primary seaweed producers in indonesia. in 2006, nusa tenggara barat province produced 32.000 ton of seaweeds and in 2011, the production of seaweed has reached 400.000 ton of seaweeds. lampung, and bangka belitung also have a great number of seaweed productions. several species of seaweed are cultivated at the regions, but mostly only three types of seaweed are cultivated in indonesia (eucheuma cottonii, eucheuma spinosum, and gracilaria sp.). seaweed from those regions is mostly exported to china, denmark, spain, united states, and some other countries. the seaweeds were exported in the form of dry seaweed as a raw material for industries. the price for dry seaweed exported to several countries was very low compared with processed or semi processed products like alkali treated cottonii (atc) or refined carrageenan (rc). the government has a program to restrict exports of raw material from 2012 to increase domestic processed products. the government would control export volumes by only allowing raw seaweed exporters to export a certain amount rather than by introducing an export tax. indonesia supplies about half of the world’s raw seaweed, which is used by the food industry and as an ingredient in health products and cosmetics. the government aims to accelerate the development of the domestic processing industry by 2012 by encouraging the private sector to invest in seaweed processing (ekawati, 2010). alkali treated cottonii is one of the products which are very potential to be developed in indonesia. several atc factories have already existed in indonesia like in bali, malang, and south sulawesi. the information about the quality of atc made from eucheuma cottonii is still very limited, though the information is very i mportant especiall y f or seaweed processing companies. this research was carried out to get important information about the quality of atc made from eucheuma cottonii collected from belitung and lampung in sumatera island and from nusa tenggara barat in eastern part of indonesia. 2. material and methods 2.1. seaweed materials seaweed used in this research was collected from three different regions of indonesia (belitung, lampung and nusa tenggara barat), and bought from local seaweed traders or exporters. the dried seaweed was sorted to remove filth or any non-algal materials such as rope, sand, and other materials found in the seaweed. this process is also aimed to remove other seaweed species than e. cottonii. after the sorting process, the seaweed was then packed into plastic bag and sent to the processing laboratory of research center for marine and fisheries product processing and biotechnology in jakarta. 2.2. methods raw material characterization. to characterize the raw materials, two important parameters were evaluated, namely clean anhydrous weed (caw) and the percentage of the impurities. besides, the description of raw materials is thoroughly observed including observation of the size and color of the thallus, the dryness of the seaweed, whether the seaweed is dirty or clean, whether there is still much salt stick in the thallus, and the age the seaweed when harvested. alk ali treat ed co tton ii pr oduct ion and characterization. the processing of atc was started by cleaning the dry seaweed to remove the impurities like sand and salt. the clean seaweed was then placed in a water bath containing of 8% of koh solution for alkali treatment. the temperature was set at 80oc for 2 hours. the sample was washed 3 times with fresh water, chopped around 1 cm long and sun dried. the drying process was done for 3 days. for the purpose of analysis, the product was ground and filtered with 60 mesh filter to make atc powder. the flow diagram of atc processing is presented in figure 1. the quality parameters of atc being evaluated were yield (fmc corp., 1977), gel strength (marine colloids, 1978), moisture content (aoac, 1995), total 119 squalen bulletin of marine & fisheries postharvest & biotechnology, 8 (3), 2013, 117-127 copyright © 2013, squalen bmfpb, issn 2089-5690 ash content (aoac, 1995), acid insoluble ash content (fmc corp., 1977), sulfate content (fmc corp., 1977), gelling and melting point (marine colloids, 1978). the analysis was done in 3 replicates and the data were statistically analyzed by one way analysis of variance using spss 15 package. 3. results and discussion 3.1. raw materials characterization results of raw material analysis of eucheuma cottonii collected from belitung, nusa tenggara barat, and lampung are presented in table 1. the important parameters observed in dry seaweed or raw materials are caw and impurities. these parameters indicate the purity of raw materials that can be used for the processing information. the fao standard for caw of raw materials is less than 30 %. if the caw value is higher than the standard it can be assumed that the raw materials contains many other materials like sand and salt that stick in to the seaweed. the caw test was conducted to see the dry solid content of pure seaweed and as indicator of impurities stick on the seaweed plant (seaplant, 2008). the caw results for raw materials from 3 regions were different one another (table 1). all the caw results indicated that the raw materials from those three regions still have a lot of sand and salt stick in to the seaweed. these values do not meet the requirement of fao standard ie. less than 30 %. the caw value of the raw materials from belitung is the highest. meanwhile the caw value of raw materials from nusa tenggara barat is the lowest. this condition can be affected by the technique of drying process applied by the farmers. most of the seaweed farmers put the seaweed in to the uncovered ground or above the sand to do the drying process. several farmers who already known the good technique to dry the seaweed usually use plastic to cover it from sand or they used para-para made from bamboo or wood to prevent the contact between seaweed and the sand. the impurities values indicated that there were still some materials such as plastic rope or other seaweed species in the raw materials. the seaweed from nusa tenggara barat has the lowest value of caw but it has the highest value for the impurities. normally, it can be happen if the farmers do not pay attention on the purity of the raw materials. the sorting process is not conducted properly. government effort to improve the quality of raw materials in the seaweed production sites has been done through research institutions, extension officers and regional offices. improved methods of cultivation, harvesting time, and handling have been extensively disseminated to the farmers throughout the production figure 1. flow diagram of atc processing dried eucheuma cottonii washing alkali treatment (8% koh ; 80 o c ; 2 hours) washing chopping sun drying atc 120 squalen bulletin of marine & fisheries postharvest & biotechnology, 8 (3), 2013, 117-127 copyright © 2013, squalen bmfpb, issn 2089-5690 regions. this effort is very important to improve the quality of the raw material, to gain the additional value of the seaweed and finally increase the farmer’s income. beside caw and impurities, an observation of raw materials also has been done to get descriptive information about the condition of raw materials from belitung, lampung and, nusa tenggara barat. the description of seaweed from 3 different regions was shown in table 2. the moisture content of atc resulted in this experiment as presented in figure 2 was ranged from 6.62±0.12% to 10.42±0.09%. figure 2 showed that the highest moisture content of atc was obtained by the atc product made from seaweed collected from belitung, while lowest moisture content of atc was by the atc product made from seaweed collected from lampung. all the products had met the moisture content standard made by fao in 2007 (12 %). moisture content of atc from those 3 regions was significantly different (p<0.05). the moisture content of product can be affected by several factors. the drying technique is one of the factors that can affect the moisture content. the drying technique used in this research was by solar drying. the period of time needed to dry the product was different between one and another because it all depends on the weather condition. one of other factors that might influence the water content of atc product is the heat intensity during sun drying. the moisture content of atc is important as the lower the water content, the longer the shelf life or in other words the slower the product to deteriorate. 3.2. ash content table 1. the characteristics of eucheuma cottonii from the three regions table 2. the description of seaweed from 3 different regions seaweed resources impurities (%) belitung 62.57±12.45 5.78±1.45 nusa tenggara barat 44.54±1.69 14.25±1.29 lampung 51.62±0.01 5.36±0.21 clean anhydrous weed /caw (%) source figure description of the sample thallus: huge and enough to be harvested color: yellowish white and and dark brown filth: small amount of sand and soil salt: small amount of salt dryness: dry harvesting period: 45 days thallus: huge and enough to be harvested color: dark brown and yellowish black filth: a lot of sand and soil salt: a lot of salt dryness: dry harvesting period: 45 days thallus: huge and enough to be harvested color: black and dark brown filth: small amount of sand and soil salt: small amount of salt dryness: dry harvesting period: 45 days belitung nusa tenggara barat lampung 121 squalen bulletin of marine & fisheries postharvest & biotechnology, 8 (3), 2013, 117-127 copyright © 2013, squalen bmfpb, issn 2089-5690 ash content analysis was conducted to determine the general mineral content contained in atc product. there are 2 major types of ashing, first, dry ashing and second, wet ashing (marshall, 2010). the ashing type used in this study was dry ashing where water and volatile compound vaporized by heating at 500600 oc. ash content value of a food indicates the large amount of minerals contained in these foodstuffs (apriyantono et al., 1989). sudarmadji et al., (1996) states that the minerals contained in a material can be differentiated into two kinds of salts, which are organic and inorganic salts. chemicals that evaporate during combustion process such as water and other volatile substances will be oxidized to produce co 2 . seaweed contain high mineral such as na, k, cl, and mg. the ash content of atc from different seaweed resources can be seen in figure 3. the ash content of atc resulted in this experiment as presented in figure 3 ranged from 19.82 ± 0.04% to 24.33 ± 0.10%. figure 3 showed that the highest ash content of atc was obtained by the atc product made from seaweed collected from belitung, while lowest ash content of atc was by the atc product made from seaweed collected from lampung. fao standard (2007) for the ash content of carrageenan is 15-40%, meanwhile the food chemical codex standard (1981) for the ash content is 35 % maximum. the ash content of atc made in this research has fulfilled the entire standard mentioned above. statistical analysis indicated that the ash content of atc product made from seaweed collected from 3 regions was significantly different (p < 0,05). the origin of the raw materials and life cycles of the seaweed can influence the amount of ash content in atc product. seaweed is one of the commodities that have high mineral content in it. basmal et al. (2003) reported that seaweed is one of the raw materials used in the industry that has high mineral content such as na+, k+, ca2+, dan mg2+. meanwhile wenno et al. (2012) indicated that the longer life cycles of the seaweed, the higher the ash content of carrageenan produced. the amount of mineral content in seaweed also can be affected by the environmental condition of the area where the seaweed was planted. the water movement supplies nutrients to the seaweed. it also figure 2. the moisture content of atc from different seaweed resources. figure 3. the ash content of atc from different seaweed resources. 122 squalen bulletin of marine & fisheries postharvest & biotechnology, 8 (3), 2013, 117-127 copyright © 2013, squalen bmfpb, issn 2089-5690 helps the seaweed to absorb nutrients, cleaning the dirt, and establish the exchange of co 2 and o 2 . the absorption of nutrients through the entire members of the plant is not causing the nutrients to decrease. this means that the nutrients in the sea are still sufficient, even excessive to the need of seaweed because of good circulation, a run-off from land and water movement (indriani & sumiarsih, 1991). 3.3. acid insoluble ash content acid insoluble ash is material that is insoluble in acid condition (hydrochloric solution). acid insoluble ash content indicates the existence of residual contamination of mineral or metal that cannot be dissolved in acid in a product, such as silica (si), which is found in nature as quartz, stone, and sand. the effect of seaweed resources on the acid insoluble ash of atc can be seen in figure 4. the acid insoluble ash content of atc resulted in this experiment as presented in figure 4 was ranged from 0.02 ± 0.01% to 0.06 ± 0.03%. figure 4 showed that the highest acid insoluble ash content of atc was obtained by the atc product made from seaweed collected from belitung, while lowest acid insoluble ash content of atc was by the atc product made from seaweed collected from nusa tenggara barat. fao standard (2007) and food chemical codex standard (1981) standard for the acid insoluble ash content of carrageenan is less than 1 %. the acid insoluble ash content of atc product made from seaweed collected from 3 regions has suited the standard required above. statistical analysis showed that there was no significant difference for the acid insoluble ash content of atc product made in this research (p > 0.05). factors that can affect the amount of acid insoluble ash content are the raw materials handling process and the process to produce the atc. the sorting process whether in raw materials handling process or in processing of atc is an important step avoiding the high value of acid insoluble ash content. high acid insoluble acid indicates the contamination of mineral residue or acid insoluble metal that cannot be reduced optimally during the processing (syamsuar, 2006). low values indicate that the atc chip produced were not contaminated during the process (suryaningrum et al., 2003). 3.4. yield yield is one of the important parameter. yield is the ratio between the final product, which is atc chips and the initial raw material (dried seaweed) that is used and multiplies with 100%. calculating the percentage of yield is very important because the quality of the raw materials for manufacturing atc can be seen from the value of the yield that is produced from the alkali treatment. the effect of seaweed resources on the yield of atc can be seen in figure 5. yield of atc that is produced in this research ranged from 30.01 ± 2.49% to 41.33 ± 1.04%. lampung has the highest value of yield among the other sources. the statistical analysis resulted that there was a significant difference in atc yield at different seaweed resources (p < 0.05). according to the tukey hsd test, there were a significant difference in yield of atc between belitung and nusa tenggara barat and also between belitung and lampung, but there were no significant difference between nusa tenggara barat and lampung. figure 4. the acid insoluble ash content of atc from different seaweed resources. 123 squalen bulletin of marine & fisheries postharvest & biotechnology, 8 (3), 2013, 117-127 copyright © 2013, squalen bmfpb, issn 2089-5690 the yield of atc product can be influenced by several factors such as the processing technique and the raw materials used in the process. basmal et al. (2009) states that the yield of carrageenan can be influenced by temperature and time used in the extraction process. meanwhile, istini & zatnika showed that yield of carragenan can be enhanced by the enhancement of koh concentration used in the process. chapman & chapman (1980) explained that the climate, method of extraction, harvesting time, and location of the cultivation influence the amount of yield. in addition, the water content also gives significant effect to yield, as higher water content would lead to heavier product produced in the process. the yield value of atc product can be influenced not only by processing technique but also by the type of seaweed used, the age of seaweed when it cultivated and the environmental condition of the area where the seaweed was planted. (jothisaraswathi et al., 2006). 3.5. gel strength gel strength as stated by glicksman (1983) is one of the important physical properties. one of the important properties of atc powder is the capability of reversible process of changing liquid to solid, or changes the form of solution into gel. the effect of seaweed resources on the gel strength of atc can be seen in figure 6. gel strength of atc resulted in this experiment as presented in figure 6 was ranged from 508,83 ± 7.68 g/cm2 to 602,89 ± 9.09 g/cm2. figure 6 shows that the highest gel strength of atc was obtained by the atc product made from seaweed collected from lampung, while lowest gel strength of atc was by the atc product made from seaweed collected from nusa tenggara barat. figure 6 shows that all gel strength results were within the standard of fao which is required to be larger than 400 grams/cm2. figure 5. yield of atc from different seaweed resources. figure 6. gel strength of atc from different seaweed resources. 124 squalen bulletin of marine & fisheries postharvest & biotechnology, 8 (3), 2013, 117-127 copyright © 2013, squalen bmfpb, issn 2089-5690 several factors affected the gel strength value of carrageenan. gelation of carrageenans, especially kappa, involves two separate and successive steps; coil-to-helix transition upon cooling and subsequent cation-dependent aggregation between helices (campo et al., 2009). glicksman (1983) stated that gel streng th i nc rease propor ti onal l y to 3,6anhydrogalactose but inversely proportional to the sulfate content. the conversion of c-6 in sulfate to 3,6-anhydrogalactose is clearly creating new tough component. 3,6-anhydrogalactose causes the anhydropillic behavior and increases the formation of double helix, so thus yielding high gel strength. the use of alkali in the process of making carrageenan can uplift the gel strength value. the presence of suitable cation, typically potassium or calcium, is an absolute requirement for gelation to proceed (campo et al., 2009). for both iota and kappa-carrageenans, the alkali metal ions (li+, na+, k+, rb+, cs+) are all capable of inducing gelation, but k+ and rb+ are considerably more effective than other ions in inducing gelation at much lower concentrations of both the cation and the carrageenan (funami et al., 2007). syamsuar (2006) also suggest that the other factors influenced on high gel strength are raw material condition, age of cultivation, method of extraction and also the chemical used for extraction. thus, the longer the raw material is stored, the lower gel strength resulted from the seaweed deterioration. so the faster the raw material is processed, the higher the gel strength value. 3.6. sulphate content sulphate content is used as parameter for different type of polysaccharides found in red algae (winarno, 1996). carrageenan is distinguished based on the sulfate content of the carrageenan precursor where k-carrageenan contains less than 28% (doty, 1987). typically, commercial k-carrageenan contains 22% (w/w) of sulphate, carrageenan 32% (w/w) and carrageenan 38% (w/w), although large variations can occur owing to differences between seaweed species or batches (de ruiter& rudolph, 1997). the effect of seaweed resources on the sulphate content of atc can be seen in figure 7. sulphate content of atc resulted in this experiment as presented in figure 7 was ranged from 16.06±0.05 to 36.37±1.68%. figure 7 showed that the highest sulphate content of atc was obtained by the atc product made from seaweed collected from belitung, while the lowest sulphate content of atc was by the atc product made from seaweed collected from lampung. figure 7 shows that all sulphate content results were within the standard of fao which is required to be between 15–40 %. statistical analysis indicated that the sulphate content of atc product made from seaweed collected from 3 regions was significantly different (p<0,05). there is a correlation between the sulphate content with the gel strength of atc produced in this research. the lower the sulphate contents than the higher the gel strength value obtained. experiment conducted by hakim et al. (2011) indicated that carragenan which has highsulphate content has a high gel strength value. gel strength of atc increases proportionally to the 36-a nhydr ogal a ctose con tent but d ecrea ses proportionally to the sulphate content (suryaningrum, 1988). the technique process such as heating treatment and the use of alkali in the process also give significant effect of sulphate content and gel strength of carrageenan. campo et al. (2009) stated that by heating the polysaccharide in strong alkaline media, figure 7. sulphate content of atc from different seaweed resources. 125 squalen bulletin of marine & fisheries postharvest & biotechnology, 8 (3), 2013, 117-127 copyright © 2013, squalen bmfpb, issn 2089-5690 the free 3-oh group is ionized and produces an intramolecular nucleophilic displacement of the sulphate group at position 6. another requirement for the helix formation is the ions present in solution. potassium ions are able to be introduced between double helices and, as they neutralize the charges of sulphate groups, facilitate the approach between them. potassium has also the property of stabilizing the double helix. 3.7. gelling and melting points the gelling point was the temperature at which the atc form gel. this is one of the important factors for the food industry to choose which material should be used. the food producer is able to determine the temperature of the food product by knowing the gelling point, for example jelly, to form gel so that the minimum temperature should be applied to keep the quality of the jelly. in the other hand, the melting point can be used by food industry to determine the maximum temperature the food product that can be stored before it is consumed. the food producer will be able to know the maximum or minimum temperature required to maintain the quality of the food product. the effect of seaweed origin on the gelling and melting points of atc can be seen in figure 8 and 9. the result of gelling point was ranged from 34.17± 0.61 to 35.6±0.70 oc.in the other hand, the result of melting point was ranged from 51,47±0.97 oc to 54,33±0.75 oc. based on the statistical analysis, there are no significant difference either gelling point or melting point of atc from different sources (p>0.05). this result proved that the atc source will not give any significant effect on the gelling and melting point of the atc. suryaningrum (1988) reported that the relation between melting point and the gelling point with the presence of the 3,6-anhydrogalactose are increased proportionally. the higher the presence of the 3,6anhydrogalactose, the higher the gelling and melting point. in the other hand, the relation between 3,6anhydrogalactose is inversely proportional to the figure 8.gelling point of atc from different seaweed resources. figure 9. melting point of atc from different seaweed resources. 126 squalen bulletin of marine & fisheries postharvest & biotechnology, 8 (3), 2013, 117-127 copyright © 2013, squalen bmfpb, issn 2089-5690 sulfate content. this statement also enhanced by syamsuar (2006) who declares that the sulfate presence tends to make the polymer in the form of sol. the forming of sol polymer will make the gelling process hard to be formed. 4. conclusion the quality of seaweed from lampung, belitung, and nusa tenggara barat was different one another. the seaweed from belitung has better quality with the high percentage of caw and low percentage of impurities compared to the seaweed from lampung and nusa tenggara barat. all atc product made from seaweed collected from the three regions meet the fao standard for carrageenan. the seaweed from lampung produce the best quality of atc product in terms of gel strength, yield, and sulphate content compare to those from belitung and nusa tenggara barat. references anonymous, (2004). seaweed harvest. retrieved from http://www.ausaid.gov.au/publications/focus/sep04 / focus_sept04_14.pdf. accessed at 10 october 2013. anonymous. 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(2010). indonesia plans to limit seaweed e x po rts f ro m (2 0 12 ). r etrieved fro m h ttp :// w w w.th e j ak a rtag lo b e .c o m / b u sin ess /in d o n es iaplans-to-limit-seaweed-exports-from-2012/366886. accessed at 2 november 2010. fm c co rp, marine c ollo ids. (19 77). ca rrag eena n. marine colloid monograph number one. springfield new jersey, usa: marine colloid d ivision fmc corporation. f oo d an d ag ricu lture organization (fa o). (2 00 7). c arra ge e n an . p repared at th e 68 th j e cfa & published in fao jecfa monographs 4. p.1-6. food chemical codex (fcc). (1981). carrageenan (p.7475). national academy press washington. funami, t., hiroe, m., noda, s., asai, i., ikeda, s., & nishinarib, k. (2007). influence of molecular structure imag e d with ato mic fo rc e mic ro s c o py o n th e rh eo lo gic a l b e ha v ior o f c a rra g e en a n a q u eo u s systems in the presence or absence of cations. food hydrocolloids, 21, (617–629). glicksman, m. (1983). food hydrocolloids, ii (pp. 74-83). florida: crs press, inc. hakim, a. r., singgih wibowo, arfini, f., & rosmawaty, p. (2011). effect of medium extraction ratio, temperature of precipitation & potassium chloride concentration on quality of carrageenan. journal of marine & fisheries postharvest & biotechnology, 6(1), 1–11. indriani, h. & sumiarsih, e. 1999. aquaculture, processing and marketing of seaweed (7th edition). jakarta: pt. penebar swadaya. istini, s. & zatnika, a. 1991. optimization of semirefine ca rrag ee na n proc es s from e uc he uma co tton ii. proceedings of the scientific meeting of seaweed post harvest technology. book ii. jakarta: research and development center for fisheries. indonesian agency for agricultural research and development. ministry of agriculture, republic of indonesia. international finance corporation. (2006). seaweed fa rmin g in in do n e s ia. mo n ito r: me a s u rin g development results in ifc. issues 7. p. 4. jothisaraswathi , s. b. & babu, r. r. (2006). seasonal stud ies on algin ate an d its c om p asitio n ii : tu rb in ariac on o id es (j . a g .) k u tz. (f u c ales, phaeophyceae). journal of applied phycocolloid. 18, 161-166. marine colloids. (1978). raw material test laboratory s ta n da rt pra ctise . m arin e c o llo id s f m c c o rp . springfield, new jesey. usa. marshall, r. d. (2010). ash analysis. food analysis (pp. 106-108). west lafayette: springer. . 127 squalen bulletin of marine & fisheries postharvest & biotechnology, 8 (3), 2013, 117-127 copyright © 2013, squalen bmfpb, issn 2089-5690 seaplant. (2008). laboratory test procedures for raw-dried s ea we ed a n d s e mi-re fin e d c a rra ge e n a n fro m eucheuma and kappaphycus. retrieved from http:// w ww.seaplan t.net/bimp eag a/im ag es/d ow nloads/ spnf_hb2h%201008%20v3%20ltp.pdf. sudarmadji, s., haryono, b., & suhardi. 1996. analysis of food and agriculture (p. 79-91). yogyakarta: liberty collaborated with pau food and nutrition ugm. suryaningrum, t. d. 1988. study on the quality properties of seaweed commodity types of eucheuma cottonii and eucheuma spinosum (p. 181). thesis. postgraduate programme, bogor agricultural university. bogor. suryaningrum, t. d., murdinah, & erlina, m. d. 2003. effect of alkali treatment and volume of extraction solution to the quality of carragenan from e.cottonii. journal of marine and fisheries postharvest and biotechnology, 9(5), 65-75. syamsuar, 2006. characteristics of carrageenan from eucheuma cottonii at different harvesting time, koh concentration and extraction time. thesis. postgraduate programme, bogor agricultural university. bogor. wenno, m. x., thenu, j. l., & lopulalan, c.g.c. 2012. c h ara c teris tic s o f k a p pa c a rra g e en a n fro m kappaphycus alvarezii at different harvesting times. s h ort co m m un ic atio n. j o u rn a l o f ma rin e a n d fisheries postharvest and biotechnology, 7(1), 61– 68. winarno, f. g. 1996. seaweed processing technology (p. 112). jakarta: pustaka sinar harapan. winarno, f. g. 1997. chemistry of food and nutrition (p. 309). jakarta: pt gramedia pustaka umum. 508 resource limit is reached resource limit is reached the website is temporarily unable to service your request as it exceeded resource limit. please try again later. 51 protein dari biota laut dan potensinya dalam industri yang menggunakan teknologi nano-silika ekowati chasanah*) abstrak peran protein dalam proses pembentukan biosilika yang terjadi di dalam tubuh beberapa biota laut telah menginspirasi peneliti untuk mempelajari dan meniru teknologi tersebut untuk aplikasi industri. dengan proses biologi biasa, protein dari spikula spons dan dinding sel diatom dapat berfungsi menjadi cetakan dan mendirect proses biosilika berskala nano. isolasi dan identifikasi protein tersebut, yang dilanjutkan dengan uji coba proses biosilika secara in vitro, memperlihatkan potensi protein tersebut sebagai biokatalis/agen biologi dalam sintesis silika. sintesis silika secara kimia, seperti dalam produksi bahan resin, katalis, dll, selama ini dikenal merupakan proses yang tidak ramah lingkungan dan boros energi (diperlukan suhu, ph dan tekanan tinggi). karena itu, eksplorasi dan riset lebih lanjut mengenai protein ini dirasa sangat penting mengingat bahan dasar silika, termasuk didalamnya teknologi nano, telah dipergunakan dalam berbagai bidang industri termasuk diantaranya industri pangan, elektronika, otomotif, dan lain-lain. perkembangan riset tentang protein ini, baik yang telah dilakukan oleh peneliti luar negeri maupun yang dilakukan di indonesia, serta aplikasi teknologi nano-silika akan disampaikan pada makalah ini. kata kunci: biosilika, biota laut, protein *) peneliti pada balai besar riset pengolahan produk dan bioteknologi kelautan dan perikanan pendahuluan pembentukan material inorganik dibawah kontrol organisma atau biomineralisasi merupakan fenomena umum di alam. silika merupakan biomineral nomor dua paling banyak di alam dan hanya kalah oleh biogenik caco 3 . biomineralisasi silika di alam didominasi oleh biota akuatik sederhana termasuk diantaranya oleh organisme bersel tunggal (diatomae, radiolaria dan synurophyta) dan organism a multiselular (spons) (sumper & kroger, 2004). biota akuati k l aut tersebut tel ah di kenal m em i l i ki kemampuan mengasimilasi materi inorganik seperti silikon dari lingkungan perairan dan membentuk polimer silika dengan berbagai disain unik untuk memperkuat sel dalam tubuhnya ataupun sebagai bagian dari struktur tubuhnya. kemampuan alami biota ini telah menarik peneliti dan pemerhati industri silika terutama di bidang teknologi nano, seiring dengan perkembangan riset bidang biomimetics, yaitu riset mengenai teknologi pembentukan material dengan sistem biologi atau biomolekul. selama ini, pembuatan peralatan berskala nano dan berstruktur mikro seperti transistor untuk microchip merupakan pekerjaan yang mahal dan demanding process. proses sintesis silika yang selama ini dilakukan di industri memerlukan kondisi proses yang boros energi dan tidak ramah lingkungan seperti suhu, ph dan tekanan tinggi serta menggunakan surfaktan yang dapat mencemari lingkungan. karena itu, studi biosilika, pembentukan polimer silika pada makhluk hidup, menjadi menarik dan sangat intensif dipelajari untuk mengatasi pada proses sintesis silika untuk industri, terutama yang berskala nano. review ini akan mengulas perkembangan terakhir riset biosilika pada diatom dan spons, potensi pengembangan dalam industri serta hasil riset di indonesia. diatom dan pembentukan biosilika diatom adalah mikroalga yang dapat ditemukan pada lingkungan akuatik, dan pertama dikenal sejak lebih dari 180 juta tahun yang lalu. menurut ahli biologi molekuler tanaman chris bowler, berdasar studi filogenetik molekuler dan morfologi, diatom diduga berasal dari eukariot yang diserang oleh eukariot fotosintetik seperti alga merah (bradbury, 2004). diatom merupakan alga bersel tunggal dengan ukuran mikro (1– 500 mm) dengan dinding sel (frustul) kaku yang berisi silika. mikroorganisma bersel tunggal ini membentuk struktur dinding sel dengan pola unik yang berukuran dari nano sampai mikrometer yang disebut juga biosilica nanopatterns, yang sangat spesifik untuk tiap spesies. karena itu, diduga proses biomineralisasi pada diatom tersebut dikontrol secara genetik, dengan perkiraan jumlah spesies lebih dari 100.000 jenis, sehingga pola nano yang unik untuk spesies-spesies tersebut menginspirasi proses pembentukan silika secara biologis (biosilifikasi) pada diatom untuk pengembangan teknologi nano. berdasar kesimetrisan dinding sel, maka diatom squalen vol. 2 no. 2, desember 2007 52 e. chasanah dibedakan dalam 2 grup besar yaitu pennate dan centric diatom. untuk keperluan struktur nano, maka tipe centric, yaitu diatom yang memiliki kesimetrisan secara radial yang sangat berpotensi (parkinson & gordon, 1999). biosilika diatom terutama terdiri dari silika (amorphous hydrated sio 2 ) dengan sebagian kecil makromolekul organik yang sejak lama diduga ikut berperan dalam mengontrol pembentukan silika dengan pola nanonya. sintesis silika pada diatom terjadi di dalam silica deposition vesicle (sdv) yang terdapat pada organ intra sel yang dikelilingi oleh membran silicalema. pembentukan struktur tiga dimensi gel silika di dalam sdv tersebut dipicu oleh ph rendah. prekursor pembentukan biosilika dalam sdv sampai dengan saat ini masih terus dipelajari, tetapi keberadaan asam monosilikat di lingkungan perairan, biasanya dalam kisaran 1– 100µm, secara jelas merupakan sumber utama pembentukan silika. asam monosilikat ditransportasikan ke dalam sel diatom melalui protein transporter yang berfungsi jika ada na+ (disebut sit), tetapi mekanisme penyimpanan asam silikat yang bersifat larut tersebut dan model tranportasinya ke dalam sdv belum diketahui. dari diatom cylindrotheca fusiformis, 3 famili protein dinding sel telah berhasil diisolasi dan dikarakterisasi, yaitu frustulins (ca+ binding protein), pleuralins dan sillafins. selain itu, poliamin berantai ekstrim panjang (lcpas) dengan distribusi panjang rantai yang sangat spesifik pada tiap spesies ditemukan sebagai bagian penting pada biosilika diatom tersebut. frustulin dan pleuralins tidak berperan dalam pembentukan silika, namun hanya akan berasosiasi dengan biosilika setelah terdeposit di permukaan sel, sedangkan sillafins dan poliamin berperan dalam polimerisasi silika secara in vitro dari larutan asam silika. sillafins terikat secara erat dalam biosilika, karena itu, sillafins hanya dapat dilarutkan setelah dinding sel terlarutkan dengan hidrogen fluorida (hf). ketika c. fusiforis dilarutkan dalam anhidrous hf, 3 polipeptida yaitu sillafins–2 (17 kda), sillafins–1b (8 kda) dan sillafins–1a (4 kda) berhasil dideteksi bersama dengan materi non protein, poliamin, yang memiliki berat molekul <3 kda. dari analisis sekuen protein, sillafins a merupakan campuran 2 peptida yang sangat mirip, yaitu sillafins–1a 1 , – 1a 2 yang memiliki tingkat kemiripan tinggi dengan sillafins–1b. yang sangat menarik, 3 peptida tersebut berisi 4 (sillafins– 1a 1 , –1a 2 ) dan 6 (sillafins– 1b) lisin yang dimodifikasi. lisin-lisin hasil modifikasi kovalen yang dikandung dalam sillafins yang merupakan polikation peptida tersebut, adalah lisin pertama, berisi poliamin yang terdiri dari 6-11 ulangan unit n-metil-propilamin. lisin kedua diidentifikasi sebagai –n-n-dimetil-lisin. modifikasi ini akan berpengaruh terhadap aktivitas presipitasi silaffins (sumper & kroger, 2004). spons dan proses biosilifikasi spons dari perairan laut memiliki struktur silikon dioksida berukuran nano pada spikulanya. dari 4 klas spons (phylum porifera), 2 klas diantaranya, yaitu klas demospongiae dan hexactinellida, memiliki spikula yang mengandung silika. klas demospongiae ditandai oleh adanya organisasi seluler dan spikula tipe monoaxonic atau tetraxonic, sedangkan klas hexactinellida ditandai oleh organisasi sincitial dan spikula tipe hexaradiate. dua jenis spikula yang diproduksi yaitu megascleres yang menyusun bentuk utama tulang spons, dan microscleres yang sangat bervariasi dalam bentuk dan ukuran dengan fungsi pen yokon g, ya i tu t i dak seut am a/sepent i ng megascleres. bentuk/tipe, ukuran, dan pola spikula dalam tubuh spons selama ini telah digunakan untuk identifikasi spons, karena diatur secara genetik. tetapi ternyata, kondisi lingkungan juga mempengaruhi keberadaan tipe spikula, sehingga sering ditemukan variasi jumlah, tipe dan ukuran spikula pada jenis spons yang sama (hooper, 2000) pembentukan silika sebagai the building blocks of the skeleton dimulai dengan diekspresikannya enzim silicatein. ketika müller dan grupnya meneliti proses pembentukan spikula pada spons suberites domuncula dengan menggunakan m ikroskop, pembentukan spikula disebutkan merupakan proses yang cepat. spikula dapat tumbuh 5 micron per jam, yang didahului oleh pertumbuhan pada bagian dalam filamen aksial protein silicatein. spikula ini diproduksi dalam sel yang bernama sclerocytes yang berisi filamen organik aksial, dimana silika secara periodik akan didepositkan sampai bentuk dan ukuran akhir dicapai. pada beberapa spesies, filamen aksial ditemukan bebas pada mesohyl dan keberadaannya dihubungkan dengan sekresi ekstraseluler spikula (adamson et al., 2004). secara umum, pertumbuhan spikula diasumsikan merupakan proses 2 dimensi yaitu pertambahan panjang dipengaruhi oleh perpanjangan filamen, sedangkan pertam bahan lebar ditandai oleh penambahan silika. pada klas demospongia, filamen aksial organik yang berfungsi sebagai template (cetakan) diperankan oleh protein yang disebut silicatein. sampai dengan saat ini terdapat 3 jenis protein silicatein yang berhasil diisolasi dari berbagai spons. protein-protein ini memiliki kemiripan dengan enzim cathepsin l (shimizu et al., 1998). dari perbandingan sekuens antara silicatein dan cathepsin l, ternyata pada cathepsin l terdapat 6 53 gambar 1. posisi relative gusus samping ser26 dan his165 pada struktur 3 dimensi protein silicatein dari hasil modeling secara teoritis. yang disebut catalytic triad dari cathepsin yang dibentuk oleh residu cys26, his165 dan asn184, pada protein silicatein, 2 dari residu ini yaitu his165 dan asn184 diko nservasi, sed an gk an c ys 26 menjadi ser26 (sumber : croce et al., 2004). residu sistein yang dihubungkan dengan jembatan disulfide yang dikonservasi juga pada silicatein sehingga diduga keduanya memiliki struktur tiga dimensi yang serupa. croce et al. (2004) memodelkan sisi katalitik silicatein seperti pada gambar 1. gambaran struktur dasar biosilika pada spons digambarkan oleh peneliti joanna aizenberg dari lucent technologies dalam morse, (2006), yang meliputi: struktur dasar dari bulatan-bulatan nano silika (gambar 2a), yang selanjutnya bulatan-bulatan seperti glas (glassy) ini disusun seputar serat protein dan diatur secara sentris, lapis demi lapis. setiap lapis silika dipisahkan dari lapisan silika lain oleh selapis tipis protein (gambar 2b). struktur ini disebut spikula. spikula dibungkus secara bersama, lapis demi lapis (gambar 2c). perekat berbahan silika menahan satuan ini seperti terlihat pada gambar 2d. hasil analisis ftir terhadap peristiwa biosilika ini secara jelas menunjukkan bahwa molekul protein pada filamen aksial berinteraksi dengan gugus si-oh pada matriks inorganik melalui ikatan hidrogen. kemiripan struktur antara silicatein dengan cathepsin l , lebih lanjut dapat dibuktikan dari prevalensi struktur β-sheet pada protein spikula yang dianalisis (β-sheet adalah struktur sekunder protein, selain α-heliks). dari simulasi interaksi antara sisi aktif silicatein dengan asa m ort hosi l i kat atau dal am bent uk an i on terdeprotonasi, rotasi gugus samping serin dan histidin melalui ikatan hidrogen dapat membawa 2 unit asam orthosilikat secara bersama, dan pembentukan dimer (oh) 3 si-o-si(oh) dari segi energi sangat dimungkinkan untuk terjadi (croce et al., 2004) biosilika dan potensi aplikasinya dalam industri apa yang telah dilakukan makhluk hidup seperti spons dan diatom dalam memperkuat struktur tubuh mereka melalui proses biosilifikasi, menginspirasi banyak peneliti. telah diuraikan pada bagian terdahulu bahwa peneliti berhasil mengidentifikasi protein yang bertanggung jawab atas konstruksi pembentukan spikula baik pada tingkat atom maupun molekular. pro tei n i ni sel an j utny a be rhasi l di u j i p ul a kemampuannya untuk membentuk polimer silika secara in vitro dalam kondisi lingkungan yang sangat ringan seperti pada proses biologi biasa. proses yang digunakan sangat jauh dari proses sintesa silika yang biasa dilakukan, dengan melibatkan suhu sangat tinggi, penggunaan surfaktan yang tidak ramah lingkungan serta boros energi. fabrikasi polimer silika berskala nano dibawah kendali protein silicatein dapat diarahkan untuk menghasilkan beberapa jenis bahan semikonduktor, termasuk diantaranya titanium dioksida, yang dapat dilakukan prosesnya pada suhu kamar tanpa adanya bahan kimia berbahaya (caustic). titanium dioksida sangat efisien dalam mengkonversi cahaya menjadi energi dan ini telah digunakan dalam berbagai keperluan elektronika. tentu saja apabila proses pembentukan silika secara biologis (biosilifikasi) ini dapat dilakukan pada skala industri, maka akan gambar 2. strukur dasar biosilika spons (sumber : morse, 2006). squalen vol. 2 no. 2, desember 2007 54 e. chasanah sangat menguntungkan, karena sampai dengan saat i ni se m i kondukto r m asi h di produksi d engan menggunakan suhu tinggi dan bahan kimia berbahaya. teknologi struktur nano ternyata telah ada dalam kehidupan kita sehari-hari. ehrmann & kuzma (2005) melaporkan dalam bidang industri farmasi, teknologi nano telah banyak digunakan dalam berbagai produk pengantar obat sesuai target (drug delivery system via patch), kulit buatan manusia (man made skin), diabetic insulin biocapsules dan terapi kanker menggunakan targetted radioactive biocapsules. pada bidang olah raga, berbagai produk bola seperti bola tenis yang lebih awet, bola golf yang melayang lebih lurus, bola bowling yang lebih keras dan raket tenis yang lebih kuat telah dihasilkan melalui teknologi nano dengan performansi yang lebih baik dari teknologi konvensional. pada industri otomotif, teknologi nano telah digunakan untuk mengisi lubang-lubang yang sangat kecil secara lebih efektif sehingga mobil menjadi lebih mengkilat catnya, dan lain-lain. peralatan yang berhubungan dengan mata dan optical devices lain, melalui sentuhan teknologi nano, telah menghasilkan peralatan menjadi lebih bersih, lebih kering dan lebih kuat. celana yang water proof, dan baju atau sepatu yang memberikan rasa dingin pada musim panas dan menghangatkan pada musim dingin, serta kaos kaki yang tidak bau, juga telah dihasilkan melalui teknologi nano. dalam bidang elektronik, teknologi nano diaplikasikan untuk membuat komputer yang lebih cepat dan lebih powerfull, juga untuk flask drives, kamera digital, cell phone, lcd, led, flexible electronics, dan lainlain. dalam industri pangan, teknologi nano berbahan silika telah digunakan untuk menghasilkan kemasan aktif, penjernih/filter, antikoagulan, dan bahan dasar chip untuk biosensor. pasar produk ini sangat potensial dengan lonjakan penjualan produk kemasan makanan dan minuman berstruktur nano yang sangat signifikan sejak tahun 2004. perusahaan pangan raksasa seperti kraft, altria dan unilever, sebagaimana dilaporkan oleh lux research, telah menggunakan smart packaging hasil teknologi nano silika karena lebih bersifat inert, tahan terhadap suhu tinggi dan oksigen, mampu mencegah serangan bakteri dan virus serta berpenampilan menarik (el amin, 2005 dalam trianawati, 2006). riset tentang protein yang berperan dalam biosilika di indonesia riset mengenai eksplorasi biota laut indonesia untuk biosilika belum banyak dilakukan. tiga riset dasar tentang ekspl orasi protein yang dapat mengkatalisis polimerisasi silika in vitro serta karakterisasi protein tersebut yang diisolasi dari beberapa spons dan 1 jenis diatom dari indonesia telah dilakukan. dari spons asal binuangen dan p. nias, telah diperoleh protein dari spikula yang memiliki berat molekul sebesar 21,4 kda. selain itu, beberapa protein dengan berat molekul 84 kda; 37 kda; 26,6 kda; 18,1 kda dan 16 kda juga berhasil diisolasi. protein tersebut memiliki panjang kurang lebih 0,1 mm dan berdiameter 10 µm. protein yang diisolasi dari binuangen dapat menginisiasi polimerisasi teos (tetraethoxyorthosilicate) sebanyak 15,9 µmol setelah 12 jam reaksi pada suhu ruang dan ph netral (nurjanah, 2007). protein serupa silicatein juga berhasil diisolasi dari spons yang berasal dari p. nias dengan berat molekul 32 kda; 27 kda dan 23 kda, sedangkan protein yang berperan dalam biosilika pada spons dari mataram memiliki berat molekul 15,5 kda dan 18 kda. dari pengujian protein tersebut terhadap inhibitor spesifik menggunakan phenyl methyl sulfonyl fluoride (pmsf), protein-protein yang diisolasi ini diduga memiliki salah satu sisi aktif berupa serin. hasil isolasi protein dari kelima spons menunjukkan bahwa kemampuan melakukan polimerisasi tertinggi adalah sebesar 144 µmol/ml dengan lama reaksi 12 jam. dari studi kinetika, protein tersebut mengikuti kinetika michaelis-menten dengan nilai km sebesar 47,59 µmol/ml dan vmax sebesar 4,7µmol/jam (trianawati, 2007). protein seperti sillafins dari dinding sel diatom chaetoceros gracilis tel ah di isolasi dan diuji kemampuan melakukan polimerisasi silika dengan substrat teos. dari pengujian tersebut diperoleh yield 0,34% atau 64µg/g sampel dan didapatkan 4 pita protein dalam sds-page, masing-masing memiliki berat molekul 44,65 kda; 42,18 kda; 23,88 kda dan 12,07 kda. protein tersebut mampu melakukan polimerisasi teos sebesar 239,74µmol/ml, teos monomer dalam waktu reaksi 24 jam (manurung et al., 2007). penutup peranan protein dalam proses biosilifikasi menjadi semakin penting seiring dengan potensi aplikasi protein tersebut dalam memproduksi berbagai produk berbahan baku silika yang bersifat ramah lingkungan. protein tersebut bekerja seperti enzim dan menjadi cetakan, dengan pola/tipe yang spesifik, tergantung dari jenis atau spesies biotanya, yaitu diatom dan spons tersebut (bersifat genetik). temuan tersebut mengindikasikan bahwa, eksplorasi terhadap protein dari biota laut menjadi sangat penting untuk mendapat protein dengan kemampuan biosilifikasi skala nano dengan pola unik atau pola tertentu seperti yang kita kehendaki untuk aplikasi industri nantinya. 55 hasil riset tentang biosilifikasi pada spons dan diatom di indonesia memang masih bersifat sangat awal dan jauh untuk aplikasi. hasil ini dapat dijadikan pancingan riset selanjutnya, mengingat potensi aplikasi biosilika dalam memenuhi kehidupan seharihari cukup besar. kekayaan biota laut indonesia dengan megabiodiversitas hayati sangat tinggi, sangat berpotensi dalam menghasilkan berbagai pro tei n uni k yang berp eran dal a m pro ses pembentukan biosilika skala nano. daftar pustaka adamson, d.h., dabbs, d.m., morse, d.e. and aksay, i.a. 2004. non-peptide, silicatein á inspired silica condensation catalyst. polymeric materials: science & engineering, 90, 239 bradburry, j. 2004. nature’s nanotechnologist : unveiling the secrets of diatoms. plos biol. 2004. october, 2 (10) : 306 croce, g., frache, a., milanesio, m., marchese, l., causà, m., viterbo, d., barbaglia, a., bolis, v., bavestrello, g., cerrano, c., benatti, u., pozzolini, m., giovine, m. and amenitsch, h. 2004. structural charac terizatio n o f silic eou s spic u les from m arin e sponges. biophys. j. 86 (1) : 526-534 ehrmann, b. and kuzma, t. 2005. real world a p plic a tio n of n a no te c hn o lo gy. c en ter fo r n ano tec hn o lo g y e d u catio n & tech n o log y. pensylvania state university. latest úpdate on june 17, 2005 hooper, j.n.a. 2000. sponguide : guide to sponge collection and identification (version august 2000). http://www.qmuseum.qld.gov.au manurung, a.i., suhartono, m.t., syah, d. and pratiwi, a.r. 2007. catalyzing formation of silica structure isolated fro m in d o n esian m arine d iato m chaetoceros gracillis. proceeding of international seminar and workshop marine biodiversity and their potential for developing bio-pharmaceutical industry in indonesia. morse, d.e. 2006. marine bio-nanotechnology : high performance materials from sponge silicatein. california seagrant college program. research profiles nurjanah, s., welson, suhartono, m.t. and chasanah, e. 2007. preliminary analysis of silicatein like protein from the indonesian sponge catalyzing silica polymerization in vitro. proceeding of international seminar and workshop marine biodiversity and their potential for developing bio-pharmaceutical industry in indonesia p ark in son , j . an d g o rd on , r . 1 9 9 9. b eyo n d micromachining : the potential of diatoms. tibtech, 17 : 190-196 shimizu, k., cha, j., stucky, g.d. and morse, d.e. 1998. silicatein α : cath epsin l-like protein in sponge biosilica. proc natl acad sci, u s a. , may 26; 95(11): 6234–6238. sumper, m. and kroger n. 2004. silica formation in diatoms : the function of long chain polyamines and silaffins. j. mater. chem., 14 : 2059-2065 trianaw ati, l. 2 007 . karak te ris as i p ro te in se ru pa silicatein d ari sp on ge asa l perairan n ias d an lombok. thesis s2, sekolah pasca sarjana ipb, bogor. squalen vol. 2 no. 2, desember 2007 508 resource limit is reached resource limit is reached the website is temporarily unable to service your request as it exceeded resource limit. please try again later. 508 resource limit is reached resource limit is reached the website is temporarily unable to service your request as it exceeded resource limit. please try again later. 63 squalen vol. 6 no.2, agustus 2011 *) peneliti pada balai besar riset pengolahan produk dan bioteknologi kelautan dan perikanan, balitbang kp, kkp; jl. ks. tubun petamburan vi, slipi, jakarta pusat 10260; email: ksp_jamal@yahoo.com penggunaan alat pres hidraulik untuk memisahkan cairan (sap) rumput laut eucheuma cottonii jamal basmal*) abstrak fungsi alat pres hidraulik dalam penanganan rumput laut segar adalah untuk mengeluarkan sebagian cairan/sap dari dalam talus untuk mempercepat proses pengeringan. model alat pres hidraulik yang dikembangkan di balai besar riset pengolahan produk dan bioteknologi kelautan dan perikanan (bbrp2bkp) mempunyai kemampuan daya tekan sebesar 30 ton yang bekerja dengan bantuan tenaga listrik 4 kw h. pada penggunaan gaya tekanan 10 ton/900 cm2 dalam waktu 10 menit yang diberikan pada rumput laut segar sebanyak 10 kg telah dapat dikeluarkan sap sebesar 20,6% dan cairan ini mengandung auksin sebesar 2.000 ppm, giberelin (ga3) sebesar 1.500 ppm, zeatin sebesar 1.200 ppm, dan kinetin sebesar 1.000 ppm. dalam budidaya pertanian, sap telah digunakan secara komersial untuk meningkatkan pertumbuhan tanaman dan memperbanyak produksi buah. abstract: usage hydraulic press to separate sap from eucheuma cottonii. by jamal basmal function of hydraulic press in fresh handling of seaweed is to separate sap from its thallus to fasten its drying process. a device model of hydraulic press has been developed at the research center for marine and fisheries product processing and biotechnology with a pressure energy (force power) of 30 tons at 4 kwh. during the use of force power of 10 tons/900 cm2 for 10 minutes for 10 kg of seaweed, 20.6% of sap was obtained. sap is known to contain 2,000 ppm of auxin, 1,500 ppm of giberelin (ga3), 1,200 ppm of zeatin and 1,000 ppm of kinetine. in horticulture, it has been applied to promote growth rate in plants and to increase the production of fruits. keywords: hydraulic press, sap, eucheuma cottonii pendahuluan mutu bahan baku rumput laut untuk industri sangat dipengaruhi oleh kualitas bibit dan perairan tempat rumput laut dibudidayakan, selain itu juga dipengaruhi ol e h um ur pa nen dan t ekni k pen anga nan pascapanennya. kandungan karaginan dalam talus rumput laut eucheuma cottonii akan mencapai nilai maksimum pada umur panen 45 hari, baik yang dipanen dengan cara dipotong sebagian talusnya maupun yang dilepaskan dari tali pengikat (anon., 2005). pada umur panen 45 hari nilai kekuatan gel akan mencapai maksimum. menurut plashchina et al. (1980) dan pekcan & tari (2008) nilai kekuatan gel merupakan faktor penentu kualitas rumput laut. hal ini disebabkan karena rumput laut dan ekstraknya banyak diaplikasikan dalam produk pangan dan non pangan, terutama rumput laut penghasil karaginan. mengingat tingginya permintaan rumput laut untuk kebutuhan industri dalam negeri maupun untuk memenuhi permintaan ekspor, maka penyediaan bah an b aku r um put l au t ber kual i tas dan berkesi nam bungan m enj adi sanga t penti ng. sehubungan dengan hal tersebut di atas, direktorat jenderal perikanan budidaya, kementerian kelautan dan perikanan berupaya mendukung penyediaan bahan baku dimaksud melalui program peningkatan produksi perikanan budidaya dengan sasaran produksi sebesar 10 juta ton berat basah pada tahun 2015. dari jumlah tersebut, diperkirakan 70% diantaranya adalah jenis rumput laut penghasil karaginan dan 30% sisanya adalah rumput laut penghasil agaropektin/ agar-agar. permasalahan yang timbul dalam penanganan rumput laut segar adalah proses pengeringan yang lama karena umumnya rumput laut dikeringkan dengan menggunakan sinar matahari. untuk rumput laut jenis e. cottonii dibutuhkan waktu hingga 2–3 hari untuk mencapai kadar air 35–38% sesuai dengan standar sni 2690.2:2009. sedangkan untuk rumput laut penghasil agaropektin, persyaratan tingkat kekeringan adalah 15–18%. efek pemanasan global yang terjadi pada akhir dekade ini telah menyebabkan perubahan iklim global sehingga sulit memprediksi musim. hal ini menyebabkan waktu pengeringan dengan sinar matahari menjadi tidak menentu. teknik yang umum digunakan untuk menarik sap dari dalam talus rumput laut adalah menggunakan 508 resource limit is reached resource limit is reached the website is temporarily unable to service your request as it exceeded resource limit. please try again later. 508 resource limit is reached resource limit is reached the website is temporarily unable to service your request as it exceeded resource limit. please try again later. 44   *)    peneliti  pada  balai  besar  riset  pengolahan  produk  dan  bioteknologi  kelautan  dan  perikanan;  email:  ekowati_ch@yahoo.com pengembangan produk kitooligosakarida dari limbah industri perikanan udang secara enzimatis: prospek dan kendala ekowati chasanah*) abstrak sejak  diketahui  memiliki  berbagai  kelebihan,  seperti  kelarutan  dalam  air  dan  aktivitas  biologis yang  lebih  baik  dari  bentuk  polimernya,  oligosakarida  kitosan  atau  disebut  kitooligosakarida menarik  perhatian  peneliti  dan  kalangan  penggunanya  yaitu  industri.  kitooligosakarida  memiliki aplikasi  yang  luas  dalam  bidang  farmasi,  pangan,  dan  pertanian.  produksi  kitooligosakarida yang  memenuhi  tuntutan  fungsi  biologisnya  sangat  tergantung  pada  deraj at  deasetilasi  dan panjang  rantainya.  produksi  secara  enzimatis  selain  menawarkan  produk  kitooligosakarida dengan  panjang  rantai  yang  spesifik  juga  bersifat  ramah  lingkungan  dan  aman.  salah  satu enzim  yang  dapat  diandalkan  dan  bersifat  efisien  sebagai  penghasil  kitooligosakarida  adalah enzim  kitosanase.  limbah  hasil  perikanan  udang  dan  krustasea  lain  merupakan  bahan  baku produk  kitin  dan  turunannya,  termasuk  kitooligosakarida  serta  sumber  enzim-enzim  pendegradasi kitin.  beberapa  hasil  riset  balai  besar  riset  pengolahan  produk  dan  bioteknologi  (bbrp2b) berupa  isolat-isolat  bakteri  penghasil  enzim-enzim  pendegradasi  kitin  salah  satunya  adalah kito sanase,  tekn ik  prod uksi  d an  informasi  karakteristik  enzim  serta  sebag ian  bioaktivitas kitooligosakarida  yang  dihasilkan.  hasil  penelitian  tersebut  memberikan  gambaran  akan  peluang memproduksi  kitooligosakarida  di  indonesia  dan  pengembangan  industri  nutrasetikal  berbasis kitoo lig osakarida.  s alah  satu   kendala  yan g  m asih  d irasa  adalah   belum   ad anya  legalitas penggunaan  kitooligosakarida  untuk  produk  makanan  dan  keperluan  farmasi  meskipun  produkproduk  nutrasetikal  berbahan  baku  kitooligosakarida  sudah  beredar  secara  luas.  aplikasi kitooligosakarida  dan  mikroba  penghasil  enzim  pendegradasi  kitin  sebagai  biokontrol  tanaman pertanian  termasuk  perkebunan,  merupakan  salah  satu  terobosan  yang  bisa  dicoba  untuk dikembangkan    lebih  dulu  karena  tidak  memerlukan  banyak  persyaratan  sebagaimana  aplikasi untuk  produk  makanan. abstract: development of enzymatically produced chitooligosaccharide from shrimp industrial waste: opportunity and challenge. by: ekowati chasanah since identified having better properties such as solubility in water and bioactivities compared to its polymer form, chitooligosaccharide has obtained great attention either from researcher and industries. wide application of chitooligosaccharides, from pharmaceutical, food and agriculture, is very depending on the deacetylation degree and the size of oligosaccharide. enzymatic production is more dep endable in producing specific, safe and en vironmentally friendly chitooligosaccharide. chitosanase is one of chitin degrading enzymes group, having important role in production of chitooligosaccharide. shrimp and crustacean waste which are abundant in in do n e s ia a re imp orta n t ra w ma te ria l fo r ch itin a n d its de riv ativ e p ro d uc ts in c lu din g chitooligosacharide and as a source of chitin degrading enzymes including chitosanase. rcmfppb h a s a c olle c tio n of p o te n tial c h itin de g rad in g mic rob e s fo r p ro d u c in g fun c tion a l chitooligosaccharides for food, pharmaceutical and biocontrol applications. there is a big opportunity for indonesia as chitooligosaccharide producer as we have relatively big amount of raw material from shrimp and crustacea’s waste, and local enzyme. one of the problem is the legality of chitooligosaccharide for food and pharmaceutical products has not been available yet. another non food application such as biocontrol can be developed first since this product does not need strict regulation as for food. keywords: chitooligosaccharide, shrimp waste, enzymatic pendahuluan kitooligosakarida  adalah  potongan  kitin  dan kitosan yang memiliki rantai 20 atau kurang dengan berat molekul lebih besar dari 3900 dalton (terbojevich & cosani, 1998). kitin dan kitosan merupakan polimer alami  yang  bersifat  biodegradable,  dan  banyak diaplikasikan  dalam   berbagai  industri  seperti pertanian, farmasi, pengolah limbah, pangan, dan lain lain. kitin dan kitosan memiliki berat molekul pada 45 squalen vol. 5 no. 2, agustus 2010 kisaran 100 kda–1000 kda. secara alami kitin ada di kulit  udang,  krustasea,  dan  serangga  lain  dalam bentuk matriks dengan protein, mineral, dan materi lain.  kitin juga secara alami ada di zooplankton yang keberadaannya di laut sangat tinggi. sekitar 3% bobot kering zooplankton adalah berupa kitin (båmstedt, 1980 dalam einbu, 2007) ki t i n  di ekst rak  d ari   l i m bah   den gan  c ara dekalsifikasi menggunakan larutan hcl, deproteinasi dengan larutan naoh dan dekolorisasi dengan 0,5% larutan kmno4 dan asam oksalat. sedangkan kitosan merupakan  deasetilasi  dari  polimer  kitin  yang diperlakukan  dengan  larutan  naoh  pekat  (sekitar 50% b/v) dan dipanaskan pada suhu tinggi selama beberapa jam. secara alami, kitosan ditemukan di alam pada dinding sel kapang (fungi) yang tergolong dalam kelas zygomycetes, chlorella sp., dan pada insekta (muzzarelli; 1977; jeauiaux, 1978; plassard, 1997). kitin merupakan polimer yang tidak larut air dan asam lemah, sedang kitosan adalah kitin dalam bentuk terdeasetilasi sebagian (lebih dari 50%) maupun yang 100% bebas asetil, memiliki sifat lebih baik dari kitin dalam kelarutan dan sifat fungsionalnya. kitosan larut dalam asam lemah, dan memiliki aplikasi yang lebih luas dibanding kitin karena memiliki gugus aktif yang lebih banyak. meskipun demikian, dengan sifatnya yang hanya larut dalam asam lemah, maka aplikasi kitosan juga memiliki keterbatasan dalam industri. modifikasi kitosan secara kimia dengan menambah gugus  hidroksil  (suka  air)  secara  kimia  sehingga menjadi karboksilmetil kitosan yang bersifat larut air, menjadi  salah  satu  strategi  dalam  memanfaatkan kitosan secara lebih luas (fawzya et al., 2008). strategi memotong kitosan menjadi potongan yang lebih  pendek  yang  disebut  kitosan  oligomer  atau kitooligosakarida merupakan salah satu strategi untuk mendapatkan kitosan larut air. keuntungan lain adalah bahwa  dalam  bentuk  potongan  dengan  panjang tertentu, kitosan memiliki aktivitas biologis yang lebih baik dibanding dalam bentuk polimernya. semakin tinggi derajat deasetilasinya (semakin sedikit gugus aseti l   ami no)  m aka  sem aki n  bagus  akti v i tas biologisnya karena gugus aktif yang dimiliki bertambah yaitu gugus positif. ki tool igosakarida  dapat  diproduksi  secara enzimatis, kimiawi maupun iradiasi. produksi secara kimiawi dan iradiasi bersifat acak dan tidak terkontrol dengan mayoritas hasilnya adalah glukosamin, bentuk monomer kitosan (won-seok et al., 2002; liu et al., 2007). sebaliknya, penggunaaan agen biologi seperti enzim bersifat spesifik dan terkontrol sesuai dengan spesifisitas masing-masing enzim yang digunakan. dari segi keamanan, penggunaan enzim dapat lebih diandalkan  dan  bersifat  lebih  ramah  lingkungan. namun karena biaya produksi dengan enzim  sangat mahal sehingga harga kitooligosakarida tidak dapat bersaing dengan harga produk sejenis lainnya. karena itu eksplorasi enzim lokal  sangat diperlukan untuk menekan biaya produksi. aplikasi kitooligosakarida lap oran  hasi l   ri se t  ten tang  apl i k asi kitooligosakarida untuk bidang kesehatan dan pangan telah  banyak  dipublikasikan.  panjang  rantai  dan derajat  deasetilasi  merupakan  faktor  yang  sangat penting pada aktivitas biologis kitooligosakarida. efek anti m i kroba  akan  m eni ngkat  sei ring  dengan peningkatan derajat deasetilasi (dd) tetapi menurun seiring dengan panjangnya rantai.  derajat deasetilasi yang efisien sebagai antimikroba adalah lebih tinggi dari  80%,  khusus  untuk  bakteri  diperlukan  derajat deasetilasi  lebih  dari  97%  (dag  et al.,  2007). kitooligosakarida dengan panjang rantai 4 (tetramer) memiliki aktivitas antibakteri terkuat dibanding polimer kitosan dan kitooligosakarida dengan panjang lebih dari 4  (guo-jane et al.,  2000;  sagoo et al.,  2002; chung et al., 2004; chasanah et al., 2008), sedangkan kitooligosakarida yang memiliki rantai panjang seperti hexamer, heptamer, dan oktamer dilaporkan memiliki aktivitas  immunostimulant,  mereduksi  kolesterol plasma dan mencegah penyakit hati pada penderita kecanduan alkohol (hennen, 1996). kitooligosakarida juga dilaporkan berpotensi untuk digunakan sebagai antidiabetes (hyean-woo et al., 2003; liu et al., 2007) karena kitooligosakarida dapat meningkatkan toleransi glukosa, sekresi insulin dan menurunkan trigliserida. pemberian kitooligosakarida 100 mg/l pada sel primer pankreas  yang dikulturkan  mampu  meningkatkan sekresi insulin secara kontinyu dari hari ke-6 sampai ke-14 (liu et al., 2007). dalam bidang peternakan, kitooligosakarida ketika diberikan sebagai bagian dari pakan ayam broiler, dilaporkan memiliki kemampuan untuk meningkatkan performansi  kualitas  daging  ayam  broiler  dengan meningkatkan  sel  darah  merah  dan  lipoprotein densitas  tinggi  dalam  darah  serta  menginduksi penurunan kadar lemak (zhou et al., 2009). kitooligosakarida yang memiliki rantai  2-8 unit monomer dilaporkan memiliki aktivitas antikapang beberapa  pathogen  tanaman  seperti  fusarium oxyporam, phomopsis fukushi, allernaria alternata (hirano & nagao, 1989). hasil penelitian wiœniewskaw ron a  et a l.  (2 007)  m enunj ukka n  ba hwa kitooligosakarida mampu menghambat secara total pertumbuhan virus lucerne mosaic (aimv) dan dalam 46 e. chasanah skala  yang  lebih  rendah  menghambat  tobacco mosaic virus  (tmv)  yang  resisten.  lebih  lanjut dilaporkan bahwa kitooligosakarida pada konsentrasi 0,5% juga menghambat bakteri tanaman clavibacter michganensis subsp.  michganensis  dan  erwinia carotovora subsp. carotovora. hasi l   ri set  b brp2b   m eng enai   pote nsi kitooligosakarida menggunakan isolat dari  lingkungan lokal indonesia (enzim lokal) dapat dilihat pada tabel 1. kerjasama  bbrp2b  dengan  balai  penelitian bioteknologi  perkebunan  indonesia menghasilkan beberapa isolat yang berpotensi sebagai biokontrol kapang  patogen  terhadap  tanaman  perkebunan. pengujian  antagonis  14  isolat  kitinolitik  koleksi bbrp2b terhadap ganoderma sp., kapang patogen tanaman kelapa sawit, memperlihatkan bahwa 3 isolat berpotensi untuk dikembangkan sebagai biokontrol tanaman  tersebut.  ketiga  isolat  yang  berpotensi tersebut berasal dari limbah udang (kpu 2123), dari terasi (t5a1) dan dari spons laut (5a) (gambar 1). penguj ian  l ebi h  l anjut  akan  di lakukan  untuk mengetahui aplikasi isolat ini. hasil-hasil  riset  di  atas  menunjukkan  bahwa ki t ool i g osaka ri da  sanga t  ber poten si   un tuk dikembangkan karena memiliki potensi aplikasi yang sangat baik pada berbagai industri. pada tingkat uji coba  terbatas, potensi  kitooligosakarida  dalam meningkatkan kualitas dan keamanan bahan pangan telah banyak diakui kehandalannya. enzim kitinolitik dari isolat bakteri koleksi bbrp2b laut merupakan sumber terbesar bagi kitin dan mikroba pengurai kitin. kehadiran mikroba penghasil enzim  pendegradasi  kitin  sangat  berperan  untuk proses  daur  ulang  ketika  kulit  krustasea  yang mengandung kitin dibuang pada saat proses moulting yang  berj um lah  rel ati f  sangat  besar  di   l aut. keberadaan  mikroba  pendegradasi  kitin  di  laut didapatkan dari air dan sedimen, juga dari beberapa biota laut seperti spons laut. berbagai limbah industri perikanan berupa kulit seperti kulit udang pun secara alami  sangat  berpotensi  sebagai  sumber  mikroba penghasil enzim pendegradasi kitin (chasanah et al., 2009). tabel  2 memperlihatkan isolat-isolat penghasil enzim  kitinolitik  yang  berasal  dari  limbah  udang, produk  perikanan  tradisional  terasi,  dan biota  laut spons. secara alami, kitosanase dapat ditemukan pada beberapa organisme termasuk  actinomycetes, fungi, tanaman,  dan  bakteri.  di  dalam  tanaman,  enzim kitosanase dapat digunakan sebagai alat pertahanan terhadap fungi patogen, sedang pada mikroorganisme, enz i m   i ni   berf ungsi   san gat  p enti n g  da l am keseimbangan alam, atau sistem daur ulang seperti yang disampaikan di bab terdahulu. tabel 1. aktivitas biologis kitooligosakarida yang diproduksi menggunakan isolat bakteri yang diisolasi dari indonesia no isola t asa l isola t aktivita s kitooligosa ka rida s um be r 1 a cinetob acter s p.    k pu 218 lim bah udang  (k ulit) ak tivitas pengham batan  terhadap sel hela lemah chas anah et al.,  2009 m enghambat staphyloc occ us aureus  dan ps eudom onas aerugenos a; pos itif  m enghambat s el hela fawz y a et al.,  2009a;  fawz y a et al.,  2009b              m enghambat beberapa k apang  patogen kelapa  sawit w idias tuti & chas anah,  2010 3 a erom onas m edia k lu 11.16 lim bah udang                          (k ulit udang) antibak teri (ps eudom onas aerugenos a ); anti k apang  perus ak  produk  perik anan chas anah et al ., 2010 4 b ac illus lic heniformis  m b-2 sum ber air panas   m anado antibak teri (s.typhym urium , p. aeruginos a, s. aureus  dan e. coli ), antik ank er (hela, a549),  im m unostim ulant chas anah et al. (2006;  2008); w ahy uni  et al., 2007) 2 s tenotrophom onas m altophilia kp u  2123 lim bah udang                (k epala udang) 47 squalen vol. 5 no. 2, agustus 2010 gambar 1. uji antagonis isolat bakteri koleksi bbrp2b dengan ganoderma sp.,kapang patogen penyebab penyakit  pada  kelapa  sawit  berturut  turut  dari  kiri  kekanan:  isolat  kpu  2123 dari limbah udang,t5a1 dari terasi, dan 5a dari spons laut. te m p. (o c) ph 1 spons  laut k bj 12sb 30 7 5 p ratitis, 2006 2 spons  laut tpp  11 sa 30 7 7 p ratitis, 2006 3 spons  laut tlp 14 la 30 7 6 p ratitis, 2006 4 spons  laut  3 37 7 1 uria et al ., 2005 5 spons  laut 18-1 37 7 1 uria et al ., 2005 6 spons  laut  t1 37 7 1 uria et al ., 2005 7 spons  laut 15a 37 7 2 uria et al ., 2005 8 spons  laut 34b 37 7 1 uria et al ., 2005 9 teras i jb 4 37 n.a 2 zilda et al , 2006 10 teras i t5a1 37 n.a 1 noviendri et al ., 2006 11 lim bah udang k pu 218 25 5 30 jam chas anah et al ., 2009 12 lim bah udang k lu 115 n.a n.a 2 chas anah et al ., 2009 13 lim bah udang k lu 1116 n.a n.a 2 chas anah et al ., 2009 14 lim bah udang k pu 2124 30 n.a 1 chas anah et al ., 2009 15 lim bah udang k pu 2123 n.a n.a 1 chas anah et al ., 2009 16 lim bah udang k lu 1121 n.a n.a 1 chas anah et al ., 2009 17 lim bah udang k lu 116 n.a n.a 1 chas anah et al ., 2009 sum be r pusta kano. s um be r isola t kode kondisi optim a l e nzim w a ktu produksi (ha ri) keterangan:  n.a:  belum  dianalisis  (sumber  :  fawzya  &  wibowo,  2009). tabel 2. waktu produksi optimal enzim pendegradasi kitin dari beberapa isolat lokal www.ibriec.org uji antagonis isolat  penghasil kitinase terhadap ganoderma sp. kitinase kitinasekitinase sumber  foto:  happy  widiastuti. 48 e. chasanah produksi kitooligosakarida kitooligosakarida dapat diproduksi melalui 2 cara yaitu secara non biologis dan biologis. secara non biologis,  kitooligosakarida  dibuat  dengan  cara hidrolisis kitin dan kitosan dengan bahan kimia seperti naoh dan asam klorida pekat maupun dengan iradiasi (w o n-seo k  et al.,  2002) .  sec ara  u m um kitooligosakarida yang diproses dengan cara ini tidak dapat  digunakan  sebagai  sumber  bahan  bioaktif karena dikhawatirkan adanya kontaminasi senyawa kimia yang bersifat toksik.  penggunaan bahan kimia pada produksi kitooligosakarida selain dikhawatirkan menghasilkan  senyawa  toksik  dan  resiko  bahaya yang lebih tinggi terhadap konsumen dan lingkungan (polusi)  yang  tidak  dikehendaki,  ternyata  juga menghasilkan  rendemen yang lebih rendah. hal ini karena sebagian besar hasil hidrolisis secara kimia adalah bentuk monomer glukosamin dan atau n asetil glukosamin (liu et al., 2007).  kitooligosakarida yang diproses secara enzimatis memiliki harga yang jauh lebih  tinggi  dibanding  yang  diproses  secara  non enzimatis (einbu, 2007). produksi kitooligosakarida secara biologi dengan menggunakan enzim lebih dipilih karena menghasilkan produk yang lebih aman, tidak menimbulkan masalah lingkungan,  dan  bersifat  lebih  spesifik  dengan rendemen kitooligosakarida yang tinggi. karakteristik kitooligosakarida  yang  dihasilkan  seperti  panjang rantai,  distribusi  derajat  deasetilasi  dan  lain-lain sangat  dipengaruhi  oleh  jenis  enzim  dan  waktu hidrolisis apabila digunakan enzim kasar. produksi kitooligosakarida sangat tergantung pada aktivitas dan lingkungan optimum bekerjanya enzim. salah satu contoh produksi kitooligosakarida ketika digunakan enzim kasar isolat klu 11.16 dan kpu 21.23 sebesar 8 u/g kitosan dengan waktu reaksi 3 jam (untuk kpu 21.23) dan 2 jam (klu 11.16) menghasilkan oligomer berukuran 2-6. analisis dilakukan dengan kromatografi pelat  tipis  (tlc)  menggunakan  standar  oligomer ukuran  1-6  unit  glukosamin.  campuran  oligomer tersebut memperlihatkan potensinya sebagai senyawa antikapang aspergillus flavus dan aspergillus niger yang diisolasi dari produk perikanan (chasanah et al., 2010). kitooligosakarida juga dapat dibuat dengan cara m el akuka n  i ra di asi   pol i m er  k i tosa n  den gan menggunakan sinar co-60 gamma. penggunaan dosis tinggi (lebih dari 100 kgy) akan menyebabkan warna coklat  pada  produk.  penurunan  viskositas  kitosan akan terdeteksi secara nyata ketika digunakan dosis sampai dengan 10 kgy. pada penggunaan dosis 100 kgy,  hasil  kitooligomer  yang  didapatkan  berupa campuran  dimer,  trimer,  dan  tetramer  dengan komposisi berturut-turut sebesar 3,6; 3,0 dan 1,8% (won-seok et al., 2002). pel uang dan kend ala p rodu ksi kitooligomer untuk industri potensi bahan baku data produksi udang baik dari perikanan tangkap maupun hasil  budidaya dalam  kurun waktu 2006– 2008 tercatat sejumlah 554.774–646.512 ton, dan dari jumlah  tersebut,  jumlah  yang  diekspor  sebesar 169.328–170.583 ton. produksi rajungan antara tahun 2006–2007  tercatat  sebesar  26.686–30.421  ton (anonim, 2009). udang pada umumnya diekspor dalam bentuk  beku  tanpa  kepala  dan  kulit,  begitu  juga rajungan  diekspor  dalam  bentuk  kaleng  daging rajungan rebus (nasution et al., 2004). karena itu, dari jumlah ekspor tersebut, potensi limbah berupa kulit dan kepala udang diperkirakan sebesar 67.731 –68.233  ton,  dan  dari  industri  rajungan  memiliki potensi  limbah  20.015–22.815  ton.  dari  limbah tersebut,  maka  potensi  kitin  yang  dapat  diekstrak cukup besar yaitu sekitar 21.936–22.762 ton apabila kita mengacu  pada  cho et al. (1998) bahwa pada cangkang atau kulit krustasea terdapat kitin sebesar 20-30%. peluang pengembangan peluang untuk mengembangkan produk bernilai tambah dari  limbah ini sangat besar,  di  antaranya menjadi kitosan dan produk turunan kitosan seperti kitooligosakarida yang memiliki nilai tambah sangat besar.  melihat  peluang  ini,  direktorat  jenderal pengolahan dan pemasaran hasil perikanan telah melakukan identifikasi wilayah atau identifikasi lokasi pendirian pabrik kitin/ kitosan pada tahun 2006 (anon., 2006). beberapa pabrik kitin kitosan skala ukm telah didirikan di beberapa daerah, namun eksistensi pabrik tersebut  tidak  berlangsung  lama.  kendala  yang dihadapi  di  antaranya  adalah  harga  yang  kalah bersaing dengan kitosan luar negeri dan proses yang menggunakan air dalam jumlah besar (komunikasi pribadi). sampai dengan saat ini ada 2 pabrik kitin/ kitosan yang masih aktif yaitu pt. biotech surindo dan  cv.  kimindo  surya  utama.  produk  yang dihasilkan  oleh  pt.  biotech  surindo  di  antaranya adalah kitosan yang memiliki bm rendah antara 40.000 –60.000 sebagai pengawet makanan (chito fresh), sedangkan cv kimindo menghasilkan produk  pupuk organik cair (chito-farm). kitooligosakarida yang diproses secara enzimatis, murni  dan  terkarakterisasi  dengan  baik    berharga 10.000 usd/kg, sementara yang diproduksi secara non  enzimatis  di  pasar  berharga  50–100  usd/kg, hampir sama dengan harga kitosan yaitu 40–100 usd/ kg  (einbu et al.,  2007).  produksi kitooligosakarida dapat  terkarakterisasi  dengan  baik  jika  dilakukan dengan menggunakan enzim kitosanase yang dapat 49 squalen vol. 5 no. 2, agustus 2010 memecah secara spesifik. karena karakteristik enzim sangat dipengaruhi oleh banyak hal, di antaranya oleh mikroba sumber enzim serta lingkungan tempat hidup mikroba  yang  mendukung  fungsi  enzim  tersebut, maka  isolasi  dan  penapisan  enzim  tersebut  tetap diperlukan untuk mendapatkan enzim lokal dengan karakteristik pemecahan yang sesuai dengan target. ketersediaan  enzim  yang  diisolasi  sendiri  dari lingkungan  lokal  indonesia  (enzim  lokal)  sangat diperlukan  untuk  m enghem at  bi aya  produksi. berbagai produk yang berbahan dasar kitosan dan kitooligosakarida  telah  dipasarkan  secara  luas  di indonesia  dan  di  luar  negeri,  di  antaranya  berupa kapsul  sebagai  produk  pelangsing  dan  produk kesehatan lainnya. kendala salah satu permasalahan dalam komersialisasi kitosan  dan  produk  turunannya  serta  aplikasinya dalam berbagai produk adalah isu legalitas. sebagai pengawet produk pangan, kitosan telah diijinkan dan digunakan di jepang dan negara asia lain. sedangkan di  negara-negara  barat  produk  ini  hanya  diijinkan sebagai bahan pembantu pengolahan dan tidak bisa digunakan  secara  langsung sebagai  pengawet.  di indonesia sendiri, penggunaan kitosan dalam produk makanan  telah  diatur  dengan  dikel uarkannya keputusan  kepala  badan  pengawas  obat  dan makanan  ri  no  hk.00.05.52.6581.  dalam  aturan tersebut kitosan dapat digunakan sebagai bahan baku dalam produk pangan tetapi tidak digolongkan sebagai pengawet dalam produk pangan. dalam ijin tersebut, kitosan tidak berfungsi sebagai zat fungsional  dan tidak  dapat  diklaim  sebagai  klaim  gizi  dan  klaim kesehatan. dari aturan ini jelas bahwa produk turunan kitosan  berupa  kitooligosakarida    belum  diijinkan untuk digunakan dan diklaim sesuai aktivitas biologis atau belum masuk dalam lingkup perijinan. meskipun  beberapa  riset  yang  mendukung keamanan  aplikasi  kitooligosakarida  telah  banyak dilakukan, antara lain kemampuan kitooligosakarida untuk  mengikat  materi  lain  selain  lemak  seperti mikronutrisi dan mikroelemen serta efeknya terhadap komposisi flora  usus, tetapi secara umum penelitian tentang efek kitooligosakarida terhadap kesehatan manusia  perlu  lebih  banyak  dilakukan.  hal  ini diperlukan untuk mendukung keamanan konsumsi kitooligosakarida apabila akan dikembangkan sebagai bahan  baku  produk  konsumsi  seperti  pangan  dan nutrasetikal. penutup limbah  perikanan  udang  yang  cukup  tinggi, memiliki potensi untuk dikembangkan sebagai produk oligomer  kitosan  di  indonesia.  dengan  beberapa kelebihannya, luasnya aplikasi dan nilai jual yang jauh lebih tinggi dari bentuk polimernya yaitu kitosan, maka produk ini sangat berpeluang untuk dikembangkan. hal -hal   yang  perl u   di si apkan   sege ra  un tuk pengembangan produk berbasis kitooligosakarida ini adalah legalitas aturan penggunaannya untuk produk pangan dan farmasi. peluang pengembangan produk ki t ool i gosak ari d a  dan   apl i kasi   enzi m -en zi m pendegradasi kitin untuk produk non pangan seperti produk biokontrol patogen tanaman ekonomis tinggi perlu segera diuji lebih lanjut sehingga dapat membuka peluang pengembangan produk benilai tambah dari limbah perikanan udang dan krustasea lainnya. daftar pustaka a n on im .  2 0 0 6.  l a po ra n ditje n p e mas a ra n d a n pengolahan hasil perikanan 2009. departemen kelautan  dan  perikanan. anonim. 2009.   statistik ekspor hasil perikanan.  ditjen p emasaran   d an  p en go lah an   h asil  p erik anan . kementerian  kelautan  dan  perikanan  2009. chasanah, e., fawzya, n., wibowo, s., patantis, g., dan a riyan ti,  s.d .  2 01 0 .  l a po ra n in te rn ke ma ju a n penelitian. departemen  kelautan  dan  perikanan. chasanah,  e.,  ilmi,  m.,  dan  mangunwardoyo,  w.  2009. iso lasi  bakteri  kitin olitik  d ari  limbah  pen golahan u dan g .  j u rn a l p as c a p an e n da n b iote k n o lo g i kelautan dan perikanan.  4  (1). chasanah,  e.,  meidina,  and  suhartono,  m.t.  2008. antibacterial  potency  of  chitosan  oligomer  produced b y  b a c illu s lic h e n ifo rmis   m b-2   c h ito san ase. indonesian fisheries research journal.  14  (2):  91– 95. chasanah,  e.,  hariyadi,  p.,  witarto, a.b.,  hwang,  j.k., and  suhartono, m.t. 2006. biochemical characteristic of  chitosanase  from  the  indonesian  b. licheniformis mb-2. journal of molecular biotechnology.33 (2): 93– 102. c h o,  y.i.,  n o,  h .k.,  and   m eyers,  s .p.  1 9 9 8 . p hysic o ch emic al  ch aracteristic s  and   fu n ctio nal properties  of  various  commercial  chitin  and  chitosan products. j. agric food chem.  46:  3839–3843. chung, y.c., su, y.p., chen, c.c., jia, g., wang, h.i., wu, j.c.g.,  and  lin,  j.g.  2004.  relationship  between an tib ac terial  ac tivity  o f  c hito san   an d  su rfac e characteristics  of  cell  wall.  acta pharmacol  sin  25: 932–936. dag, y.w.,  zhou,  p.,  yu, j., pan, x., wang, p., lan, w., an d    tao,  s .  2 0 0 7 .  a n timic ro bial  effec t  o f chitooligosaccharides  produced  by  chitosanase  from pseudomonas cuy8. asia pac. j. clin. nutr; 16 (suppl 1): 174–177. einbu, a., 2007. characterisation of chitin and a study of its a c id-c a taly s ed h y dro ly sis .  p h d   t h esis, department  of  biotechnology.  faculty  of  natural science  and  technology.  norwegian  university  of science  and  technology.  trondheim.  75  pp. fawzya, y.n.,  noviati,  r.,  sutrio,  dan  wibowo,  s.  2008. p eng aru h   d easetilasi  d an   alk alin asi  terh ad ap 50 karakteristik  karboksimetil  kitosan.  jurnal perikanan: journal of fisheries sciences  x (1): 64–75. fawzya,  y.n.  and  w ibowo,  s.  2009a.  exploration  of ind onesia  marine  ch itino lytic   enzymes  and  th eir application. indonesian marine and fisheries product processing and biotechnology,  jakarta. fawzya,  y.n.,  pratitis,  a.,  dan  chasanah,  e.  2009b. karakterisasi  enzim  kitosanase  dari  isolat  bakteri kpu  2123  dan  aplikasinya  untuk  produksi  oligomer k ito san.  j u rna l p a sc a p a n e n d a n b io te k n olo g i kelautan dan perikanan  4 (1):  69–78. g uo -jan e,  t.,  yu o n,  w.z .,  and   hu ey,  s.w .  2 0 00 . antibacterial  activity  of  chitooligosaccharide  mixture prepared  by  cellulosa  digestion  of  shrimp  chitosan and  its  application  to  milk  preservation.  j. food protection  63  (6):  747–752. hennen, w.j. 1996. chitosan. woodland publishing inc. p.o. box 160. pleasant grove, ut 84062. 31 pp hirano, s. and nagao, n. 1989. effects of chitosan, pectic acid, lysozyme and chitinase on the growth of several phytopathogens.  agric. biol. chem  53:  3065–3066 hyean-woo, l.,  yoon-s prun, p.,  jong-w han, c.,  sangyeop, y., and woon-seob, s. 2003. antidiabetic  effects o f  c h ito san   o lig o sacc h arides  in  n eo n atal strep tozo to cin -ind u c ed   n o nin su lin -dep en den t diabetes  mellitus  in  rats.  biol. pharm. bull. 26 (8) 1100–1103. j eau iau x,  c .  19 7 8 .  d istrib u tio n   and   q u antitative importance  of  chitin  in  animals. proceddings of the first international conference on chitin/chitosan. p. 5– 10. liu b. , wan-shun liu, bao-qin han, yu-ying sun. 2007. anti  diabetic  effects  of  chitooligosaccharides  on p an c reatic   islet  c ells  in   strep to zo to c in -in d u ced diabetic  rats.  world j. gastroenterol february  7;  13 (5):  725–731. muzzarelli, r.a.a. 1977. chitin.  pergamon press. oxford, uk. 309 pp. nasution,  z.,  tazwir,  dan  zilda,  d.s.  2004.  potensi, pemanfaatan  dan  nilai  ekonomi  limbah  udang  dan rajungan   di  propinsi  lampung.  warta pe nelitian perikanan indonesia.  edisi  pasca  panen  dan  sosial ekonomi.  10  (7):  2–5. plassard,  c.  1997. assay of  fungal  chitin  and  estimation of  mycelial  biomass.  in chitin handbook.  edited  by r.a.a.  muzzarelli  and  m.g,  peter.  european  chitin society, lyon. sagoo,  s.,  board,  r.,  and  roller,  s.  2002.  chitosan inhibits  growth  of  spoilage  microorganisms  in  chilled pork products.  food microbiol.  19:  175–182. terbojevich,  m.  and  cosani,  a.  1997.  molecular  weight d etermin atio n   o f  ch itin   an d   ch itosan .  in c h itin handbook. edited by mozzarelli, r.a.a. and martin g. peter.  european  chitin  society. wahyuni,  s.,  witarto, a.b.,  syah,  d,  zakaria,  f.r.,  and suhartono,  m.t.  2007.  activity  of  chitooligomers produced  by  enzymatic  hydrolysis  upon  proliferation of lymphocyte and a549 (lung carcinoma) cancer cell line.  international seminar and workshop: marine biodiversity and their potential for developing biopharmaceutical industry in indonesia.  jakarta,  may 1 7-18 th  2 0 06 .  r esearc h   cen ter  fo r  p rod u c t processing  and  biotechnology. w idiastuti,  h.  and    chasanah,  e.  2010.  screening of a n ta g o n is tic o f c hitino ly tic b a cte ria to ward s ganoderma sp.  presented  at  international  oil  palm conference  (iopc),  june  3,  jogyakarta. wiœniewska-wrona, m.,  niekraszewicz, a., ciechañska, d.,  pospieszny1,  h.,  and  orlikowski,  l.b.  2007. b iolo g ical  p ro p erties  o f  c hito san   d egrad atio n products. polish chitin society, monograph xii, 2007. won-seok, c.,  kil-jin, a.,  dong-w ook  l.,  myung-woo, b.,  and  hyun-jin,  p.  2002.  preparation  of  chitosan oligomers  by  irradiation.  polymer degradation and stability 78  (3): 533–538.      zhou,  t.x.,  chen, y.j.,  yoo,  j.  s.,  huang, y.,  lee,  j.h., jang, h.d., shin, s.o., kim, h.j., cho, j.h., and kim, i.h.  2 0 09 .  effec ts  o f  ch ito o lig o sac c h arid e su pp lem en tatio n   o n  p erform an ce,  b lo o d characteristics,  relative  o rgan  weigh t,  and  m eat quality  in  broiler  chickens.  poult sci    88:  593–600. e. chasanah 508 resource limit is reached resource limit is reached the website is temporarily unable to service your request as it exceeded resource limit. please try again later. 508 resource limit is reached resource limit is reached the website is temporarily unable to service your request as it exceeded resource limit. please try again later. 508 resource limit is reached resource limit is reached the website is temporarily unable to service your request as it exceeded resource limit. please try again later. 508 resource limit is reached resource limit is reached the website is temporarily unable to service your request as it exceeded resource limit. please try again later. squalen: buletin pascapanen dan bioteknologi kelautan dan perikanan (squalen: bulletin of marine and fisheries postharvest and biotechnology) vol.10 no. 1, may 2015 publisher : research and development center for marine and fisheries product processing and biotechnology, research and development agency for marine affairs and fisheries (rdcmfppb) managing director : head of research and development center for marine and fisheries product processing and biotechnology editor in-chief : dr. ekowati chasanah (biotechnology of marine and fisheries, rdcmfppb, ina) editors : 1. dr. achmad poernomo (food safety, agency of marine and fisheries research and development, ina) 2. dr. bagus sediadi bandol utomo (fisheries product development, rdcmfppb, ina) 3. dr. puspita lisdiyanti, m.agr.chem (microbiology, indonesian institute of science, ina) 4. dr. noer khasanah (marine biotechnology, university of gadjah mada, ina) 5. dr. antonius herry cahyana (natural product chemistry, university of indonesia, ina) 6. prof. dr. jose alberto ramirez de leon (food processing, universidad autonoma de tamaulipas, mexico) 7. prof. dr. anthony d. wright (marine natural product chemistry, university of hawaii, usa) peer reviewers : 1. prof. dr. santiago valdez hurtado (food processing, universidad de sonora, mexico) 2. prof. dr. rijkelt beumer (food microbiology, agrotechnology and food sciences, w ageningen university the netherlands) 3. prof. dr. nurul huda (food processing, university sains malaysia, malaysia) 4. prof. dr. hari eko irianto (fisheries product processing, ina) 5. prof. dr. djagal wiseso marseno (faculty of agriculture technology, university of gajah mada, ina) 6. prof. dr. rosmawaty peranginangin (fisheries product processing, rdcmfppb, ina) 7. prof. dr. jörn piel (bacterial natural products, institute of microbiology, eth zurich) 8. dr. nahla e.e. omran (zoology department, faculty of science, tanta university, tanta, egypt) 9. prof. dr. joko santoso (fisheries and marine, bogor agricultural university, ina) 10. dr. ir. alim isnansetyo (marine natural product, university of gajah mada, ina) 11. dr. supriyadi (food processing and flavor, university of gajah mada, ina) 12. dr. singgih wibowo (fisheries product processing, rdcmfppb, ina) 13. dr. tati nurhayati (fisheries product, bogor agricultural university, ina) 14. dr. siswa setyahadi (agricultural, indonesian institute of science, ina) copy editor : 1. tuti hartati siregar, m.biomedsc 2. dr. agustinus robert uria 3. dr. dedi noviendri 4. sinta nurwijayanti, ma 5. muhamad darmawan, mt 6. nurrahmi dewi fajarningsih, m.biotech (adv.) 7. diah ikasari, m.biotech administration address : research and development center for marine and fisheries product processing and biotechnology k.s. tubun petamburan vi, jakarta, 10260 phone/fax : +62 (21) 53650157/+62 (21) 53650158 e-m ail : squalenbulletin@kkp.go.id squalenbulletin@gmail.com citation is permitted with acknowledgement of the source squalen: buletin pascapanen dan bioteknologi kelautan dan perikanan (squalen: bulletin of marine and fisheries postharvest and biotechnology published periodically three times a year i.e. may, august, and december by research and develo pm en t center for m arine and f isheries prod uc t pro cessin g an d biotec hn olog y. accredited number: 631/au2/p2mi-lipi/03/2015 issn: 2089-5690 e-issn: 2406-9272 mailto:squalenbulletin@kkp.go.id mailto:squalenbulletin@gmail.com i foreword we would like to present a new volume of the squalen, bulletin of marine and fisheries postharvest and and biotechnology volume 10 number 1, 2015. this volume contains the latest research in the field of marine and fisheries postharvest and processing as well as biodiscovery and biotechnology. there are articles associated to screening and identification of bioactive and chemical components of marine organisms namely: identification and antimicrobial activity of marine streptomyces from geographically different regions of indonesia; chemical composition and fatty acid profile of some indonesian sea cucumbers. this also includes mini-review titled capturing natural product biosynthetic pathways from uncultivated symbiotic bacteria of marine sponges through metagenome mining. other articles related to fish processing are screening of significant variables for sliced frying fish ball using plackett-burman design; mechanical properties and biodegradability of acid-soluble chitosan-starch based film. we would like to acknowledge dr. uju sadi; dr. supriyadi; dr. nahla e.e. omran; and prof. dr. jörn piel as reviewers in this edition, the authors, and the editorial board members who have involved in the publication process for their passion and hardwork related to manuscript submission, processing, and editing. we hope that this issue will bring valuable information for the readers in the related area. nonetheless, we realize that the improvement of publication is still needed. therefore, comments as well as suggestions are welcomed. editors iii squalen buletin pascapanen dan bioteknologi kelautan dan perikanan (squalen: bulletin of marine and fisheries postharvest and biotechnology) vol. 10 no. 1, may 2015 issn: 2089-5690 content page foreword ................................................................................................... ....................... i content ................................................................................................... .......................... iii mechanical properties and biodegradability of acid-soluble chitosan-starch based film novalia rachmawati, radestya triwibowo, and roni widianto ..................................................... 1-7 screening of significant variables for sliced frying fish ball using plackett–burman design syamdidi and theresia dwi suryaningrum ................................................................................. 9-15 identification and antimicrobial activity of marine streptomyces from geographically different regions of indonesia rofiq sunaryanto and anis herliyani mahsunah ........................................................................ 17-25 chemical composition and fatty acid profile of some indonesian sea cucumbers yusro nuri fawzya, hedi indra januar, rini susilowati, and ekowati chasanah ............................ 27-34 capturing natural product biosynthetic pathways from uncultivated symbiotic bacteria of marine sponges through metagenome mining: a mini-review agustinus robert uria ............................................................................... .............................. 35-49 e-issn: 2406-9272 authors guidelines squalen: buletin pascapanen dan bioteknologi kelautan dan perikanan (squalen: bulletin of marine and fisheries postharvest and biotechnology) 1. scope: research paper, short communication and reviews in the field of postharvest, food safety and environment, and biodiscovery of marine and fisheries. 2. language: english. it is preferable that manuscripts are professionally edited. the abbreviated name or expression should be cited in full at first usage, followed by the accepted abbreviation in parentheses. metric si units should generally be used. chemical formulas and solutions must specify the form used, e.g. anhydrous or hydrated and the concentration must be in clearly defined units. common species names should be followed by the latin binomial (italic) at the first mention. 3. manuscript: should be prepared max. 15 pages, double-spaced (except for title, tables, figures, and bibliography which is prepared in single-spaced) in microsoft w ord on a4 paper, font (arial 10). research paper manuscripts should contain the following sections : a. title: brief, concise and informative, reflecting the manuscript material. avoid the abbreviation and formula where possible. author’s name, institution, and address are written under the title. b. abstract: a concise and factual abstract is required. the abstract should state briefly the purpose of research, the principle method, result and conclusion.non-standard or uncommon abbreviation should be avoided, but if essential they must be defined at their first mention in the abstract itself. abstract should not exceed 250 words. c. key words: contain 3 to 5 words, referred from agrovocs. d. introduction: state the background and objective(s) of the work, avoiding a detailed literature survey or a summary of the results. e. material and methods: provide sufficient detail to allow the work to be reproduced. methods already published should be indicated by a reference; only relevant modification should be described. f . results and discussion: results should be clear and concise. discussion should explore the significance of the results of the work, not repeat them. avoid extensive citation and discussion of published literature. -tables: short, clear and must be able to stand alone. table should have a title and number insequence and are placed above the table, font (arial 10). -figures and graphics: prepared in appropriate pattern usable for black and white versions of all the color illustrations. graphics should be prepared in ms excel. figure should have a title and number insequence. figure and graphics title and their explanation or notes are placed below the figures or graphics, font (arial 10). -pictures/photograph: prepared in contrast color or b/w. pictures/photographs should have a title and number insequence. tiff: color or grayscale photographs (halftones) always use a minimum of 300 dpi; bitmapped line drawings use a minimum of 1000 dpi; combinations bitmapped line/halftone (color or grayscale) a minimum of 500 dpi is required. g. conclusion: concise considering the title, objectives and research results. h. acknowledgement: collate acknowledgements, if any, in separate sections at the end of the article before the reference and do not, therefore, include them on the title page, as a footnote to the title or otherwise. i. bibliography: follow the apa (american psychological association) style referencing. references in the text should cite the authors’ names followed by the date of their publication, unless there are three or more authors when only the first authors’ name is quoted followed by et al. e. g. smith et al. (1999) or jones & smith (2000). references at the end of the paper should be listed in the alphabetical order. please ensure that every reference cited within the text is also present in the reference list (and vice versa). latest and primary references at least 80% are strongly advised. a. journal with doi; b. journal without doi; c. thesis or dissertation; d. book chapters houbraken, j. a., frisvad, j. c., & samson, r. a. (2010). taxonomy of penicillium citrinum and related species. fungal diversity, 44(1), 117-133. doi: 10.1007/s13225-010-0047-z. yu, z., lang, g., kajahn, i., schmaljohann, r., & imhoff, j. f. (2008). scopularides a and b, cyclodepsipeptides from a marine sponge-derived fungus, scopulariopsis brevicaulis. journal of natural products, 71(1), 1052-1054. prigent, s. (2005). interactions of phenolic compounds with globular proteins and their effects on food related function al properties. ph.d. thesis. w ageningen university, the netherlands. lanier, t. c., carvajal, p., & yongsawatdigul, j. (2005). surimi gelation chemistry. in j.w. park (ed) surimi and surimi seafood, p. (pp. 435-489). boca raton, fl: crc press taylor and francis group. review paper, manuscript should be written as a continuous articles sub headings that ref lect the matter discuss ed, not necessarily the same as the sub heading of the research paper (abstract, introduction, discussion, conclussion, acknowledgement, ref erences) 4. submission of manuscript: a soft copy of the manuscript should be submitted electronically by open journal system (ojs) at website: www.bbp4b.litbang.kkp.go.id/squalen-bulletin. manuscript should be uploaded as w ord (.doc) files (not writeprotected) or/ and ms excel for the figures or graphics. the text file must contain the entire manuscript including title page, abstract, keywords, text, references, tables, and figures. all the manuscripts submitted to ‘squalen: buletin pascapanen dan bioteknologi kelautan dan perikanan (squalen: bulletin of marine and fisheries postharvest and biotechnology)’ will be reviewed by peer reviewers and editors. 5. manuscript which does not meet the above requirements will be rejected. 6. editor provides reprint for the author(s). http://www.bbp4b.litbang.kkp.go.id/squalen-bulletin. 508 resource limit is reached resource limit is reached the website is temporarily unable to service your request as it exceeded resource limit. please try again later. 81 squalen bull. of mar. and fish. postharvest and biotech. 15(2) 2020, 81-90 www.bbp4b.litbang.kkp.go.id/squalen-bulletin squalen bulletin of marine and fisheries postharvest and biotechnology issn: 2089-5690 e-issn: 2406-9272 copyright © 2020, squalen bmfpb. accreditation number: 21/e/kpt/2018. doi: http://dx.doi.org/10.15578/squalen.v15i2.438 antibacterial activities of the extracts of sponge-associated fungus trichoderma longibrachiatum against pathogenic bacteria sri sedjati1,3*, ambariyanto ambariyanto1,2, agus trianto1,2, endang supriyantini1, ali ridlo1, muhammad syaifudien bahry2, gita wismayanti1, ocky karna radjasa1, and erin mccauley4 1department of marine sciences, faculty of fisheries and marine sciences, diponegoro university, indonesia 50275 2integrated laboratory, diponegoro university, jl. prof. soedarto, tembalang, semarang, jawa tengah, indonesia 50275 3marine science techno park jl. undip, desa teluk awur, tahunan, jepara, jawa tengah 59427 4department of chemistry and biochemistry, california state university dominguez hill, 1000 e. victoria street, carson, california, usa 90747 abstract this study aims to explore the antibacterial potential of a sponge-associated fungus trichoderma longibrachiatum isolated from ternate waters, north maluku, eastern indonesia. various culture media were used to stimulate the production of secondary metabolites in t. longibrachiatum. the isolate was cultured in various media for 6-9 days. then, the antibacterial activities of the ethyl acetate extracts were assayed against pathogenic bacteria of multi-drug resistant (mdr) strain (pseudomonas aeruginosa, staphylococcus aureus, klebsiella pneumoniae, and bacillus cereus). the results showed that all extracts had similar profiles on the thin layer chromatography. however, two of the most potent extracts were produced from the pca and mea media for 9 days. these extracts inhibited methicillinresistant s. aureus (mrsa) (12.48 mm and 12.27 mm); b. cereus (12.11 mm and 12.12 mm); k. pneumoniae (12.40 mm and 10.76 mm); and p. aeruginosa (11.59 mm and 8.69 mm) at concentrations 500 g/disc. in conclusion, the fungus t. longibrachiatum that was cultured in pca and mea media had the potential to produce antibacterial compounds against mdr pathogens and both had similar compounds. meanwhile, the ethyl acetate extracts from fungus cultured in the tpa and ta media were inactive against all tested bacteria. keywords: sponge-associated fungus, trichoderma longibrachiatum, antibacterial activity, pathogen *corresponding author. e-mail: sedjati69@gmail.com article history: received: 11 march 2020; revised: 13 may 2020; accepted: 22 june 2020 1. introduction indonesian waters are reservoir of various spongeassociated fungi, which are potential to produce antibacterial compounds. the ethyl acetate extract of aspergillus niger biomass associated with haliclona fascigera sponge from mandeh island waters (west sumatra) has been reported to inhibit the growth of staphylococcus aureus and escherichia coli (handayani et al., 2015). aspergillus flavus, from the sam e wat ers, asso ci ate d wi t h th e spo nge acanthrostrongylophora ingens also produced antibacterial compounds against bacillus subtilis, s. epidermidis, salmonella typhosa, and e. coli (handayani & aminah, 2017). aspergillus sydowii associated with axinella sp. from riung island waters (east nusa tenggara) produced ethyl acetate extracts, that were active against s. aureus and e.coli “strain” multi-drug resistant (mdr) bacteria (widyaningsih et al., 2018). furthermore, triododerma saturnisporum associated with sponges from pandang island waters (north sumatra) produced ethyl acetate extract with antibacterial activity against mdr s. enterica, extended spectrum beta-lactamase (esbl) of e. coli and k. pneumoniae (sibero et al., 2018a). trichoderma reesei from the same waters, associated with cinachyrella sp. produced antibacterial against of mrsa, mdr s. hemolyticus, s. enterica, and esbl of e. coli (sibero et al., 2018b). in our previous researc h, we i so l ate d pse udoalt eromo nas http://www.bbp4b.litbang.kkp.go.id/squalen-bulletin http://dx.doi.org/10.15578/squalen.v15i2.438 mailto:sedjati69@gmail.com 82 s. sedjati et al. /squalen bull. of mar. and fish. postharvest and biotech. 15 (2) 2020, 81-90 maricaloris, a. nomius, eurotium rubrum, and penicillium sp. from the sponge reniera sp. all of the bacteria and fungi were active against mdr s. aureus and e. coli (trianto et al., 2019b). thomas et al. (2010) and nalini et al. (2018) reported various groups of secondary metabolites compounds of marine fungi with antibacterial activities. other studies hav e successfully isolated antipathogenic bacterial compounds from marine fungi. brevianmides from a. versicolor inhibited bacille calmette-guerin (bcg) (song et al., 2012); meanwhile scleroderolide, citrinin, and penicillosides b from penicillium sp. inhibited mrsa, wild type of s. aureus, vancomycin-resistant e. faecium, and e. coli (li et al., 2019; subramani et al., 2013; murshid et al., 2016). furthermore, spiculisporic acid f from a. candidus inhibited p. solanacearum and s. aureus (wang et al., 2015); asperchondols b from aspergillus sp. inhibited s. aureus and e. faecalis (liu et al., 2016), and heterocornol from pestalotiopsis heterocornis inhibited s. aureus and b. subtilis (lei et al., 2017). several trichoderma sp. species have also been studied and have the potential to produce antibacterial compounds. ethyl acetate extract of trichoderma sp. was reported to exhibit antibacterial activities against pathogens (e. coli, p. aeruginosa, s. aureus, and b. cereus). the fungus isolated from the mangrove ecosystem (rhizosphere mangrove) has been shown to produce secondary metabolites that can be dev eloped to ov ercome the crisis of bacterial resistance (narendran & kathiresan, 2016; basiriya et al., 2017). similar results were also shown by synytsya et al. (2017), the only difference is that the antibacterial compounds in the study were derived from petroleum ether and ethanol extracts. data from several other researchers showed the potential of antibacterial compounds produced by trichoderma sp., such as trichodermaquinone against mrsa (khamthong et al., 2012), trichodins a & b, and pyridoxatin against s. aureus and s. epidermidis (wu et al., 2014). li et al. (2019) stated that trichoderma sp. is a producer of various secondary metabolites with diverse biological activities. this fungus is commonly found and distributed throughout the world, spreading in many ecosystems, including the ocean. despite their promising bioactivities, not all spongeassociated fungi can be cultured. to overcome this problem, an appropriate solid media can be used for the adaptation phase of the fungus from its original environment to the condition in the laboratory. solid media have been shown to increase the production of secondary metabolites or enzyme secretion from the fungus. on the other hand, cultivating a fungus in liquid media has been considered reducing the efficiency of metabolite production, although conventionally, this method has been widely applied to produce beneficial natural compounds (barrios-gonzález & tarragócastellanos, 2017). fungal media must contain the sources of carbon (c), nitrogen (n), macro-elements, and micro-elements. there are two general categories of culture media, natural and synthetic. natural media is composed of natural ingredients, such as plants, seeds, wood/stems, and other natural materials (basu et al., 2015), ri ce, grits, oatmeal, wheatgerm (vandermolen et al., 2013), cassava, potato, taro (wongjiratthiti & yottakot, 2017). the advantage of this natural media is the ease of preparation; although the chemical composition is not measurable. alternatively, the use of synthetic media allows the accurate measurement of chemical composition in the fungi extract. the only drawback of this method is its cost-prohibitive. this study therefore aims to explore the potenti al of sponge-associ ated fungus t. longibrachiatum as a producer of antibacterial compounds against the mdr pathogens. the isolated fungus was cultivated in various alternative media to stimulate the production of secondary metabolites. 2. material and methods 2.1. fungus isolate the fungus was isolated from a sponge obtained from falajava beach, ternate island, north maluku, indonesia (coordinates 00o47’09.12'’ n; 127o 23’21.76'’ e) and was coded as te-pf-03.1. molecul ar identification of its rdna sequences indicated the fungus as trichoderma longibrachiatum (trianto et al., 2020). 2.2. morphological characterization of the fungus morphological characterization confirmed that the sample fungus was t. longibrachiatum. the isolate was cultured using malt extract agar (mea) (merck) media for seven days at 27 oc to observe its colony shape. microscopic characteristics were determined based on slide culture method (bath, 2017; sibero et al., 2017) with 100-1000 times magnification. 2.3. pathogenic bacteria the tested pathogenic bacteria were mdr strain, consisting of p. aeruginosa, s. aureus, k. pneumoniae, and b. cereus. these test bacteria were obtained from the laboratory of diponegoro national hospital (rsnd) and collections belonging to the authors’ peer group, the integrated laboratory of diponegoro university, semarang. 83 2.4. preparation of antibacterial activity the preparation of antibacterial activity was initiated by antagonistic testing using the agar plug method (trianto et al., 2017; sibero et al., 2018a). the fungus was cultured in mea for seven days at 27 oc. the mycelium was taken out approximately at the size of a 1-cm diameter circle with a cork borer. the test bacteria of p. aeruginosa, s. aureus, k. pneumoniae, and b. cereus mdr strain were cultured using mueller hinton agar (mha) media. preparation of test bacteria was conducted using a 24-hour-old bacteri al suspension with a density equivalent to 1.5 x108 cfu/ ml (remel mcfarland equivalence), which was inoculated with the swab method. the mycelium with a 1-cm diameter circle was plugged to the test bacterial culture, and the petri dish was closed and then incubated for 24-48 h at 37 oc. the antibacterial activity was indicated by the formation of inhibition zones around plug fungus. 2.5. cultivation of fungus the fungus was subcultured using mea media. the mycelium was taken out from the media using a circular loop needle with a 2-mm diameter and inoculated in new media. the media used were mea as standard media and some modified media (solid, 20 ml/petri dish), the ph was near neutral (around 5.60-6.50), with 50-60 ‰ salinity. the fungus, was incubated for 6,7,8, and 9 days (24 hours in dark and static conditions) at 27 oc as described by hamad et al. (2014). the modified media were prepared based on the methods of wongjiratthiti & yottakot (2017) and trianto et al. (2019a) with some modifications. modified media used include: fructose fish-infusion agar (ffa), glucose fish-infusion agar (gfa), tofu pulp-infusion agar (tpa), tempeh-infusion agar (ta), cassava-infusion agar (ca), and pepton cassavainfusion agar (pca). the followings are steps of media preparation. the mea was prepared by dissolving 48 g media in 1 l of sterile seawater (30 g malt extract, 3 g peptone, 15 g agar) (merck’s protocol). the ffa was prepared by boiling 100 g of indian mackerel (rastrelliger sp.) in 1 l of sterile seawater for 30 min. the boiled water was filtered, then the filtrate was added with 20 g fructose and 15 g agar (volume of the total solution on all media was 1 l in sterile seawater). the gfa was prepared as the ffa, but the fructose was replaced by glucose. the tpa was prepared by boiling 100 g tofu pulp/waste in 1 l of sterile seawater for 30 min and filtered, then the filtrate was added with 15 g agar. the ta was prepared by boiling 100 g of small pieces of tempeh in 1 l of sterile seawater for 30 min and was subsequently filtered. after that, the filtrate was added with 15 g agar. the ca was prepared by boiling 100 g of cassava for 30 min in 1 l of sterile and filtered seawater. after filtration, the filtrate was added with 15 g agar. the pca was prepared as the ca, but the filtrate was added with 3 g peptones and 15 g agar. 2.6. extraction and profiling of secondary metabolites the culture media and the mycelia were macerated with methanol (1:1, v/v) and concentrated with a rotary evaporator under vacuum at 35-40 oc until the remaining volume was 25%. then, the extract was partitioned using distilled water and ethyl acetate (1:1, v/v). the ethyl acetate (ea) layer was concentrated using the rotary evaporator (40 oc) to provide the crude extract. chemical profiling of the ea extract was conducted using thin-layer chromatography (tlc) on tlc plates (merck silica gel plate 60 f254). after development, the tlc was visualized with uv light (254/366 nm) and subsequently sprayed with reagent; 2% vanillin-h2so4, 0.25% ninhydrin in acetone, and 1% ferric (iii) chloride in methanol (harborne, 1973; sen et al., 2012; trianto et al., 2019a). the plates were then heated at 110 oc for 2-3 min. 2.7. antibacterial activity test bacteria were cultured using mha media with the initial density equivalent to 1.5 x108 cfu/ml, and the bioassay was tested using 24-hour-old bacteria. the antibacterial bioassay was conducted by determining the diameter of the inhibition zone using disc diffusion based on trianto et al. (2017). inhibition zones were measured after a 24-hour incubation period at 37 oc. extracts in methanol solvent were tested against pathogenic bacteria using discs (oxoid, 6 mm diameter) with 500 µg/disc concentration. the positive control used was chloramphenicol (oxoid, 30 µg/disc). 2.8. proximate analysis of culture media the proximate analysis of the culture media was conducted using the aoac method (1990). the moisture content was measured by the oven drying gravimetric method. the determination of total ash content was carried out by the furnaces combustion gravimetric method. the protein content was measured by the kjeldahl method, and the crude fat content was measured by solvent extraction (soxhletation) gravimetric method. the carbohydrate content was calculated by reducing 100% with the total of moisture, ash, lipid, and protein content. s. sedjati et al. /squalen bull. of mar. and fish. postharvest and biotech. 15 (2) 2020, 81-90 84 3. results and discussion fungus identification was determined as t. longibrachiatum based on morphological features and molecular tests (trianto et al., 2020). fungus t. longibrachiatum cultured in mha media for seven days (27 oc) produced a yellowish-green colony (figure 1a). the morphological details of the fungus can be seen under the microscope as in figure 1b-f. the hyphae had a partition and conidiophores emerging from its hyphae’s phialide. elongated conidiophores had many short branches leading to phialides. these phialides’ tip rounded to form conidia. these characteristics confirmed the morphology of t. longibrachiatum species as reported by others researchers. t. longibrachiatum markers, according to richter et al. (1999) and gams and bissett (2002), typically have a conspicuous pigment, green-yellowish, at least when it was first isolated. the conidia have a green color, smooth surface, and ellipsoidal to obovoid shape. the main branches of conidiophores are long, producing short branches, ended with phialides, and the tips rounded to conidia. similar observation was also reported by samuels and ismaiel (2012). the longibrachiatum clade of trichoderma character shows that the phialides arise singly along the branches of the main axis and solitary phialide arises within a short distance of the main axis or its branches. the morphological identification of the fungus thus confirmed it as t. longibrachiatum. the fungus t. longibrachiatum was cultured on modified media (ffa, gfa, tpa, ta, ca, and pca) and mea as the standard media. the fungus was able to produce ethyl acetate extracts (ee) in all media, as shown in figure 2. the fungus produced the highest extract yields on day 6 in ffa, gfa, and ca; on day 7 in mea and pca; on day 8 in ta; and on day 9 in tpa. the highest secondary metabolites production occurred when the fungal growth phase was at the stationary phase. this result confirms the results of gliseida et al. (2013), arumugam et al. (2014), and sibero et al. (2018a) that the production of secondary metabolites compound would increase when entering the stationary phase. in this research, the fungus entered the stationary phase by day 6-9. among all the of the tested media, fungus cultured in the gfa produced the highest ee yield, i.e., 27 mg/20 ml media on day 6. the fungus produced higher ee in all modified media than that in mea during the stationary phase. the phase was not always reached on the same day in each media. the abilities of fungus to grow, multiply, and secrete secondary metabolites are influenced by the nutrient content of their culture media. macronutrients contained in the media are presented in table 1. all media contained sources of c and n, which were sufficient for fungal life during cultivation and could stimulate biosynthesis of secondary metabolites. indian mackerel contains 72.24% water content, 19.14% protein, 8.19% lipid, and ash 1.42% (fresh wei ght) (sonav ane et al ., 2017). it contains macrominerals (na, k, and ca), microminerals (fe and zn), and vitamin b12 (nurilmala et al., 2015). s. sedjati et al. /squalen bull. of mar. and fish. postharvest and biotech. 15 (2) 2020, 81-90 figure 1. microscopic morphology of fungus te-pf-03.1 ((b) 100x, (c-f) 1000x magnification): (a) colony in mea (7 days of incubation, 27 oc), (b) pustules, (c) partitioned hyphae, (d) conidiophores, (e) conidia, and (f) phialides 85 meanwhile, the fresh cassava contains water 60%, starch 35%, crude fiber 2.5%, protein 1%, lipid 0.5%, and ash 1% (prabawati et al., 2011). cassava has high mineral content, containing the macro elements i.e. na, k, mg, ca, and cl, and the microminerals i.e. mn, zn, v (danso et al., 2001) as well as vitamins b, and c (depkes ri., 1995). table 1 showed that all media contained high ash content, so it could be assumed that the needs of the mineral for fungal growth were fulfilled. source of c could be derived from carbohydrate, protein, and lipid, but n could only be obtained from protein. the production of biomass and secondary metabolite reaches a peak when the source of n is high, but c is low, so the c/n ratio is very crucial (lee et al., 2008; dinarvand et al., 2013). minerals and vitamins in the chemical reaction of fungi metabolism play a role an energy source or signal transduction, enzym e acti v i ty, heme-protein, cytochrome, and redox reaction (walker & white, 2005). compounds in ee from cultured fungus could be tra ced th rough the sp ot pro f i l e o n the tlc chromatogram (figure 3). the blue fluorescent spots when exposed to uv light indicates the presence of organic compounds with double bonds (diene/polyene or conjugation) (mohammed, 2018). compounds that react positively with vanillin reagents will generate certain colored spots (varying colors) characterizing the presence of carbonyl functional groups (e.g., ketones and aldehydes); such as phenolic, flavonoids, terpenoids, steroids, fatty acids/essential oils, or high molecular weight alcohol groups. vanillin reagents are sensitive to steroids (harborne, 1973; jork et al., 1990). s. sedjati et al. /squalen bull. of mar. and fish. postharvest and biotech. 15 (2) 2020, 81-90 figure 2. yield of ethyl acetate extracts of t. longibrachiatum on different days of incubation (6-9 days) note: mea = malt extract agar; ffa= fructose fish-infusion agar; gfa = glucose fish-infusion agar; tpa = tofu pulp-infusion agar; ta = tempeh-infusion agar; ca = cassava-infusion agar; pca = pepton cassava-infusion agar media water ash lipid protein carbohydrate mea 90.49 3.92 0.29 0.39 4.91 ffa 91.96 3.65 0.68 0.15 3.56 gfa 92.57 3.45 0.38 0.12 3.48 ca 93.72 4.01 0.77 0.08 1.42 pca 93.86 3.55 0.45 0.17 1.97 table 1. macronutrient composition of fungus culture media (% wet basis) note: mea = malt extract agar; ffa= fructose fish-infusion agar; gfa = glucose fish-infusion agar; tpa = tofu pulp-infusion agar; ta = tempeh-infusion agar; ca = cassava-infusion agar; pca = pepton cassavainfusion agar e xt ra ct y ie ld ( m g/ 20 m l) m ed ia days of incubation 86 the fungus that cultured in mea produced three spots (rf1=0.67; rf2=0.42; rf3=0.31), which are reactive to uv 365 nm, but only rf1 was reactive to 2% vanillin-h2so4. similar tlc patterns were also observed from the extracts of fungus grown in the other media. visualization under uv 365 nm and 2% vanillin-h2so4 indicated that the first spot (rf1) might contain conjugated compounds (e.g., diene, polyene or benzene ring) which possess a ketone or aldehyde group. this spot was relatively nonpolar since it interacts most strongly with its mobile phase. two other spots (rf2 & rf3) only positively absorbed uv 365 nm, indicated that both spots contained conjugated compounds. all of the three spots were not of nitrogen (e.g., amines, peptides, or alkaloids) or phenolic (e.g., phenolic acids, polyphenols, flavonoids, lignans) compounds, since they were not reactive to ninhydrin and ferric chloride reagents (harborne, 1973). som e of t he com pounds conta i ned i n t. longibranchiatum extract have been researched. extract of ethyl acetate from marine-derived fungus t. longibrachiatum from jiaozhou bay, qingdao, chi na co ntai n ed two st ructu ral seri es of sesquiterpenes and cyclodepsipeptides, both have antifungal activity (du et al., 2020). other studies reported that the extract of ethyl acetate of t. longibrachiatum associated with the sponge haliclona sp. from sulawesi contained vertinoid polyketides (sperry et al., 1998). the vertinoids (also called sorbicillinoids) are hexaketide secondary metabolites in which the cyclization has taken place on the carbox yl ate terminus. sev eral of them hav e antibacterial activity, such as rezishanone a-d, sorbicil li nol, 2,3-di hydrosorbi cil li n as wel l as soh i rno nes a & c (ha rned & vol p , 2011). sorbicillinoids are important hexaketide metabolites derived from fungi because it has a v ariety of bioactivities including antioxidant, cytotoxic, antiviral and antimicrobes. based on the tlc spot profiles, it was assumed that all ees contained three compounds (figure 3). in comparison to previous studies, the tlc profiles may indicate the presence of terpenoids (such as sesqui terpenes) and/or pol yketi des (such as sorbicillinoids). in the antibacterial assay, it was found that all ees formed inhibition zones, except ees from tpa and ta media culture. the results of the antibacterial assay against four test bacteria are presented in table 2. extracts produced from pca and mea culture media (9 days of incubation) were the most potent. extracts from pca and mea media inhibited the growth of the tested pathogenic bacteria: mrsa (12.48 mm and 12.27 mm), b.cereus (12.11 mm and 12.12 mm), k. pneumoniae (12.40 mm and 10.76 mm), p. aeruginosa (11.59 mm and 8.69 mm), respectively. the ee of ca culture media was inactive against p. aeruginosa and b. subtilis, whereas those of ffa and gfa culture media were inactive only against b. subtilis. the fungus from all culture media contained similar compounds, but only mea and pca produced extracts with the highest antibacterial activities. it was suspected that the concentrations of the secondary metabolite were probably higher in both extracts than in the other investigated extracts so that they could inhibit the growth of the tested bacteria. s. sedjati et al. /squalen bull. of mar. and fish. postharvest and biotech. 15 (2) 2020, 81-90 figure 3. tlc profiles of ethyl acetate extracts of t. longibrachiatum cultivated in various media using nhexane: ethylacetate (4:1) as mobile phase and their chromatogram spot visualization (a) uv 365 nm, (b) 2 % vanillin-h2so4 (a) (b) note: mea = malt extract agar; ffa= fructose fish-infusion agar; gfa = glucose fishinfusion agar; tpa = tofu pulp-infusion agar; ta = tempeh-infusion agar; ca = cassavainfusion agar; pca = pepton cassava-infusion agar 87 4. conclusion sponge-associated fungus t. longibrachiatum from falajava beach, ternate island had the potential to produce anti bacteri al com pounds agai nst p. aeruginosa, s. aureus, k. pneumoniae, and b. cereus. the fungus t. longibrachiatum was successfully grown in various media (mea, ffa, gfa, tpa, ta, ca, and pca). the fungus produced similar secondary metabolites, as indicated by both the number and characteristic spots on the tlc plate. pca and mea were two of the best culture media for the production of secondary metabolites that exhibited antibacterial activity against four pathogenic test bacteria with inhibition zone diameters ranging from 7.88-12.48 mm. s. sedjati et al. /squalen bull. of mar. and fish. postharvest and biotech. 15 (2) 2020, 81-90 table 2. inhibition zones of ethyl acetate extracts of t. longibrachiatum against pathogenic bacteria note: values are means (n=3 )± sd; mea = malt extract agar; ffa= fructose fish-infusion agar; gfa = glucose fishinfusion agar; tpa = tofu pulp-infusion agar; ta = tempeh-infusion agar; ca = cassava-infusion agar; pca = peptone cassava-infusion agar k. pneumoniae p. aeruginosa mrsa b. subtilis mea 6 days 8.43±0.23 9.63±1.11 10.07±0.45 7.88±0.01 7 days 8.90±0.00 9.63±0.00 10.01±0.00 7.89±0.00 8 days 8.85±0.00 9.68±0.00 9.69±0.57 8.02±0.30 9 days 10.76±0.00 8.69±1.01 12.27±0.00 12.12±0.00     ffa     6 days 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 7 days 9.81±0.80 10.73±0.12 10.96±0.37 0.00±0.00 8 days 11.21±0.90 10.91±0.06 10.35±0.15 0.00±0.00 9 days 10.12±1.00 8.61±0.05 8.79±0.00 0.00±0.00     gfa     6 days 8.08±0.31 0.00±0.00 0.00±0.00 0.00±0.00 7 days 7.13±0.00 7.90±0.00 0.00±0.00 0.00±0.00 8 days 8.15±0.00 8.33±0.00 0.00±0.00 0.00±0.00 9 days 8.02±0.00 7.34±0.10 8.60±0.51 0.00±0.00     ca     6 days 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 7 days 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 8 days 8.85±0.20 0.00±0.00 8.95±0.48 0.00±0.00 9 days 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00     pca     6 days 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 7 days 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 8 days 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 9 days 12.40±0.00 11.59±0.55 12.48±0.00 12.11±0.00 chloramphenicol (30 µg/disc) 29.7±1.32 26.55±1.26 31.37±2.05 24.43±0.93 day of incubation/ media the diameter of inhibition zone (in mm excluding disc) 88 acknowledgments this research is part of the final project of doctoral education program at the department of marine sciences, diponegoro university. research funding was partially provided from by the faculty of fisheries and marine sciences, diponegoro university, grant number: 35/un 7.5.10/ pp/2019. this work was also supported by grants from usaid peer project # 5215 (okr), especially on the sample collection. references arumugam, g. k., srinivasan, s.k., joshi, g., gopal, d., & ram alin g am , k . 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(2014). two new antibiotic pyridones produced by a marine fungus, trichoderma sp. strain mf106. ma rin e dru gs,12 (3), 1 20 8– 12 19 . d oi: 10 .3 39 0/ md12031208. 508 resource limit is reached resource limit is reached the website is temporarily unable to service your request as it exceeded resource limit. please try again later. 45 copyright © 2014, squalen bmfpb, issn 2089-5690 squalen bulletin of marine & fisheries postharvest & biotechnology, 9 (2), 2014, 45-54 correlation of demerit point score (dps) and chemical parameters on quality of fresh estuary grouper (epinephelus tauvina) during storage in ice korelasi demerit point score (dps) dan parameter kimia pada kualitas ikan kerapu lumpur (epinephelus tauvina) selama pengesan farida ariyani1*, yusma yenni, and rudi riyanto1 1research and development center for marine and fisheries product processing and biotechnology, jl. k.s. tubun petamburan vi, central jakarta 10260, indonesia, *correspondence author: idapoernomo@yahoo.co.id abstract quality changes of fresh estuary grouper (epinephelus tauvina) fish kept in ice was evaluated for sensory and chemical parameters during 21 days of storage. fish was killed by immersing in ice water (hypothermia). fresh fish was placed in insulated boxes, and covered with ice flake. the ratio of ice and fish was 2:1 (w/w) and the boxes were kept at ambient temperature. evaluation was conducted every 3 days for sensory characteristics by demerit point score /dps for appearance, eyes, gills, belly, vent, and belly cavity. moreover, total volatile base/tvb, trimethyl amine/tma , and the k value were measured until fish was rejected by panelists. the results showed that estuary grouper fish stored in ice was rejected by panelists on 21 days of storage with tvb value of 29.43 mgn%, tma value of 22.10mgn% and k value of 40.54% (max level of 60%). related to correlation between sensory and chemical parameters, it showed that demerit point score correlated well with tvb, tma and k value. keywords: dps, estuary grouper (epinephelus tauvina), ice storage, chemical characteristics abstrak p eru b ah an k u alitas k eseg aran ik an kerap u ((e p ine p h e lus ta u vin a ) selam a 2 1 hari penyimpanan dalam es telah dikaji secara sensori maupun kimiawi. ikan dimatikan dengan merendamnya di dalam air es (hypothermia). ikan segar dimasukkan ke dalam cool box dengan dilapisi es curai dengan perbandingan antara ikan dan es 1:2 b/b, selanjutnya cool box disimpan pada suhu ruang. evaluasi dilakukan setiap 3 hari terhadap parameter sensori dengan metoda demerit point score/dps (kenampakan, mata, insang, perut, anus, rongga perut), total volatile base/tvb, trimethyl amine/tma, dan k value sampai ikan dinyatakan ditolak panelis. hasil penelitian menunjukkan bahwa ikan kerapu lumpur yang disimpan dalam es telah ditolak panelis pada 21 hari penyimpanan dengan nilai tvb 29,43 mgn%, tma 22,10 mgn% and k value 40.54%. hasil penelitian juga menunjukkan bahwa dps berkorelasi positif dengan tvb, tma dan k value. kata kunci: dps, kerapu lumpur (epinephelus tauvina), pengesan, karakteristik kimia article history: received: 16 mei 2014; revised: 9 juni 2014; accepted: 3 juli 2014 available online at website: www.bbp4b.litbang.kkp.go.id/squalen-bulletin 1. introduction grouper is one of coral fish having high valuable market fishes in southeast asia. indonesia is now one of major farmed-grouper producing countries in asia. in addition, indonesia is the largest producer and exporter of grouper seed, and whole live fish for consumption (sugama, 2013). there are 7 general of grouper available in indonesia, and amongs them, fish having potency for export are including polkadot grouper/panther grouper (cromileptesaltivelis), carpet cod (epinephelus fuscoguttatus) and estuary grouper (epinephelus suillus). fish quality is clearly influenced by the freshness. loss of freshness followed by spoilage is a complex combination of microbiological, chemical and physical processes. during handling and storage, quality deterioration of fresh fish rapidly occurs and limits the shelf life of the product, since fish is one of the most highly perishable food products. preservation in ice is one of the most efficient ways of retarding spoilage. the rate of deterioration during ice storage of fish varies with species and depends on condition of catching/harvesting as well as handling, where fish handled and kept at higher temperature will be spoiled faster than that handled and kept at lower temperature 46 squalen bulletin of marine & fisheries postharvest & biotechnology, 9 (2), 2014, 45-54 copyright © 2014, squalen bmfpb, issn 2089-5690 (david and tom, 2006; ghaly et al., 2010; massaquoi et al., 2011) to estimate the qualtiy of both fresh and processed fish, it is necessary to analyse parameters reflecting sen sory, che m i cal s an d m i c robi o l ogi cal characteristics. the organoleptic evaluation of fish quality is the most easy and rapid technique and the most satisfactory method of assessing the freshness quality of fish, since the methods can be very fast, reliable, non-destructive on raw fish and no expensive instruments are needed (martinsdo´ttir, 2002). several sensory methods have been developed, including demerit point score developed by the tasmanian food research unit (branch & vail 1985). demerit point score is a descriptive, fast and simple method to determine freshness quality and useful because it evaluates those sensory parameters and attributes that change most significantly in each species during degradation processes (bremner et al., 1987). since sensory method is a subjective method, therefore to obtain representative results, it should be performed by trained panel ists. comparativ e studies to established correlation between sensory and chemical parameters such as tvb, tma and kvalue are also necessary. many studies on sensorial and chemical properties of fresh fish during storage have been published (jeyasekaran et al., 2005; siah & afif, 2007; ozogul et al., 2008; okoro et al., 2010; ariyani et al., 2011, sulieman et al., 2012; ariyani & triwibowo, 2013), however, it is necessary to evaluate the deterioration of fresh estuary grouper during storage in ice, since the changes of fish freshness during ice stoage varies with species. 2. material and methods raw material used in this experiment was obtained from live estuary grouper (average body weight : 300 – 450 g) harvested from net-cage grouper mariculture in lampung, washed with freshwater and killed by immersing in ice cold water (hypothermia) for approximately 20-30 min. ungutted whole fresh grouper of 12 kg was placed a in insulated boxes with capacity of 50 – 60 kg, covered with ice flakes in a ratio of ice and fish of 2:1 (w/w) and the boxes were kept at ambient temperature. the treatment was conducted in 7 replicates. samples were withdrawn every 3 days and evaluation was conducted for sensory characteristics by demerit point scor/dps dev eloped by the tasmanian research food unit (branch & vail, 1985), total volatile base/tvb, trimethyl amine/tma, and k value were according to miwa & ji method (1992) until fish was rejected by panelists. the changes of sensory characteristics was evaluated by 6-7 trained panels for certain attributes (appearance, eyes, gills, belly, vent, belly cavity) with score of 0 for the best quality and 3 for the worst quality. the scores for separate attributes were summed to give an overall score. the character of fresh cooked (odour, flavour and texture) grouper was analyzed using the torry scheme (huss, 1995) in which scores are given from 10 (very fresh in taste and odour) to 3 (spoiled). 3. results and discussion results of the observations and tests, indicated that the quality of fresh estuary grouper decreased significantly with the increase of storage time reflected by the changes of quality parameters. 3.1. the changes of sensory characteristics evaluation of sensory characteristics of raw fish was conducted on general appearance, eyes, gills, belly, vent, and belly cavity with total demerit point score from 0 (as minimum score) to 30 ( as a maximum score), where 0 represented best quality and a hi gher score i ndi cated poorer quali ty. assessment of cooked fish was conducted on odour, taste and texture with a minimum average score of 0 and a maximum average score of 10. the results of sensory evaluation are presented in figure 1 – 3. in the first three days of storage, there was a considerable increase in the scores obtained for general appearance, eyes and gills color, while the scores for belly, belly cavity and vent were relatively constant in this period. scores for general appearance, eyes and gills did only slightly increase in the next six days. for all sensory characteristics, except for general appearance, there was a considerable increases in scores from day 9 of storage (figure 1) the general appearance attribute actually consists of several attributes i.e., appearance, skin, scales, m ucus and f i rm ness. the scores of general appearance was increased considerably in day 3 and decreased on day 6, relatively constant up to 12 days storage and subsequently increased gradually until the end of storage. considerable increase in day 3 was due to considerable increase of score for mucus attribute, which reaching demerit point score of 1.1 with mucus characteristics thick but not sticky. in subsequent storage (on day 6), however, scores of mucus decreased significantly and almost constant in the remaining of storage, which might be due to dissolution of mucus in ice water resulted from ice melting. this resulted in decrease in general appearance score since other scores (appearance, skin, and scales) were relatively constant. 47 copyright © 2014, squalen bmfpb, issn 2089-5690 squalen bulletin of marine & fisheries postharvest & biotechnology, 9 (2), 2014, 45-54 s c o re s storage time (days) figure 1. the changes of sensory characteristics (general appearance, eyes, gills, belly, vent, and belly cavity) of fresh grouper fish during icing. figure 2. the changes of torry scores of cooked grouper fish during icing. 48 squalen bulletin of marine & fisheries postharvest & biotechnology, 9 (2), 2014, 45-54 copyright © 2014, squalen bmfpb, issn 2089-5690 figure 3. the changes of torry scores of cooked grouper fish during icing. the thick surface mucus found in the early storage is not resulted from spoilage process, but due to stress condition during handling and transportation. the live fish in this experiment was harvested from lampung waters and road transported to jakarta and was killed by immersing in ice water (hypothermia) in jakarta laboratory. when the fish in stress condition, mucus secretion is greatly stimulated increasing the thickness of the mucus blanket (esteban, 2012). hur et al. (2013) stated that high frequencies of mucussecreting goblet cells were found in the digestive tract, mainly in the anterior intestine portion and pyloric ceca. one of the compound responsible in producing m uc us i n oran ge sp otted grou per (epinepheluscoioides) is a defensin like proteins, contributing to the innate host defense in multiple ways (masso-silva & diamond, 2014). on the day 9 of storage, the belly was still firm, no discoloration, with belly cavity stain was grayish and vent was in normal condition. this could be associated with grouper skin characteristic which is firm, resulted in longer time to spoilage bacteria entering the flesh. after 18 days of storage, the belly was soft, and discoloration was detectable, belly cavity was grayish turn to yellow with dark-red bloods, vent was still normal but fishy, the gills were faded, with moderate mucus and slightly stale in odour, the eyes were slightly cloudy, slightly bloody and sunken. when storage time reaching up 21 days, the belly was soft, with detectable discoloration while the belly cavity was yellowish turn to brown, with brown blood, vent was slightly exudes and fishy smell, the gills were very faded with moderate mucus and stale in smell. when the scores of separate attribute were summed, overall scores was obtained as total dps. the minimum score (0) reflects the most fresh condition while the maximum score (30) reflects the most deteriorated condition. during storage, dps increased coincided with the decreased of the freshness of grouper fish and until 18 day of storage grouper fish was still acceptable having dps of 20.8, while on the 21 day of storage the grouper fish was considered unacceptable judged by trained panel with approximate score of 23.0 (figure 2). ozogul et al. (2008) reported that the acceptable shelf life of white gro uper was f ound to b e 16 days f or i ced corresponding with a demerit score of 20.8, while jeyasekaran et al., 2005 observed quality changes in ice-stored of reef cod (epinephelusmerra) found that on the 19th day of storage, the fish was sensorily unacceptable. the cooked grouper fish assessed by torry-scale indicated that during storage the scores decreased linearly as the storage time increased (r = 0.95) as shown in figure 3. at the time of rejection (on the 21 days of storage), the score was approximately 4.6. during the first three days of storage, the highest torry scores (10) remained constant, and the following storage the scores decreased subsequently reaching up 5.4 on day 18 and drop to 4.6 at 21 days of storage. at the beginning of the storage time the smell of cooked estuary grouper was fresh with species characteristics, the taste was sweet and juicy, and the texture was firm, elastic and springy. on the other hand, on 21 days of storage, the smell was stale, the taste was musty, slightly sour and off flavor while the texture was slightly soft and sandy like. this result supports the finding of ozogul et al. (2008) explaining that white grouper fish was rejected at 19 days in ice in term of cooked fish having score of 5.30. related to cooked fi sh, wi l d and cul ture sea bream (sparusaurata) kept in ice was also unacceptable on days 19-23 having score of 4.0 (alasalvar et al., 2002). 49 copyright © 2014, squalen bmfpb, issn 2089-5690 squalen bulletin of marine & fisheries postharvest & biotechnology, 9 (2), 2014, 45-54 according to martinsdottir et al. (2001), the average score of torry-scale of 5.5 has been used as the limit for consumption. 3.2. the changes of chemical characteristics 3.2.1. total volatile base (tvb) one of parameters widely used to evaluate fish quality is tvb, representing the sum of ammonia, dma, tma and others basic nitrogenous compounds volatile. in the early stage of storage, tvb values slowly increased and increased more rapidly in the remaining time of storage. according to etienne et al. (2005) tvb-n remains constant for the first days of storage or increases slowly but rises fast later in the spoilage since they only rise as a result of the bacterial activity during later stages of deterioration at the beginning of storage in this study, tvb value was 2.58 mgn% and on the 18th day of storage tvb value reached 24.63 mg%n as an acceptable value and increased significantly at the time of rejection reaching up 29.3 mg%n (figure 4). the maximum tvb value that unfit for human consumption is varied depending on the species, and european committee classified into 3 groups, i.e., sebastes spp. (25 mg%n), pleuronectidae family flat fish (30 mg%n), merluccidae and gadidaefamilies, cod and related species (35mg%n) (howgate, 2010). ozogul et al. (20 08) who st ored whi te grou per f i sh (epinephelusaeneus) in ice found that when the grouper was rejected in term of sensory assessment, tvb value reached up to 28.8 mgn%. on the first day of storage tvb value of white grouper was 15.4 mgn%, while tvb value on day 0 (at the beginning of storage) in this experiment was 2.58 mgn%. this difference was clearly due to different species and also different in catching and handling of fish. in this experiment, cultured fish was live transported to the laboratory, and on arrival at the laboratory, fish was killed and immediately sampled to chemical, microbiological and sensory analysis, hence the condition was very fresh. on the other hand, white grouper was caught from the sea, kept in crushed ice on board, delivered to the laboratory in dead condition and sampled on the 1 day of storage. sulieman et al. (2012) also stated that at the first day of storage total volatile nitrogen (tvn) of frozen greasy grouper was estimated at 8.4mg/100g, and after 25 days of storage the tvn increased reaching its marginal acceptable limit (29.4 mg/100g). 3.2.2. trimethylamine (tma) trimethylamine (tma) arises from the bacterial reducti on of tmao (mal le et al., 1986) and development of tma in seafood depends primarily on the content of the substrate trimethylamine-oxide (tmao) in the fish as raw material (anon.,2009). similar pattern to the changes of tvb value during storage, tma values slowly increased in the beginning of storage and increased more rapidly in the remaining time of storage (figure 4). on the 18th day of storage tma value reached 18.75 mg%n and the grouper fish was still acceptable by panelists, while on the 21th day of storage grouper fish was rejected by panelist having tma of 19.7 mgn%. ozogul et al. (2008) observed the deterioration of white grouper during icing and pointed that on the 22th day of storage,white grouper was rejected by panelis having tma value of 17.38 mgn%. different from the results of this study, as well as ozogul finding (ozoul et al., 2008), the tma value of yellow grouper (epinephelusawoara) was still low i.e., approximately 1.7 mgn% although the storage time was already 16 days (wei et al., 2010). this is due to the different handling and processing used for both experiments. the yellow grouper used in wei’s study was in fillet form and vacuum packed in polyethylene bag, while estuary grouper used in this study was in a whole form, ungutted and kept in figure 4. the changes of tvb and tma values of fresh grouper fish during icing. 50 squalen bulletin of marine & fisheries postharvest & biotechnology, 9 (2), 2014, 45-54 copyright © 2014, squalen bmfpb, issn 2089-5690 boxes covered with ice flake (1:2 w/w). the lower formation of tma in fillets could be explained by the absence of viscera and head in which micro organisms that able to convert tmao into tma are concentrated. moreover, in vacuum pack, oxygen present in the bag was limited resulted in lowering bacterial activity to convert non protein compound into biogenic amine including tma. relatively high content of tma during icing in this experiment could be due to higher present of tmao as well as bacterial reduction of tmao in estuary grouper although those parameters were not analysed in this experiment. this was also stated by kodom et al. (2014), that tma content in fresh grouper was relatively high reaching up 12-16 mgn%. regarding to the ratio of tma and tvb content in estuary grouper fish, where tma content was lower than that of tvb content, it is noted that degradation of the fish beyond the reduction of tmao producing tma as well as volatile compounds such as ammonia as proposed by kodom et al. (2014). 3.2.3. k value k value is objective index of fish freshness, calculated from the results of adenosine tri phosphate (atp) degradation ( hiyama et al., 1972; anon., 2009). the k-value gives a relative freshness rating based primarily on the autolytic changes which take place during post mortem storage of the muscle. a short coming of the k-value as a freshness index is that it varies between species owing to differences in rates of atp degradation. it also varies with post-mortem time and temperature, storage conditions, handling conditions and method of killing (olafsdóttir et al., 1997). k value in this experiment increased slowly in the early time of storage up to 9 days of storage and increased moderately to at the remaining of storage (figure 5) reaching up 40.54%. the sample was considered unacceptable by trained panelists although this value was still below the rejection level (60%) proposed by ehira and uchiyama (1987). during 3 days storage, k value was in the level of 5-6% supporting the aleman statement that fish immediately after being killed have k value of 5% (aleman et al., 1982). additional time of storage (612 days storage) increased k value into the level of 11.5 -16.5% and less than 20%, while in the remaining of storage (15-21 days of storage) considerable increased of k value was observed reaching up to 40.54% after 21 days. this phenomenon could be explained that in the early of storage, the amount of atp in the flesh is still abundant, while the amount of atp breakdown products is still low on the other hand, at the end of storage, the amount of atp was depleted while the amount of atp breakdown products such as inosine and hypoxanthine were abundance. this could be understood since the k value is defined as the ratio of the sum of inosine and hypoxanthine (hxr + hx) to the sum of atp and related catabolites consisting of adp, amp, imp, hxr and hx (atp + adp + imp + hxr + hx) expressed as a percentage (ehira and uchiyama (1987). in wild white grouper (e.aeneus) stored in ice, the maximum k value was 90%stored f or 22 days, whi le at the l im it of acceptability k value was proximately 81%, stored for 16 days (ozogul et al., 2008). k value of yellow grouper (epinephelusawoara) stored under vacuum packaging at 0 increased fast with time and reached 54.91% at the end of 15 days storage, keeping the middle level of freshness (wei et al., 2010). siah & ariff (2007) demonstrated that the k-value of fillets of fresh brackishwater grouper packed in plastic bag rose at a fairly moderate rate reaching over 60% from an figure 5.the changes of k value of fresh grouper fish during icing. 51 copyright © 2014, squalen bmfpb, issn 2089-5690 squalen bulletin of marine & fisheries postharvest & biotechnology, 9 (2), 2014, 45-54 initial value of 12% after 11 days of storage, while those packed in modified atmosphere packaging reached 60% after 18 days of storage. there were some variations of k value among fish species and also between individuals of the same fish species, due to differences in rates of atp (hattula, 1997, olafdottir et al., 1997). in addition, the method of killing also have an effect of the k value on the same species related to degraded products, i.e., inosine and hypoxanthine (urbieta & ginés, 2000; ahimbisibwe et al., 2010). 3.2.4. sensory characteristics in relation to chemical parameters sensory evaluation was in good correlation with the results of the chemical assessment for estuary grouper stored in ice. the relationship between dps and chemical parameters is shown in figure 6–8. when tvb and tma values increased, dps increased reflecting decrease in fish quality. dps was correlated well with tvb as well as tma with polynomial pattern where the increment rate of dps was constant, while the rate of increment of either tvb or tma was slower in the beginning of storage and more rapid for subsequent storage. this finding support previous study revealing both tvb-n and tma level which did not increase much during the early stages of deterioration but followed by an exponential increase in the later period of storage (sykes et al., 2009), therefore both tvb and tma are not a reliable indicator of early changes in quality because they only rise as a result of bacterial activity during later stage of deterioration (ozogul & ozogul, 2000).similar polynomial pattern was also obtained in correlation between dps and k value where the k value increased with slow rate during the first 9 days of storage and increased with relatively high rate during the following storage, while the increment rate of dps occurred linearly during the whole of storage period. this indicates that k value is considerably affected by the early autolytic activity. figure 6. correlation between dps and tvb value of grouper fish during icing. figure 7. correlation between dps and tma value of grouper fish during icing. 52 squalen bulletin of marine & fisheries postharvest & biotechnology, 9 (2), 2014, 45-54 copyright © 2014, squalen bmfpb, issn 2089-5690 4. conclusions based on the results of the experiments, it could be concluded that the results of sensory evaluation was in good agreement with the results of chemical assessment for estuary grouper stored in ice, where dps correlated well with tvb, tma as well as k value. in relation to the panelist’s acceptance, estuary grouper fish stored in ice was rejected by panelists on 21 days of storage with tvb value of 29.43 mgn%, tma value of 19.70-22.10 mgn% and k value of 40.54%. references ahimbisibwe, j. b., inoue, k., shibata, t. , & aoki, t. 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(2012). determination of quality and shelf life of three marine fishes (coral trout, greasy grouper and red mouthed bream) based on total volatile nitrogen test (tvn). j. life sci. biomed. 2(5), 187-191 sykes, a. v. oliveira, a. r.,domingues, p. m., cardoso, c .m., a n drad e, j . p., & nu n es, m . l. (2 0 09 ). assessment of european cuttlefish (sepia officinalis, l.) nutritional value and freshness under ice storage using a developed quality index method (qim) and b ioc h em ic al m eth o d s. f o od s c ie n c e a n d technology, 42, 424–432 uchiyama, h., ehira, s. & kato, n. (1972). analytical m eth o d s fo r estim atin g fresh n ess o f fish . in : utilization of marine products. japan; otca; 206-214 u rbieta, f.j., & g in és r . 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(abstract). journal of fisheries of china, 34(8), 12851294. 54 squalen bulletin of marine & fisheries postharvest & biotechnology, 9 (2), 2014, 45-54 copyright © 2014, squalen bmfpb, issn 2089-5690 appendix 1. score sheets of dps for a whole grouper 0 very bright 1 bright 2 slightly dull 3 dull 0 firm 1 soft 0 firm 1 slightly loose 2 loos e 0 tras parent not slim y 1 slightly s lim y 2 slim y 3 very slim y 0 prerigor 1 rigor 2 post-rigor 0 clear 1 slightlycloudy 2 cloudy 0 norm all 1 slightly s unken 2 sunken 0 vis ible 1 not vis ible 0 no blood 1 slightly bloody 2 very bloody 0 characteristics red 1 slightly dark, slightly faded 2 very dark, very faded 0 transparent not slim y 1 moderate 2 exes sive 0 fres h oily, m etallic, seaweed 1 fis hy 2 stale 3 spoilt 0 absent 1 detectable 2 moderate 3 exes sive 0 firm 1 soft 2 burs t 0 norm al 1 slightly break, exudes 2 excess ive, opening 0 fres h 1 neutral 2 fis hy 3 spoilt 0 opales cent 1 greyish 2 yellow-brown 0 red 1 dark red 2 brown belly cavity stains blood belly discoloration firm ness vent condition sm ell eyes clarity shape irish blood gills colour mucus sm ell parameter quality caracteristics scores description general appearance appearance skin scales surface m ucus firm ness qualen bulletin of marine and fisheries postharvest and biotechnology published online: 30 december 2022 page 168 of 176 1 jakarta technical university of fisheries, jl. aup no.1 pasar minggu, south jakarta 12520, indonesia 2 r es ea rch ce nt er f o r ma ri ne a nd lan d bioindustry, national research and innovation agency, ds.teluk kodek, desa malaka, kec. p emen a ng , k ab u pa t en lo mb o k ut a ra , mataram 83352, indonesia *corresponding author: aef.permadi@politeknikaup.ac.id received: 31 october 2022 accepted: 22 december 2022 published: 30 december 2022 academic editor: dr. tatty yuniarti ©squalen bulletin of marine and fisheries pos th arve st a nd b io te ch no lo gy, 2 0 21 . accreditation number:148/m/kpt/2020. iss n: 20 89-56 90, e -iss n: 24 06-92 72. https://doi.org/ 10.15578/squalen.731 o pe n acces s squalen bulletin water soluble chitosan from green mussel (perna viridis) shells and its use as fatabsorber in cookies aef permadi1*, rufnia ayu afifah1, dita ambar kartika apriani1 and farida ariyani2 abstract green mussel chitin can be converted by h2o2 into water-soluble chitosan (wsc). this can subsequently be utilized for a variety of different purposes, such as a fat binder. this study examines how different h2o2 concentrations (13, 21.5, and 30%) affected the properties of wsc (yield, moisture content, ash content, degree of deacetylation, and solubility in water and acid). moreover as well as how wsc (8%, 9%, and 10%) affected the hedonic scores, proximate composition, and fat binding capacity of weight-loss cookies. a single factor completely randomized design and single-factor anova were used to analyze the data, followed by duncan’s additional testing as necessary. the results showed that water-soluble chitosan was impacted by h2o2 concentration in that its yield and ash content decreased, its color changed to a brownish, and its solubility in acid and moisture content all increased. according to de garmo’s effectiveness index test, 30% h2o2 concentration resulted in the best wsc. the addition of wsc did not affect the hedonic quality, protein, moisture, or carbohydrate contents of the cookies, but it did have an impact on the ash and fat contents. the ability of all cookie samples in all treatments to bind fat in liquified butter and peanut oil validates the use of cookies containing wsc in body weight loss research. keywords: water-soluble chitosan, fat-binding capacity, weight-loss cookies introduction chitin, which is found in the cell walls of fungi and the exoskeletons of insects, crustaceans, and molluscs, is the second most abundant polysaccharide in nature after celluloses. the crystallinity and insolubility of chitin limit its use in industry. chitin, therefore, may be converted into chitosan, chitooligosaccharides, and glucosamine, which enhances its biological qualities and expands its uses in the textile, culinary, medicinal, and cosmetic industries. due to their adaptable biological properties, chitin and chitosan are both significantly different in economic significance. the medical and technical applications of chitosan are however restricted because of its low solubility and physiological ph (imtihani et al., 2021). this is especially due to the large molecular weight that makes it difficult to dissolve in water (du et al., 2009). hydrogen per oxide (h 2 o 2 ) could be an additive producing water-soluble chitosan. a study by du et al. (2009), showed that hydrogen peroxide has good potential to convert crude chitosan into water-soluble chitosan. several researchers, namely lestari et al. (2018); chamidah et al. (2019); sudianto et al. (2020); and xia et al. (2013), have used hydrogen peroxide in making water-soluble chitosan from crab and shrimp shells. chitosan is one of the natural ingredients that can reduce fat levels in the blood and is also used in functional food supplements for the prevention or treatment of diseases bacterial, antidiabetic, antioxidant, anticancer, anti-inflammatory, and hypocholesterolemic properties (chiu et al.,2020). in animal studies the dietary supplemented chitosan appears to attach to negatively charged lipids, limiting their absorption through the digestive tr act and lower ing ser um cholesterol (zacour et al., 1992; deuchi et al., 1995; ormrod et al., 1998). chitosan was assumed to be able to bind the fat and cholesterol in the body. therefore, the blood cholesterol could decrease (imtihani et al., 2021; kurniasih et al., 2016; pavinatto et al., 2005; zhou et al., 2006; idacahyati et al., 2020). chitosan mailto:aef.permadi@politeknikaup.ac.id https://doi.org/ squalen bull. mar. fish. postharvest biotech. (2022) 17(3): 168-176 aef permadi et al., page 169 of 176 can also reduce the workload of the liver and reduce the work pressure of other organs due to excess fat (pratiwi, 2014). jin et al. (2017) in his research stated that the potential for fat binding by soluble chitosan will be greater because it is more flexible than rigid chitosan. epidemic over weight a nd obesity a re major challenges to public health (rahmad, 2019). sety et al. (2021) explain that obesity is an excessive or abnormal accumulation of fat in the body. the mechanism of accumulation of fat in the body is caused by the unbalanced amount of energy intake and energy output (kemenkes ri, 2018). consumption of functional food that can reduce energy intake or improve fat metabolism is one of the methods that can be chosen for dietary purposes. one of the food products that can be used as an alternative functional food for people with obesity and diabetes mellitus is cookies (novidahlia et al., 2015), referred to as weight loss cookies. these functional properties can be achieved by substituting the main ingredients, namely wheat flour with other ingredients that have high fiber such as oatmeal, use low-calorie sugar, and ingredients that ha ve fat a bsor ption ca pa city such as chitosa n. according to jin et al. (2017), water-soluble chitosan was an excellent ingredient for functional food to treat obesity due to its ability to absorb, bind and trap cholesterol, fat, sterols, and triglycerides in the diet. in clinical trials, chitosan was found to have anti-obesity benefits and to be able to lower blood pressure, cholesterol levels, and body weight in obese/overweight people as well as in animal models fed a high-fat diet (huang et al., 2019). in addition, liu et al. (2021) demonstrated that high-molecular-weight chitosan was able to increase lipid excretion and inhibit lipid absorption in induced obese rats. a meta-analysis of human trials using chitosan revealed better weight loss with chitosan than with a placebo (ernst & pittler, 1998). bahijri et al. (2017) reported that chitosan improved lipid profile, insulin sensitivity, and oxidative stress and might be useful in controlling the weight of wistar rats fed with a high-fat/high-cholesterolcontaining chitosan diet. there are, however, debates in the literature regarding the ability of chitosan to absorb fat or to reduce weight (mhurchu et al., 2005). green mussels (perna viridis) are one of the indonesian fishery commodities tha t have high economic value and are abundantly found in coastal waters, mangrove areas, and river estuaries. the indonesian mussels (blood clams, green mussels, simping oysters, pearl mussels, and mussels) production in 2020 was 34,427 tons valued at rp. 30,256,914,000 (kkp, 2020). mussels shells, including those from gr een mussel shells, contain chitin (dana rto & distantina, 2016; wulandari et al., 2020), and can be further converted into chitosan using hydrogen peroxide and used for certain health purposes. however, there is limited information available in the literature on the studies of the conversion of their chitin to water-soluble chitosan using hydrogen peroxide. the purpose of this study was to examine the effects of various h 2 o 2 concentrations on the properties of water-soluble chitosan derived from green mussel shells and to examine the possible use of water-soluble chitosan as a fat absorber in weight-loss cookies. material and methods materials the green mussel shells were collected at a green mussel meat picking operation in ketapang, tangerang, and were tr anspor ted to the la borator y of aup polytechnic in south jakarta which took 2 hours driving. the shells were cleaned upon arrival, dried, and ground to 200 mesh. other materials used for producing water-soluble chitosan (wsc) and cookies include calico fabric sheet with a dimension of 150 cm x 60 cm and 232 g/m density, 60 cm x 60 cm filter paper with a maximum pore size of 10-15 m, hydrogen peroxide 50% (supelco merck brand), acetic acid (merck brand), iodized table salt (dolphin brand), palm sugar (nourish indonesia brand), backing powder (koepoe brand), baking soda (koepoe brand), nhexane for analysis, sulfuric acid 95-97%, sodium hydroxide (mer ck brand), methyl r ed indica tor, hydrochloric acid 37% for analysis (merck brand), boric acid (merck brand), di-sodium tetraborate decahydrate for analysis, peanut oil (golden nut brand), butter (menara brand), ethanol 96% and diethyl ether (merck brand). methods green mussel shell meal was initially converted into crude chitosan using danarto and distantina’s method, (2016), and then converted into water-soluble chitosan (wsc) following the method of du et al. (2009). the modification was on the concentrations of hydrogen peroxide. a 2% acetic acid solution was used to dissolve the chitosan in a 1:20 ratio, and different amounts of h 2 o 2 (13, 21.5, and 30%) were added in a 1:2 ratio. the mixture was then heated for four hours at 40 °c in a water bath. the solution was subsequently neutralized with 10% naoh, filtered through the calico fabric, and the filtrate was then added with twice as much 96% ethanol. this mixture was then incubated at 10 °c for 24 hours before drying in an oven at 50 °c for three hours. the experiments were replicated three times. the weight-loss cookies were made based on squalen bull. mar. fish. postharvest biotech. (2022) 17(3): 168-176 the method of zakiyah & handayani,( 2021), while the addition of wsc (0, 8, 9, and 10 g) to 100 g of cookie dough r efer r ed to ma eza ki et a l., ( 1993) . the formulation of cookies is depicted in table 1. the wsc wa s ana lyzed for yield, degr ee of deacetylation using potentiometric titration procedure no iii of czechowska-biskup et al. (2012), solubility in water and acid (tungtong et al., 2012), moisture content (bsn, 2015a), and ash content (bsn, 2010). all tests were done in triplicates. the properties of weight-loss cookies tested in the study included hedonic (bsn, 2015b), moisture-content (bsn, 2015a), ash content (bsn, 2010), protein content using the kjeldahl method (bsn, 2006), fa t content (bsn, 2017), carbohydrate content (by difference), and fat binding capacity (jin et al., 2017) by simulating gastrointestinal conditions in human using peanut oil and liquified butter as standard fats. cookies with and without the addition of wsc from mussel shells were tested. the calories (kcal) in the cookies were calculated per 100 grams using the atwater method (4-9-4) as described by lestari et al. (2017). data analysis one way anova and duncan’s advanced test (spss ibm 25 software) were used to analyze the effect of hydrogen peroxide concentration on the properties of water-soluble chitosan, and to analyze the effect of water-soluble chitosan on the properties of weight loss-friendly cookies. determination of the best concentration of water-soluble chitosan was carried out using the effectiveness index test method (de garmo et al., 1984), by which the parameters included in the analysis were solubility in water and acid, fat binding capacity, degree of deacetylation and yield. results and discussion properties of wsc from green mussel shells the properties of wsc for each treatment are presented in table 2. appearance the appearance of water-soluble chitosan in each treatment was relatively the same. however, regarding the color in figure 1, the control was more white. this may be caused by the maillard reaction during the chitosan depolymerization process as is also reported by tian et al. (2004) that in the ftir spectrum of water-soluble chitosan there is an absorbance at 777.5 cm-1 which indicates a maillard reaction between cho 2 , 5-anhydro-d-mannose and –nh2 of chitosan. the chitosan depolymerization process began with the dissolution of chitosan in a 2 % acetic acid solution and was carried out in a water bath. this led to an increase in ph due to the binding of h+ in solution with nh23 (nucleophile) to form nh + (electrophile). referring to hodge (1953) in yue, (2014) the increase in the reactivity of the amine group by deprotonation in a higher ph solution can lead to the maillard reaction. however, the protonated free amino group can also enhance the steric barrier effect for the maillard reaction. in addition, oxygen due to the open reaction is also able to slow down and even inhibit the maillard, reaction (katsuno et al., 2013). therefore, the color of watersoluble chitosan in each treatment was not profound and only produced a slightly brownish color. w0 w8 w9 w10 oatmeal 25.07.00 25.07.00 25.07.00 25.07.00 low-protein wheat flour 12.09 12.09 12.09 12.09 palm sugar 14.03 14.03 14.03 14.03 butter 21.04 21.04 21.04 21.04 egg 15.06 15.06 15.06 15.06 skim milk 07.01 07.01 07.01 07.01 vanilla extract 00.05 00.05 00.05 00.05 salt 00.05 00.05 00.05 00.05 baking powder 00.05 00.05 00.05 00.05 baking soda 00.05 00.05 00.05 00.05 wsc 0 8 9 10 proportion (g) ingredients note: w0: 0% wsc/control; w8: 8% wsc; w9: 9% wsc; w10: 10% wsc wsc: water soluble chitosan table 1. weight-loss cookies formulation h0 h1 h2 h3 figure 1. chitosan (h0/control) and water soluble chitosan (h1-h3) (h0: h2o2 0%/control; h1: h2o2 13%; h2: h2o2 21.5% and h3: h2o2 30%) aef permadi et al., page 170 of 176 squalen bull. mar. fish. postharvest biotech. (2022) 17(3): 168-176 yield the yield of water-soluble chitosan for each treatment was calculated based on the percentage of final weight after precipitation by ethanol to the initial weight (5 g) as shown in table 2. the results of the anova test and duncan’s test showed that differences in hydrogen peroxide concentration resulted in significantly different yields (p<0.05), where the higher the concentration, the lower the yield. according to tian et al. (2004), the molecula r weight of chitosa n decr ea ses as the concentration of hydrogen peroxide used increases, and the lower the molecular weight of chitosan, the lighter and smaller the molecular size. this prevents chitosan from being precipitated by ethanol and released during the filtration process (tanasale, 2019). degree of deacetylation the percentage loss of acetyl groups in chitosan is indicated by the degree of deacetylation. the proportion of d-glucosamine and n-acetyl-d-glucosamine in chitosan determines the degree of deacetylation (saleh, 2017) . ta ble 2 displa ys da ta on the degr ee of deacetylation of water-soluble chitosan for each treatment. the anova test results revealed that differences in hydrogen peroxide concentration resulted in significantly different degrees of deacetylation (p<0.05), with the higher concentration resulting in a lower degree. when the reaction is carried out under a cidic conditions, mor e a mino gr oups become protonated groups, increasing chitosan solubility and assisting in raising the ph of the solution. this shows that the more free amino groups in the polysaccharide chain, the easier –nh2 reacts with h 2 o 2 to break the chitosan chain (tian et al., 2004). solubility the w sc solubility wa s expr e ssed in two parameters, namely solubility in acid and solubility in water. the data presented in table 2 shows that differences in hydrogen peroxide concentration resulted in significantly different solubility in acid and water (p<0.05). it might be due to the hcl being the corrosive acid so the chitosan was easily soluble. whenever more acetyl groups in wsc were cut or deacetylated, the molecular weight decreased causing chitosan more soluble in water (tian et al., 2004). therefore the value of the solubility of chitosan will be directly proportional to the degree of deacetylation. based on table 2, it can be seen that the highest solubility of wsc at the degree of deacetyla se wa s 75.08% . depolymer iz a tion decreases molecular weight and shortens chitosan molecular chains (tanasale et al., 2016). santoso et al. (2020) state that an increase in the degree of deacetylation may contribute to a reduction in the molecular weight of chitosan. according to chamidah et al. (2019). chitosan with low molecular weight makes chitosan ha ve a high solubility in water. meanwhile, according to lu et al. (2004), the degree of deacetylation of chitosan will determine the solubility of chitosan in water and chitosan will dissolve in water with a degree of deacetylation of 46.7-64.4%. moisture and ash contents the moisture content of water-soluble chitosan is presented in table 2. the addition of hydrogen peroxide significantly (p<0.05) affected the moisture content. the moisture content of chitosan is influenced by the relative humidity of the air around its storage area table 2. properties of wsc from green mussel shells note: h0: h2o2 0%/control; h1: h2o2 13%; h2: h2o2 21.5% and h3: h2o2 30% *different letters that follow the figures in the same row denote a significant difference (p<0.05) parameters h0 h1 h2 h3 sni 7949-2013 appearance brownish white brownish white brownish white brownish white pale brown to white yield 44.79±1.44a 37.40±0.14b 33.13±1.06c degree of deacetylation 78.01±0.35d 72.37±0.07c 73.59±0.14b 75.08±0.12a min 75% solubility: ·  acid 92.92±0.11d 93.59±0.09c 95.77±0.56b 97.27±0.27a min 99% ·  water 21.77±0.03d 76.39±0.58c 86.92±0.23b 91.72±0.40a moisture content 0.41 ± 0.04d 2.20 ± 0.09c 2.59 ± 0.03b 3.71 ± 0.07a max 12% ash content 4.54 ± 0.03a 4.19 ± 0.06b 4.15 ± 0.08b 4.11 ± 0.07b max 5% fat binding capacity: ·  peanut oil 4.73 ± 0.34d 5.61 ± 0.02c 8.30 ± 0.35b 9.18 ± 0.13a ·  butter 4.10 ± 0.04d 4.35 ± 0.15c 6.37 ± 0.06b 7.60 ± 0.09a aef permadi et al., page 171 of 176 squalen bull. mar. fish. postharvest biotech. (2022) 17(3): 168-176 because chitosan is easy to absorb moisture from the surrounding air. chitosan polymer groups (amine, nacetyl, and hydroxyl groups) will form hydrogen bonds with h 2 o from the air (dompeipen et al., 2016). table 2 also shows that the addition of hydrogen peroxide significa ntly ( p<0.05) r educed the a sh content compared to the original chitosan, while the effect of hydrogen peroxide concentrations was not significant. the ash content of water-soluble chitosan in each treatment was relatively lower when compared to that of the original chitosan. the organic materials used in the process were thought to affect the ash content of water-soluble chitosan. the acetic acid was used to bind the amine groups of chitosan, hydrogen peroxide for depolymerization of chitosan, naoh to neutralize the solution, and ethanol to precipitate the filtrate. according to basmal et al. (2007), the ash content of water-soluble chitosan is influenced by the number of organic materials that react with chitosan during the process of converting water-soluble chitosan. fat binding capacity the molecular weight of water-soluble chitosan will change depending on the hydrogen per oxide concentration. the molecular weight of chitosan decr ea ses wit h incr ea sing hydrogen per oxide concentration. the molecular dimensions and shape of chitosan must be able to capture fat or oil droplets (muzzarelli, 1996). this is due to a high likelihood of failing to capture fat droplets of bigger size, small particles with short polymer chains are less effective at binding fat, making the chitosan-lipid complex less durable. a higher percentage of fat molecules can be maintained in the particles and medium-sized polymer chains are more flexible. due to its high degree of molecular affinity and crystallinity, the ability of chitosan to bind fat will decrease as polymer chains get longer. additionally, the capacity to bind fat may be diminished as a result of the potent intramolecular interactions and the creation of particle aggregates. the ability of different chitosan chain lengths to form micelles and capture the oil phase in precipitation varied (czechowska-biskup et al., 2005). due to their relative rigidity, long chains are not appropriate for producing micelles. the ability to trap fat molecules may be improved by shorter chains with more mobility. at neutral ph, very short chains will have a higher level of solubility. as a result, chitosan will be more likely to form stand-alone dissolved molecules than to bind fat molecules. in a ddition to molecula r weight, a nother characteristic of chitosan that can affect fat-binding capacity is the degree of deacetylation. to ascertain the relationship between the level of deacetylation and the ability of water-soluble chitosan to bind fat, pearson analysis was used. for peanut oil and butter, the coefficient of determination (r2) was respectively 0.9329 and 0.9695. the capacity of chitosan to bind fat increases with deacetylation level, resulting in a more linear relationship between the two (dimzon et al., 2013). chitosan had a higher level of deacetylation than the three samples of water-soluble chitosan, but it had a lower potential for binding to fat. this is probably because chitosan has a more rigid structure, making it less effective at trapping fat droplets. in contrast, water-soluble chitosan has a shorter chain with higher mobility, making it more successful at doing so. selection of best h2o2 concentration compared to sni 7949-2013 for chitosan (bsn, 2013), specifications for moisture and ash contents were met by all samples, while for the degree of acetylation, only h0 and h3 met the specification. to select the best h 2 o 2 concentration for the current condition, effectiveness index test (de garmo et al.,1984), wa s used. ea ch par ameter wa s given weighted scores for importance, followed by a series of calculations to result in an effectiveness index for each parameter which was the total ranked. the results are presented in table 3, showing that h3 (wsc produced with 30% h 2 o 2 ) obtained the highest total value, ie. 0.598. in the second experiment, weight-loss cookies were made using this wsc (h3). properties of weight-loss cookies the properties of the cookies tested included hedonic scoring, proximate composition, and calories; all were compared to sni 01-2973 (bsn, 1992) for appropriate parameters, of which the results are presented in table 4. h0 h1 h2 h3 solubility in water 0 0,119444444 0,142361111 0,152777778 solubility in acid 0 0.015 0.034 0.048 fat binding capacity 0,013 0 0,082638889 0,229166667 degree of deacetylation 0,18 0.068 0.022 0 yield 0,2 0.036 0.014 0 total 0,393 0,202083333 0,272916667 0,415277778 parameters effectiveness index table 3. result of the effectiveness index test note: h0: h2o2 0%/control; h1: h2o2 13%; h2: h2o2 21.5% and h3: h2o2 30% aef permadi et al., page 172 of 176 squalen bull. mar. fish. postharvest biotech. (2022) 17(3): 168-176 hedonic scores the average hedonic scores of cookies as presented in table 4 are the average scores of appearance, flavor, taste, and texture. as shown in the table, wsc did not affect the scores significantly (p<0.05). however, the scores were increased when the concentration of wcs was added at the level of 8% compared to the control treatment (wcs 0% ) and then decreased as the concentration increased. similarly to this, ghoshal & mehta, (2019) found that as chitosan levels grew from 0.1 to 2% w/w, bread’s sensory scores decreased. since chitin and chitosan are known to have inherent astringency (luck et al., 2015 and wang et al., 2021) it could be possible that the hedonic ratings of cookies in this study may have declined as the wsc increased. proximate composition water in food is a medium for bacteria (microbes) and fungi to grow and develop (natalia et al., 2019). the way to preserve food is to reduce the moisture content in foodstuffs. overall, the moisture content in each treatment has met the standard for biscuits (bsn, 1992), which is a maximum of 5%. the anova showed that there was no effect of the addition of water-soluble chitosan on the moisture content of cookies. ash content is a mixture of inorganic or mineral components contained in a food ingredient (novidahlia et al., 2015). overall, cookies in each treatment have met the standard limit of ash content for biscuits (bsn,1992), which is a maximum of 1.5%. however, cookies with the addition of water-soluble chitosan have a relatively high ash content. the ash content detected in the cookies most likely came from the added water-soluble chitosan. chemically, the added quality of wsc contained 4.11% ash. in addition, the large proportion of oatmeal in the cookie is also a contributing factor. oatmeal contains fiber consisting of the basic elements that make up plant cell walls and contain inorganic ions. fiber can act as a mineral and electrolyte binder because of the presence of free carboxyl groups in the glucuronic acid that makes up hemicellulose (schneeman, 1986, kumalasari, 2018). therefore, the higher the fiber content in cookies, the higher the ash content. the anova showed that there was a very significant difference in the addition of wsc to the ash content of cookies. the greater the mass of the addition of water-soluble chitosan in cookies, the greater the ash content of cookies. this is due to the ash content in the water-soluble chitosan itself which can increase the ash content of cookies. overall, the protein content in cookies for each treatment did not meet the standard for biscuits (bsn, 1992), which was a minimum of 9%. the low content is rela ted to pr otein loss due to heating during processing, which caused the maillard reaction. the maillard reaction occurs at a temperature above 115 °c, while cookies are baked at a temperature of w0 w8 w9 w10 sni 01 2973 hedonic score (9-scale) 7.17±0.69a 7.37±0.55a 7.30±0.69a 7.13±0.99a proximate composition (%): ·     moisture 4.24±0.1a 4.14±0.04a 4.38±0.07a 4.19±0.04a max 5 % ·     ash 0.35±0.01c 0.84±0.03b 0.85±0.02bc 0.92±0.01a max 1.6 % ·     protein 6.66±0.21a 6.80±0.06a 7.00±0.03a 6.90±0.01a min 9 % ·     fat 11.29±0.13a 12.00±0.01a 10.73±0.19b 10.96±0.02b min 9.5 % ·     carbohydrate 76.95±0.08a 76.22±0.02b 77.01±0 .23a 77.03±0.02a min 70 % total calories (kcal/100g): 441.47±0.95 440.145±0.29 432.69±1.01 434.425±0.35 min 400 protein 26.66±1.16 27.22±0.37 28.02±0.20 27.6±0.06 -  carbohydrate 307.8±0.45 304.88±0.11 308.06±1.27 308.14±0.08 -  fat 107.01±1.65 108.05±0.19 96.62±2.48 fat binding capacity : •      peanut oil 4.15±0.01d 9.26±0.04c 12.22±0.03b 13.16±0.04a •      liquified butter 4.36±0.11d 8.24±0.06c 10.36±0.04b 11.10±0.05a properties treatment table 4. properties of weight-loss cookies note: w0: 0% wcs/control; w8: 8% wcs; w9: 9% wcs and w10: 10% wcs; * different letter following figures in the same row indicates a significant difference aef permadi et al., page 173 of 176 squalen bull. mar. fish. postharvest biotech. (2022) 17(3): 168-176 160 °c for 10 minutes (widiawati & anjani, 2017). in addition, the use of low-protein flour can also be a factor causing the low protein content in cookies. the anova results showed that there is no significant difference in the addition of wsc to the protein content of cookies. based on a standard for biscuits (bsn, 1992), the standard carbohydrate content in cookies is at least 70%. so it can be concluded that cookies that have met the standard are those with the addition of 9 and 10 grams of wsc. oats and wheat flour are ingredients that contribute to the carbohydrate content of cookies. oats a r e whole gra ins, which include complex carbohydrates so they take longer to be digested by the body (utami, 2020). the anova shows that there is a significant effect (p<0.05) of the addition of watersoluble chitosan on the carbohydrate content of cookies calories of cookies the calorie of cookies is calculated as data to support the creation of weight-loss-friendly cookies. although the weight-loss cookies produced are low in calories, the quality still meets the standards of oatmeal cookies as a product with ideal nutrition which must contain a minimum of 400 kcal/100 g of energy (van tongeren & jansen, 2020). table 4 shows the total calories in cookies having met the minimum energy of cookies. fat binding capacity the test results of fat binding capacity (table 4) show that all samples can have the ability to bind fats, both fats from peanut oil and liquified butter. the addition of wsc to cookies has significantly affected the fat-binding capacity of cookies to both peanut oil and liquified butter fats (p<0.05). the greater the concentration of water-soluble chitosan, the greater the fat-binding capacity. these results are consistent with a study conducted by maezaki et al., (1993), that the addition of 99.2 grams of chitosan in 1 kg of biscuit dough was able to reduce ldl levels, triglycerides, and increase hdl levels in the respondents’ blood. based on table 4 it can be concluded that the fat-binding capacity of the sample added with wsc is greater in peanut oil than in liquified butter. peanut oil contains more monounsaturated and polyunsaturated fatty acids (sinaga, 2018), while butter contains more saturated fatty acids (li et al., 2018). the interaction of chitosan and fatty acids will be different depending on the type of acid as stated by (wydro et al., 2007) that the binding power or tr a pping power of chitosa n to monounsaturated fatty acids and polyunsaturated fatty acids is stronger than that of saturated fatty acids. conclusion wa tersoluble chitosa n is a ffected by h 2 o 2 concentration in that the color turns brownish, the yield and ash content decrease, and the increasing of a degree of deacetylation, solubility in acid and water, and moisture content. when hydrogen peroxide is used to produce wsc, a 30% concentration produced the best wsc. the hedonic results of quality, protein, water, and carbohydrate contents of cookies were unaffected by the addition of wsc. however, the ash and fat contents were affected. all cookie samples in all treatments demonstrated the ability to bind fat in liquified butter and peanut oil, which justifies the use of cookies containing wsc in body weight reduction research. further research is needed to be carried out related to the fat-binding capacity of cookies using the in vivo method in test animals (pure strain white mice) or humans. supplementary material supplementary material is not available for this article. references bahijri, s. m., alsheikh, l., ajabnoor, g., & borai, a. 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(2006). in vitro binding of bile acid s a nd triglycerid es b y selected chitosan preparations and their physico-chemical properties. 39, 1087–1092. https://doi.org/10.1016/j.lwt.2005.07.009 aef permadi et al., page 176 of 176 https:// https://doi.org/10.1088/ https://doi.org/ https://doi.org/10.1016/j.carbpol. https://doi.org/10.11648/ https://doi.org/10.1016/j.ijbiomac.2013.04.034 https://doi.org/10.1016/j.lwt.2005.07.009 qualen bulletin of marine and fisheries postharvest and biotechnology research article published online: 30 august 2022 page 85 of 94 1 faculty of science and marine environment, universiti malaysia terengganu, kuala nerus 21030, terengganu, malaysia 2 biological security and sustainability research gr ou p ( bio se s), fa cu lty of s cie nce a nd ma rin e env iro nm ent , u niv er sit i m al ays ia terengganu, 21030, kuala nerus, terengganu, m al a y si a 3 institute of marine biotechnology, universiti malaysia terengganu, 21030 kuala nerus, terengganu, malaysia 4 centre for drug research, universiti sains ma lay si a, pul au pi nan g, ge lug or, 1 180 0, m al a y si a 5 school of chemical sciences, universiti sains ma lay si a, pul au pi nan g, ge lug or, 1 180 0, m al a y si a *corresponding author: ramesh@umt.edu.my; vicky@umt.edu.my received: 23 june 2022 accepted: 5 august 2022 published: 30 august 2022 academic editor: dr. ariyanti suhita dewi ©sq ual en bul let in of mar ine an d f ish eri es po st har ves t and bi ot ech nol og y, 202 1. ac cre di tat ion n umb er: 14 8/m /kp t/ 202 0. is sn : 2 089 -5 690 , e -i ssn : 240 6-9 27 2. https://doi.org/ 10.15578/squalen.669 o pe n acces s squalen bulletin anticancer potential of three sea cucumber species extracts on human breast cancer cell line rameshkumar santhanam1,2*, nurul shahirah mohd azam1,3, ammira shafiqha abdul khadar1,3, ambrose louise1,3, gregory dominic1,3, nur shahida ahmad sofian1,3, see wee han1,3, thiruventhan karunakaran,4,5, thilahgavani nagappan1, tengku sifzizul tengku muhammad3, and sevakumaran vigneswari1,2,3* abstract sea cucumber has long been utilized as a treatment for a variety of ailments, including antibacterial, antifungal, immunomodulatory, and wound healing. as for the first time, the extracts of three sea cucumber species’ i.e, actinopyga lecanora, holothuria atra, and stichopus vastus, were compared and tested on the cytotoxicity of cancer cells using mtt and annexin v/propidium iodide assays. this study investigates the protection of sea cucumber extracts against the breast cancer cell line (t-47d). all three extracts were found to be actively declining the cancer cell progression, with the ic50 values of 6.25±0.50 µg/ml (a. lecanora), 7.5 ± 1.39 µg/ml (h. atra) and 3.25 ± 0.53 µg/ml (s. vastus). lc-ms/ ms analysis was used to identify chemical compunds in the extracts. the 1,1diphenyl-2-picrylhydrazyl (dpph), total phenolic and flavonoid contents, and anti-collagenase activity were also assessed in all the three extracts. the results demonstrated the absence of antioxidant and flavonoid chemicals in s. vastus, a. leconara and h. atra extracts. however, h. atra contained phenolic compounds (0.4794 mggae/g dw). furthermore, all tested extracts showed significant anti-collagenase activity, which supported the reduction of cancer cell proliferation. however, more research into the mechanism of action of the extract is needed before sea cucumbers therapeutic characteristics may be used to combat breast cancer. keywords: sea cucumber, cytotoxic, anti-collagenase, phenolic, antioxidant introduction br ea st ca ncer is the second la rgest cause of morta lity and the second most prevalent cancer identified in women, according to the national cancer institute in developed and developing countries (francies et al., 2020). there are about 2.26 million new cases and almost 685,000 deaths from breast cancer in 2020 (iarc globocan, 2020). some breast cancers are not inherited, but others are attributable to genetic propensity primarily due to mutations in the tumor suppressor genes of breast cancer 1 (brca1) and breast cancer 2 (brca2) genes (american cancer society, 2021) . tr ea tments such a s surger y, radiotherapy, hormone therapy, chemotherapy, other targeted treatment or even a mixed treatment are currently available to treat breast cancer (mustafa et al., 2016). the treatments used for those who have breast cancer somehow depend on the type and stages of cancer. these treatments are categorized into two, local and systemic treatments. local treatments are meant to treat tumors without affecting other body parts. the treatments are surgery and radiation therapy. as systemic treatments, the patients are treated using drugs, administered orally or intravenously. the currently available treatments seem to be less effective as it causes side effects to the patients such as nausea, hair loss, loss of appetite and vomiting (panno, 2010). since the current modern treatment mailto:ramesh@umt.edu.my; mailto:vicky@umt.edu.my https://doi.org/ squalen bull. mar. fish. postharvest biotech. (2022) 17(2): 85-94 santhanam, r., et al., page 86 of 94 has so many side effects on patients, finding a new alternative method that can kill or incapacitate cancer cells without causing harm or excessive damage to the normal cell has become the main priority. natural products play a main role in developing an anticancer cure, and marine organisms’ metabolites content is starting to gain popularity in the field of antitumor drug discovery (nagle et al., 2004). a marine organism is an excellent natural product source since 70 % of the earth comprises oceans. in recent years, more than 3000 new compounds from marine sources have been discovered to exhibit potential anticancer and antitumor properties and a number of them have gone into clinical trials (khalifa et al., 2019). although the marine resources are rich with potential chemicals, most of them are still unexplored and their function remains unknown. sea cucumbers are one of the most important marine organisms found almost in every marine environment that belongs to the class holothuroidea. it is a long worm-like organism with a gelatinous body that usually is soft-bodied echinoderm (janakiram et al., 2015). in the last two decades, sea cucumber extracts have been widely studied in the medical and pharmaceutical fields as wound healing promoters, exhibiting anticancer, antimicrobial a nd immunomodulatory properties (fredalina et al., 1999). sea cucumber may possess potential compounds for fighting cancer as they consist of protein, vitamin a, thiamine, riboflavin, niacin, calcium, iron, magnesium, zinc, and other unique molecules (janakiram et al., 2015). there are various kinds of compounds from sea cucumber such as monosulfated triterpenoid glycoside frondoside a, the disulfated glycoside frondoside b, the trisulfated glycoside frondoside c, 12-methyltetradecanoic acid, griseaside a, echinoside, cucumarioside a2–2 and fucosylated chondroitin sulfate. these compounds have been explored and reported as anticancer compounds for various types of cancer such as colon cancer, liver cancer, lung cancer and cervical cancer (wargasetia & widodo, 2022; wargasetia & widodo, 2017; sajwani, 2019). in this study, for the first time, three different sea cucumber s, namely ac tinopy ga le c anora, holothuria atra and stichopus vastus were studied in comparison to their potential cytotoxic effect against t-47d breast cancer cell line and their mode of cell dea th investiga ted. this study a imed to provide preliminary data to compare the effectiveness of the three species of sea cucumber extracts as an alternative option to tr ea t br ea st ca ncer ( figur e 1) . this preliminary data will pave the way to study further the molecular mechanisms of bioactive metabolites derived from sea cucumbers and translate them from bench to bedside in the future. materials and methods chemicals and reagents ascorbic acid, 2,2-diphenyl1-picrylhydra zyl (dpph), myricetin, epigallocatechin gallate, coomassie brilliant blue were purchased from sigma-aldrich, uk while methanol, aluminium chloride, calcium chloride, sodium chloride and porcine gelatin were purchased from r&m, uk. other than that, gallic acid, follin ciocalteu’s phenol reagent, sodium carbonate, and tris hydrochloride were obtained from merck, germany, mts assay from promega, annexinv-fitc from sigma-aldrich. sample preparation the fresh samples of a. lecanora, h. atra and s. vastus were obtained from the repository of institute marine biotechnology (imb), university malaysia ter engga nu. the ta xonomic identity of the sea figure 1. schematic summarizes the study which involves cytotoxic effect against breast cancer cell line using the extract from different species of sea cucumber. squalen bull. mar. fish. postharvest biotech. (2022) 17(2): 85-94 cucumber is identified based on its morphology and mitochondrial dna evidence. specimen id for the deposited sa mples in imb a s a. le c anora (ech1015002), h. atra (ech1015003) and s. vastus (ech1015001). briefly, the fresh samples were washed and rinsed several times with fresh water to remove the sand and salt in the sample. the sample was cut into small pieces and then dried using a freeze dryer to remove the moist and liquid. the yield of each aqueous extract was calculated as the weight of dried extract to the weight of the frozen sample in the range of 1.2– 1.5 %. sample extraction about 200 g of dried powder of sea cucumber samples were soaked in 750 ml of hexane for 24 h and were shaken at 200 rpm on an orbital shaker. then the hexane was filtered from the sample through whatman filter paper no. 2 (110ø) under gravity. the exact process was repeated three times and the hexane extract was discarded. next, the samples were soaked in absolute methanol for 24 h and kept in an orbital shaker at 200 rpm for 24 h. dissolved samples were filtered through whatman filter paper no. 2 (110 ø) and the process was repeated three times. the filtered solution was dried using a rotary evaporator (ika, germany) under vacuum pressure below 40 °c and the methanol crude extract was obtained and stored in –20 oc. cell culture and maintenance the breast cancer cell lines (t-47d) were obtained fr om the institute of pha r ma ceutica ls a nd nutraceuticals (ipharm), malaysia and stored in a liquid nitrogen tank. the medium preparation was carried out in biosafety cabinet (bsc) class ii. the medium wa s pr epa red using rpmi 1640, which wa s supplemented with 10% of fetal bovine serum (fbs), 1% (v/v) of non-essential amino acids, 1% sodium pyruvate, 1% antibiotic solution (100 u/ml penicillin and 100 g/ml streptomycin) to prevent the growth of bacteria. insulin was added to the media to activate the cell lines and the media was stored at 4 oc. initially, the cells’ vials were thawed in a 37 oc water bath and the vials were transferred to bsc class ii and wiped with 70% of ethanol. then the cells were transferred into a centrifuge tube containing a 5 ml medium and were centrifuged at 200 g for 5 mins. after that, the supernatant was removed and the pellet form was suspended in a 5 ml medium and cultured in a t-25 flask. then it was incubated at 37 oc in a 5% carbon dioxide incubator. the medium of the cell culture was then replaced every three days and the cells were monitored every day to observe their growth and contamination. subculture of cell lines the cells were observed daily under an inverted phase-contrast microscope to determine the confluence of the cells in the t-25 flask. once the cells reached 80% confluence, the old medium was discarded from the flask and the cells were washed with phosphate buffer saline (pbs) to remove any remaining waste. after that, pbs was removed from the flask, and subsequently, trypsin-edta (1x) was added to the t-25 flask and incubated at 27 °c for 10 mins to ensure the full detachment of the cells from the growth surface. after several mins, the complete medium was a dded into the fla sk a nd mixed gently for homogenization to deactivate trypsin. next, the cells were transferred into a tube containing 10 ml of complete medium. the tube containing the medium was centrifuged at 1000 rpm for 5 mins. the cells were transferred into a completely fresh medium after removing the supernatant. the number of cells was calculated using a hemocytometer. subsequently, as many as 6000 cells/well of breast cancer cells line t47d were cultured in a 96-well plate in 100 l of complete medium for each well. the plate wa s incubated in 5% co 2 for 24 h before the treatment. cytotoxicity assay the cells were treated with various concentrations of h. atra extracts (0 g/ml, 1.56 g/ml, 3.12 g/ml, 6.25 g/ml, 12.5 g/ml, 25 g/ml and 50 g/ml). positive and negative controls were also used in this treatment. one percent (v/v) dmso served as negative control and vincristine sulfate (10 µg/ml), a vinca alkaloid derived from catharanthus roseus, served as the positive control. the cells were then incubated at 37 oc for 72 h in a 5% co 2 incubator. then 20 µl of mts solution was pipetted into each well and incubated at 37 oc, 5% co 2 for 3 to 4 h and six replicates were carried out for each concentration. the viability of cells was measured using a microplate reader (glomax-multi detection system, promega) at 490 nm. then the percentage of cell viability was calculated using the following formula annexin v and propidium iodide assay annexin v and propidium iodide assays were used to detect the early stages of apoptosis. the cells were santhanam, r., et al., page 87 of 94 % squalen bull. mar. fish. postharvest biotech. (2022) 17(2): 85-94 seeded in a 96-well plate and incubated with various extract concentrations, a. lecanora (6.25 µg/ml), h. atra (7.5µg/ml) and s. vastus (3.25 µg/ml) extracts and compared with vincristine sulfate (positive control, 0.25 µg/ml) for an approximate of 12-hour time gap starting from 0 h to 36 h. the medium in each well was removed and 20 l of annexin v and propidium iodide reagents were added. cells were then incubated at 25 oc for 15 mins. next, the cells were analyzed using fluorescence microscopy techniques with an excitation wavelength of 488 nm and a detection range of 540 nm. lc-ms/ms analysis of the sea cucumber extracts an agilent 1200 series chromatograph (agilent technologies, santa clara, ca, usa) was used to perform the analysis, which was coupled to a bruker impact ii qtof mass spectrometer (bruker daltonics, bremen, germany). for chromatographic separation, a zorbax eclipse xdbc18 column (1.0x150 mm, 3.5 m, agilent technologies, santa clara, ca, usa) was employed with a zorbax sbc8 guard column (2.1x12.5 mm, 5 m, agilent technologies, santa clara, ca, usa). one percent formic acid in h 2 o (eluent a) and 1% formic acid in meoh were used as mobile phases (eluent b). the gradient program was as follows: isocratic at 60% of eluent b from start to 3 min, from 60% to 90% eluent b from 3 to 29 min, from 90% to 100% eluent b from 29 to 30 min, isocratic at 100% of eluent b to 35 min, from 100% to 60% eluent b from 35 to 38 min. after returning to the initial conditions, the equilibration was achieved after 15 min. chromatographic separation was performed at a 0.1 ml/min flow rate at 40 oc. the injection volume was 1 l. mass spectrometry detection has been performed using an esi ionization source. the mass spectra were recorded within the m/z mass range of 100–1500 and 70–1500 for ms/ms spectra (scan time 1 s) (popov et al., 2017). dpph free radical scavenging assay the free radical scavenging activity of the extracts on dpph radical was determined using the method. test samples (a. lecanora, h. atra, s. vastus) and standard (ascorbic acid) were prepared in various concentrations (0.008, 0.016, 0.031, 0.063, 0.125, 0.250, 0.500 mg/ml) with serial dilution. then, 0.1 mm dpph (1.9715 mg in 50 ml methanol) was freshly prepared. methanol was served as blank or control. in each well of the 96-well plate, 100 µl of sample and 100 µl of dpph reagent were loaded and left in the dark at room temperature. after 30 min incubation, the absorbance of the mixture was measured using a spectrophotometer (perkin-elmer, new jersey) at 517 nm and measurements were made in triplicates. the dpph radical scavenging activity was calculated using the formula: where: abs control is the absorbance of dpph radical + methanol  abs sample is the absorbance of dpph radical + sample extract/standard total flavonoid content (tfc) the total flavonoid content for all the samples was determined using the spectrophotometric method (santhanam et al., 2013). test samples (a. lecanora, h. atra, s. vastus) and standard (quercetin) were prepared in methanol with various concentrations (0.016, 0.031, 0.063, 0.125, 0.250, 0.500, 1.000 mg/ ml) using serial dilution. next, 2% of alcl 3 solution (2 g of alcl 3 in 100 ml methanol) was prepared. in each well of the 96-well plate, 100 µl of sample and reaction solution alcl 3 were loaded and samples were made triplicate. then, the reaction mixtures were incubated for an hour at room temperature. the absorbance was measured using a spectramax plus microplate reader at max 415 nm. total fla vonoid contents were determined using the quercetin standard curve and expressed as mg quercetin equivalent (qe) per gram of dry plant extract (mg qe/g). total phenolic content the total phenolic content for all the samples was deter mined using the folincioca lteu method (santhanam et al., 2013). briefly, 50 µl of extracts prepared in methanol with various concentration (0.016, 0.031, 0.063, 0.125, 0.250, 0.500, 1.000 mg/ ml) was mixed with 50 µl distilled water, 50 µl of 10% follin ciocalteu’s phenol reagent and 50µl of 1 m sodium carbonate solution in a 96-well plate. methanol was used as blank and gallic acid was used as standard. reaction mixtures were incubated for an hour in the dark at room temperature. the absorbance of the reaction mixture was measured using a spectramax plus microplate reader at max 750 nm. then, the total phenolic content was determined using the gallic acid, and the results are expressed as milligram gallic acid equivalents (gae) per gram of dry extracts. all tests were made in triplicates. dpph radical scavenging activity (%) = (abs control)] [(abs control – abs sample)]/ × 100 santhanam, r., et al., page 88 of 94 squalen bull. mar. fish. postharvest biotech. (2022) 17(2): 85-94 gelatin digestion assay the gelatin digestion assay was done according to previously described (santhanam et al., 2018) with slight modifications. agarose (2%) was prepared in collagenase buffer (50 mm tris-hcl, 10 mm cacl 2 , 0.15 m nacl, ph 7.8) and 0.15% porcine gelatin. next, the mixture is poured into the petri dish and allowed to solidify (about one h) at room temperature. after solidification, wells were made using a sterile 200 µl microtip. then, an aliquot of 25 µl of the samples (0.25, 0.50 and 1.00 mg/ ml prepared in collagenase buffer) were incubated with 25 µl of bacterial collagenase-1 (0.1 mg/ ml) for 1 h. after incubation, the reaction mixture (50 µl) was loaded into the well and further incubated overnight. epigallocatechin gallate (egcg) was used as the positive control. the degree of gelatin digestion in agarose gel was visualized by coomassie brilliant blue staining. the gelatinase inhibition activity was determined by measuring the a r ea of the light tr a nslucent z one over a blue background formed after destaining. all tests were made in triplicates. statistical analysis data was provided as mean ± sd. graphpad prism version 5 was used for statistical analysis, with a 95% confidence level * (t-test). the significant value was set at p < 0.05. results and discussion cytotoxicity e ffects of sea cucumber extracts on human breast (t-47d) cancer cells the present study evaluated the cytotoxic effects of the sea cucumber extracts such as actinopyga lecanora, holothuria atra and stichopus vastus against human breast (t-47d) cancer cells. cells were treated with various concentrations such as 0 g/ml, 0.78 g/ml, 1.56 g/ml, 3.13 g/ml, 6.25 g/ml, 12.5 g/ml, 25 g/ml and 50 g/ml of sea cucumber extr acts. six replicates were prepa red for ea ch concentration of the extracts. table 1 shows the ic 50 value of the samples against the human breast (t-47d) cancer cells. a previous study reported that the ethanolic extract of h. atra was found to be significantly cytotoxic to the t-47d breast cancer cell line with an ic 50 value of 9.6 µg/ml. tr iter pene glycosides such a s cucumechinol and philinogenin b were the chemicals responsible for the bioactivity (nursid et al., 2019). a similar pattern was observed in this study where the methanolic extract of h. atra induced cytotoxicity towards t-47d breast cancer cell line with an ic 50 value of 7.94 ± 1.39 µg/ml. the saponins, namely holothurin a5, holothur in a, echinoside a a nd 24dehydroechinoside a, isolated from the h. atra etoac/meoh (1:1) extract were also reported to possess significant cytotoxicity towards human cervix carcinoma hela cell line with the ic 50  values ranging from 1.2 to 2.5 µg/ml (grauso et al., 2019). studies on s. vastus also revealed that it has an appreciable cytotoxic effect towards mcf-7 cancer cell line with the ic 50 value of 65.14±5.59 µg/ml. triterpene tetraglycosides isolated from the sticophus was reported to possess significant cytotoxicity against mcf-7 breast cancer cell line with the ic 50 value ranges from 1.56 µm to 11.45 µm. in this research, the methanolic extract of s. vastus possessed appreciable cytotoxic towards t47d breast cancer cells with the ic 50 value of 3.16 ± 0.53 µg/ml. furthermore, studies on the extracts of actinopyga sp. reported that it has moderate cytotoxicity towards the t-47d cell lines with the ld 50 value of 87.55 µg/ml (nurshid et al., 2016). our study revealed that the methanolic extract of a. lecanora showed significant cytotoxicity towards t47d cancer cell line with the ic 50 value of 6.25 µg/ ml. generally, it is clear that the methanolic extract of all three species, namely a. lecanora, h. atra and s. vastus, potentially inhibited the growth of t-47d breast cancer cell lines. as per the literatures, it could be suggested that the cytotoxic effect induced in the t47d cancer cell line might be due to the presence of triterpene glycosides and saponins present in it (vien et al., 2018). however, further research is needed to identify the chemical constituents and demonstrate their mode of action towards cancerous cell lines. this research highlighted the cytotoxic effect of three different sea cucumber extracts obtained from the same extraction techniques against t-47d breast cancer cell lines. determination of the mode of cell death t-47d breast cancer cells were treated with the a. lecanora (6.25 µg/ml), h. atra (7.5µg/ml) and s. vastus (3.25 µg/ml) extracts and compared with vincristine sulfate (positive control, 0.25 µg/ml) and samples ic50 (µg/ml) a. lecanora 6.25 ± 0.50 h. atra 7.94 ± 1.39 s. vastus 3.16 ± 0.53 table 1. the ic50 value of the sea cucumber extracts towards human breast (t-47d) cancer cells santhanam, r., et al., page 89 of 94 squalen bull. mar. fish. postharvest biotech. (2022) 17(2): 85-94 dmso (negative control) at different time intervals 0, 3, 6 and 24 h to determine the mode of action of cell death using annexin v/propidium iodide (pi) assay. the results are shown in figure 2. only dark images were seen at zero h since the dye was freshly added to the cell lines. after 3 h of treatment, green staining of annexin v at the cells’ cellular membr ane was observed, indicating the inducement of apoptosis. at 6and 24-h, a gradual increase of distinguishable membrane boundary was detected, indicating the early stage of apoptosis. annexin is one of the family of calciumdependent phospholipid binding proteins, which bind to phosphatidylserine (ps) to identify a poptotic cells ( liz a rbe et a l., 2013) . ps is predominantly located along the cytosolic side of the plasma membrane. during the early initiation of apoptosis, ps loses its asymmetric distribution in the phospholipid bilayer and is exposed and translocated to the extracellular membrane, which is stainable by annexin v and detectable with a fluorescent microscope with the green stained (ahmed et al., 2015). during the late-stage apoptosis, loss of membrane integrity allows annexin v to bind to the cytosolic ps and the cell to uptake the propidium iodide (pi) (ahmed et al., 2015). from figure 2, it is clearly understood the cell treated with the extracts and positive control undergo early apoptosis with the green staining of annexin v at the cellular membrane of the cells. all the treated extracts undergo early apoptosis rapidly within 24 h, which is higher than the cells treated with the positive control. the results obtained are in accordance with the cytotoxic results. nurshid et al. (2019) confirmed the apoptosis induction via caspase-3 activation in t47d cells. lc-ms/ms analysis of the extracts the lc-ms/ms spectra and the mass fragmentation obtained from the sea cucumber extracts were provided in the supplementary file. chemical constituents such as 7-n, n-dimethylamino-1,2,3,4,5-pentathiocyclooctane, iophendylate, epibatidine and other unknown compounds were identified in the sea cucumber extracts (popov et al., 2017). the data were interpreted using the masshunter qualitative analysis-metlin database and compared with literature values dpph free radical scavenging activity the antioxidant is a common medicinal property in plants yet rare in animals. antioxidants help prevent or reduce cell damage caused by free radicals, which are unstable molecules produced by the body in response to environmental and other stresses (kumar et al., 2017). they are also known as “free-radical scavengers”. antioxidants can come from both natural and synthetic sources. dpph or 2,2-diphenyl-1picrylhydrazyl free radical scavenging assay is usually used to detect the antioxidant effect of an extract. the dark purplish color of the dpph free radical would be figure 2. annexin v and pi stainings of t-47d cells treated with sea cucumber extracts and positive control (vc, vincristine sulfate). images were taken within the time interval of 3, 6 and 24 h. this was done to detect the early apoptosis mode of cell death. the arrow pointed (a) indicated necrosis, while (b), (c) and (d) indicated that early apoptosis was induced. santhanam, r., et al., page 90 of 94 squalen bull. mar. fish. postharvest biotech. (2022) 17(2): 85-94 r educed to yellow when it is sca venged by the antioxidants present in the extract (dos reis et al., 2018). unfortunately, our extracts did not manage to reduce the purple color of dpph, indicating no antioxidant activity of the extracts (h. atra, s. vastus and a. lecanora). despite that, our result showed a negative value for the percentage of dpph inhibition (figure 3). the negative value also suggested that our extracts may exhibit a pro-oxidative effect. pro-oxidative indicates the poor effect of the extract by inducing more oxidative stress in the cellular environment. however, a previous study found that pro-oxidative natural products possess the ability as cancer chemopreventive, carcinogenic and chemotherapeutic agents (martin-cordero et al., 2012). it is well understood that pro-oxidant substances can generate carcinogenic consequences because they raise cellular levels of reactive oxygen species (ros). nevertheless, when pro-oxidant agents raise cellular levels of ros to lethal levels, they may cause cancer cells to be selectively killed and so be therapeutically beneficial (martincordero et al., 2012). all these effects can be obtained by substances with both antioxidant and pro-oxidant capabilities, such as curcumin (martin-cordero et al., 2012). substances such as curcumin can obtain all these effects with both antioxidant and pro-oxidant capabilities (martin-cordero et al., 2012). even though the present data showed a negative value (up to 0.5 mg/ml), increasing the extract concentration was found to have some antioxidant activity with a low percentage of dpph inhibition. a study from esmat et al. (2013) coincided with our finding where they found small dpph inhibition activity at a concentration of 0.6 mg/ml. the study found tha t h. atra sea cucumber mixed extract had a hepato-protective effect against liver fibrosis in rats. this outcome also suggests that h. atra strongly possesses anticancer properties as it can reduce hepatic damage through the discarding of the ros. total flavonoid and total phenolic content both flavonoid and phenolic compounds are known for their potent antioxidant properties (sulaiman & ba lachandra n, 2012). flavonoids ar e chemica ls commonly found in plants responsible for their vibrant hues of colors together with carotenoids. with six differ ent gr oups of flavonoids ( a nthocya nidin, flavanols, flavonols, flavones, flavonones, isoflavones), plant extract aid in cellular activity regulation and the battle against free radicals that cause oxidative stress in the body (mustafa et al., 2010). this contributes to pla nts’ a ntioxida nt, antica r cinogenic, a ntiinflammatory, and anti-mutagenic properties (panche et al., 2016). due to this reason, flavonoids have become an indispensable ingredient applied in a variety of medicinal, nutraceutical, pharmaceutical, and also cosmetics (panche et al., 2016). kartikaningsih et al. (2018) stated that a flavonoid test was not done since it is a phytochemical presence in plants, whereas h. atra is an animal species. little did we know, flavonoid phytochemicals can also be found in certain animal species (agustina et al., 2021; rapi et al., 2020). other than that, phenolic compounds also found ubiquitously in plants exhibit potent antioxidant activity regulated through redox properties acting as reducing agents, singlet oxygen quenchers, hydrogen donors, and metal ion chelating agents (gulcin, 2020; kasote et al., 2015). all the available functions allow plants to survive in different extreme environments. interestingly, h. atra was found to possess phenolic compound in just a small amount while flavonoid compound is absent (table 2). in s. vastus and a. lecanora extracts, both the phenolic and flavonoid compounds are absent. the standard calibration curve plotted to determine the total phenolic and flavonoid contents was shown in figures 4 & 5, respectively. as stated previously, both flavonoid and phenolic compounds are agents for discarding free radicals and avoiding plant stress. just like plants, some animals have their antioxidants for species independence in a harsh or extreme environment. sukmiwati et al. (2020) supported our study where their h. atra extract from cerocok beach, west sumater a, indonesia also presented a phenolic compound without flavonoid compound. however, another part of indonesia was found absent with phenolic compound, but flavonoid was present in their h. atra extract  from a different location at benteng inong, aceh besar, and panjang island, jepara (agustina et al., 2021; sibero et al., 2019). esmat et al. (2013) found both compounds figure 3. percentage of dpph inhibition of h. atra, s. vastus and a. lecanora extracts and ascorbic acid as standard. ascorbic acid showed high dpph scavenging activity, whereas h. atra, s. vastus and a. lecanora showed no antioxidant property. the data are expressed as mean ± standard deviation (n = 3). santhanam, r., et al., page 91 of 94 squalen bull. mar. fish. postharvest biotech. (2022) 17(2): 85-94 cancer (martin-cordero et al., 2012). since the other two extracts (s. vastus and a. lecanora) demonstrated the absence of phenolic and flavonoid content, it is evident that other classes of chemical constituents are present in the extracts, which could cause early apoptosis in the breast cancer (t-47d) cell lines.    gelatin digestion assay the gelatin digestion assay determines the ability of an organism to generate extracellular proteolytic enzymes (gelatinase) used to liquefy gelatin, a type of connective tissue found in vertebrates. the proteolytic enzyme hydrolyzes proteins into amino acids, which are necessary for homeostatic regulation in prokaryotes and eukaryotes (kasana, 2010). the extracts were examined for gelatinase, inhibition. wherein nature matrix metalloproteinases (mmp-2 and -9) were some examples. mmps in low concentration has a great function in proteolyze extracellular matrix component, membrane shedding, chemokine processing, and also regulate angiogenesis in wound healing (castleberry et a l., 2016; löffek et a l., 2011) . however, high concentr ations of mmp-2 a nd mmp-9 promote intravasion of tumor and neoangiogenesis that form leaky blood vessel that can lead to metastasis of cancer (adhikari et al., 2020). this condition stipulated that the high value of mmp-2 and mmp-9 is a precursor for cancer and a contributor to a malignant tumor (adhikari et al., 2020). captivatingly, our extracts showed inhibition against ba cterial collagenase, indicating the protective function against extracellular proteolytic enzyme (figure 6). interestingly, all data showed significant differences against the control showing that our extracts had inhibition activity for gelatin digestion. this in part displayed the anticancer effect of h. atra, s. vastus and a. lecanora. a higher extract concentration showed a lower dia meter of the digestion zone, indicating higher inhibition of bacterial collagenase activity. based on the similar previous study, our study where a higher extract concentration had higher inhibition activity against the enzyme (prabhu, 2021). conclusion all of the three sea cucumber extracts (a. lecanora, h. atra, and s. vastus) actively inhibit (t-47d) cancer cell growth progression by inducing apoptosis in the cell. the anticancer effect of the extract is might be due to the a ctive chemica l constituents such as saponins, triterpene glycosides other than the phenolic/ flavonoid compounds. anti-collagenase activity results supported the protective effect of the extracts against figure 5. standard calibration curve to determine the total flavonoid content in samples (n=3). figure 4. standard calibration curve to determine the total phenolic content in samples (n=3). samples tpc (mg gae/g) tfc (mg qe/g) h. atra 0.4794 ± 0.076 mg gae/g s. vastus a. lecanora table 2. concentration of flavonoid and phenolic in sea cumber extracts present in their h. atra extract originate from the red sea (hurghada). the phytochemical content of h. atra extract was different due to its different origins where their environment and food access available were different, resulting in different medicinal properties of the same species from different places (kartikaningsih et al., 2018). however, the presence of phenolic compound indicates that our extract might possess an anti-carcinogenic effect and can help inhibit cancer development. a substance with a phenolic compound is also known to have antioxidant properties. as stated before, pro-oxidant and antioxidant substances act as chemo-preventive and therapeutically beneficial to treat santhanam, r., et al., page 92 of 94 squalen bull. mar. fish. postharvest biotech. (2022) 17(2): 85-94 the cancer cell. although the results seem promising, but specific mechanism of action of the extracts still unknown and need to be explored further. acknowledgement we would like to thank the researchers from imb dr jasnizat saidin, nur asniza aziz and kamaria.h bakar for helping us with the sample collection. supplementary material supplementary material is not available for this article. references adhikari, n., amin, s. a., & jha, t. (2020). collagenases and gelatinases and their inhibitors as anticancer agents. in cancer-leading proteases (pp. 265-294). academic press. agustina, s., bella, s., karina, s., irwan, i., & ulfah, m. (2021). isolation of sea cucumbers’s (holothuria atra) secondary metabolite using column-chromatography technique. in iop conference series: earth and environmental science (vol. 869, no. 1, p. 012010). iop publishing. 1-6 ahmed, i. d., sohair, r. fahmy., amel, m. soliman., ayman, s. mohamed., & sayed, a. m. amer. 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(2022) 17(2): 85-94 qualen bulletin of marine and fisheries postharvest and biotechnology research article published online: 17 december 2021 page 110 of 118 school of bioscience and biotechnology,tokyo university of technology, 1404-1 katakura, hachioji, tokyo, 192-0982, japan * corresponding author: sekihrk@stf.teu.ac.jp received: 16 july 2021 accepted: 22 november 2021 published: 17 december 2021 ©squalen bulletin of marine and fisheries pos th a rves t a nd b i ot echn o lo gy, 2 0 21 . accreditation number:148/m/kpt/2020. is sn: 2089 -569 0, e -is sn: 2406 -927 2. doi: 10.15578/squalen.585 o pe n acces s squalen bulletin quality changes in the adductor muscle of ezo giant scallop mizuhopecten yessoensis (jay, 1857) during refrigerated storage hiroko seki abstract recently, the popularity of scallops consumption and the preference to eat them raw have been increasing worldwide. therefore, maintaining its freshness and quality is important. it is necessary to investigate the changes in quality, particularly umami-related component parameters and perform a comprehensive evaluation to assess scallop quality over time. in this study, the distinction in the abundance of microorganisms, k value, ph, color value, glycogen content, and atp-related compound levels (i.e., atp, adp, amp, imp, hxr, hx, and glutamic acid levels) were investigated to determine the quality of ezo giant scallops. the parameters were evaluated every day for six days at 4°c post mortem of the scallops. the total viable aerobic count of marine bacteria increased from 1 to 3 log cfu/g over six days, and the k value increased sharply from 18% on day 2 to 66% on day 4. the ph decreased from 7.0 on day 0 to 6.0 on day 3, but the color value did not change during the six days of observation. the amp content increased over three days and then decreased during the last three days of storage. imp was not detected; meanwhile, the glycogen and glutamic acid levels were stable during the observation. based on these results, the best recommendation is to serve the refrigerated scallops as sashimi for not more than two days and cook by the third day to preserve the quality. keywords: scallop, marine product, umami components, seafood, adductor muscle introduction in recent year s, the consumption of mar ine pr oducts ha s been increa sing a long with food distribution and the need for a highly proteinaceous diet. the global demand for marine products is expected to grow with the increasing population worldwide (fisheries agency, 2016). japanese marine products are high-quality and highly valued globally (ministry of agriculture, forestry, and fisheries, 2014). the most exported japanese marine product is scallops (84,000 tons in 2019) (policy research institute, ministry of agriculture, forestry and fisheries, 2020). in 2013, the hokkaido federation of fisheries cooperatives scallop “hanging ceremony” and “digit net fishing” fisheries’ export volume in 2013 was approximately 60,000 tons. it increased to 85,000 tons in 2018 (hakodate customs, 2019). scallops are consumed in several countries, and their export is expected to increase. currently, most marine products are exported as “frozen,” but in recent years, opportunities to export these products “raw and refrigerated” are increasing. quality assessment is critical when exporting “raw” products; consequently, accurate quality evaluation is necessary to confirm quality assurance. to accurately evaluate the qua lity of marine pr oducts, especially scallops, it is necessar y to investigate the alteration in quality-related parameters and perform a comprehensive evaluation over time. such quality-related parameters include the abundance of microorganisms, k value, ph, color value, odor, and degree of oxidation. as the lipid content in the ezo giant scallop mizuhopecten yessoensis (jay, 1857) is low, the changes in hardening, ph, k value, arginine and octopine levels over time are considered important for the quality evaluation (kimura, 2003). however, a s sca llops a r e a popula r food due to their “deliciousness,” investigating the changes in umamirelated components and analyzing quality-related parameters over time is necessary. the umami-related components in scallop are glycogen, inosinic acid ( imp) , a denosine monophosphate (amp), and glutamic acid (glu) mailto:sekihrk@stf.teu.ac.jp seki, h. page 111 of 118 squalen bull. mar. fish. postharvest biotech. (2021) 16(3): 110-118 (yamanaka, 2007). in particular, a moderate positive correlation between the detection threshold and taste preference associated with glu and imp has been reported, at 0.46 and 0.36, respectively. besides, the range of preferred glu and imp concentrations is 3.13 to 200 mm a nd 0.313 to 20 mm, r espectively (chamoun et al., 2019). therefore, this study investigated the distinction in the a bunda nce of micr oorga nisms, atp-r elated compounds, k value, ph, color value, and glycogen and glu levels to evaluate the quality of scallops during a 6-day storage trial at 4 °c. raw scallops could be contaminated by photogenic bacteria such as (vibrio sp/salmonella sp/listeria sp). hence, health hazards and food safety issues become a concern for raw consumption purposes. in the current study, the shift in total viable aerobic count (tvac) and total viable count (tvc) of marine bacteria were investigated to determine the scallop quality. material and methods sample collection in september 2020, twenty farmed ezo giant scallops, m. yessoensis (diameter 12–15 cm), were obtained from hokkaido. after harvest, the scallops were transported to the laboratory in cold storage and shelled; thereafter, the adductor muscles were separated from other tissue and stored at 4 °c. three adductor muscles were assessed daily, with microbiological and chemical parameters measured from arrival to day 6. samples that were harvested at the same places and periods were considered the same sample. changes in viable and marine bacterial counts the changes in the tvac and tvc of marine bacteria were analyzed according to seki, nakazato and hamada-sato (2017) with minor modifications. briefly, the scallop adductor muscle (2.5 g) was finely chopped and placed in a 50 ml centrifuge tube. sterile nacl solution (0.9 %; the salt industry center of japan) was added to adjust the final volume to 25 ml. the supernatant was appropriately diluted with 0.9% sterile nacl solution, and 100 µl of the diluted sample was poured onto standard agar medium (5.0 g peptone, 2.5 g yeast extract, 1.0 g glucose, and 15 g agar/1.0 l pure water; eiken chemical co., ltd.) to determine the tvac. subsequently, 100 µl of the diluted sample was poured onto a standard agar medium containing 1% nacl to determine the tvc of marine bacteria. the standard agar medium was incubated at 35 °c for 48 h, and the standard agar medium containing 1% nacl was incubated at 20 °c for 168 h. changes in atp-related compound levels and k value only atp-related compound levels and k value were measured in this study following the method of seki et a l. ( 2016) with minor modifica tions. approximately 2.5 g of scallop adductor muscle was placed in a 15-ml centrifuge tube, and 4 ml of 10% per chlor ic a cid ( fujifilm wa ko pur e chemica l cor pora tion) wa s added to extr a ct atprela ted compounds. the solids wer e r emoved by centrifugation (11,000 × g, 10 min, 5 °c) (mx 201; tomy seiko co., ltd.), and the supernatant volume was adjusted 10 ml with 10% perchloric acid. one milliliter of this solution was neutralized with koh (kanto chemical co., inc.). the precipitate was removed by centrifugation (12,000 × g, 5 min, 5 °c), and the volume of the supernatant was adjusted to 5 ml with purified water. this solution was filtered through a 0.22-µm filter (shanghai fenghan industrial co., ltd.), and the level of atp-related compounds wa s mea sured by highper for mance liquid chromatography (hplc; column: cosmosil packed column 5c18-paq, 4.6 mm i.d. × 150 mm, mobile phase: 20 mm kh 2 po 4 (fujifilm wako pure chemical corporation) solution (ph 7), flow velocity: 1.0 ml/ min, temperature: 40 °c, detector: u.v., wavelength: 260 nm, injection volume: 20 µl) (10a series; shimadzu corporation). the k value was calculated using the following formula: where hxr, inosine (tokyo chemical industry co., ltd.); hx, hypoxanthine (fujifilm wako pure chemical corporation); atp, adenosine triphosphate (fujifilm wako pure chemical corporation); adp, adenosine diphosphate (oriental yeast co., ltd.); amp, adenosine monophosphate (oriental yeast co., ltd.); imp, inosine monophosphate (tokyo chemical industry co., ltd.). changes in ph and color values the ph of samples was measured by setting a piercing ph electrode (pe-06hda; sato shouji inc.) on a digital ph meter (yk-21sp; sato shouji inc.) and piercing the electrode into the scallop adductor muscle. the color value was measured using a colorimeter (cr13; konica minolta, inc.) directly on the surface of the scallop adductor muscle and measuring the l*a*b* (%) value ܭ = (hx + hxr) (atp + adp + amp + imp + hxr + hx) x 100 squalen bull. mar. fish. postharvest biotech. (2021) 16(3): 110-118 value using the hunter lab system, where l* indicates lightness, a* redness, and b* blueness (konica minolta, 2021). changes in the glycogen content glycogen content was evaluated according to the methods described by ishihara, kodama, and yasuda (1966) and bennett, keirs, peebles, and gerard (2007) with minor modification. approximately 0.1 g of scallop adductor muscle was placed in a 15-ml centrifuge tube, and 2 ml of 10% perchloric acid was added to it for glycogen extraction. the solid matter was removed by centrifugation (12,000 × g, 5 min, 5 °c), and the supernatant was moved to another tube. thereafter, 5 ml of etha nol ( fujifilm wa ko pur e chemica l corporation) was added to the supernatant, the mixture was stir red, a nd the precipitated glycogen wa s centrifuged (12,000 × g, 5 min, 5 °c). the supernatant was removed, and the precipitate was resuspended in 50 ml of purified water. two hundred microliters of the sample solution, 12 µl of phenol (fujifilm wako pure chemical corporation) and 500 µl of sulfuric acid (fujifilm wako pure chemical corporation) were added into a new tube. the solution was incubated at 25 °c for 10 min and then shaken briefly. after incubating for another 20 min, the glycogen content was measured using a plate reader at 490 nm (imark; bio-rad laboratories, inc.) and calculated using a glycogen standard (fujifilm wako pure chemical corporation) curve. changes in glutamic acid content glutamic acid extraction was performed following the method used to determine the atp-related compound levels and k value as previously described (seki et al., 2016). the labeled glu in the sample solution was obtained by mixing 40 µl of the sample solution with 70 µl of ethanol, 20 µl of triethylamine (fujifilm wako pure chemical corporation), and 20 µl of phenyl isothiocyanate (kanto chemical co., inc.). hereafter, the mixture was allowed to react at 25 °c for 30 min (hanzawa et al., 2001). subsequently, 500 µl of acetate–sodium acetate (fujifilm wako pure chemical corporation) buffer (50 mm, ph 6.0) and acetonitrile (fujifilm wako pure chemical corporation) (97:3 v/v) were added to the sample mixture. the mixture was filtered through a 0.22-µm filter, and the glu content was measured using the hplc method. the hplc column (cosmosil packed column 5c18-ms-ii, 4.6 mm i.d. × 150 mm) was injected with 20 µl of the sample at a 1.0 ml/min flow velocity. hplc detection was at a wavelength of 254 nm at the temperature of 40 °c. the mobile phase conditions were as follows: (eluent a was composed of 50 mm acetate–sodium acetate buffer (ph 6.0):acetonitrile (97:3); eluent b was composed of acetonitrile:water (6:4); gradient: eluent b was increased from 5-100% between 0 to 16 min, decreased from 100-5% for 4 min and was kept steady for another 5 min. the glutamic acid content was calculated using the standard curve of a glutamic acid (fujifilm wako pure chemical corporation). statistical analysis data wer e obta ined ba sed on fisher ’s three principles. data change by time was analyzed using the one-way analysis of variance, and the post hoc analysis was performed to determine the difference between mean values using microsoft excel. results with p < 0.05 were considered statistically significant. results and discussion changes in the viable and marine bacterial counts figure 1 shows the changes in the tvac and tvc of marine bacteria on the scallops during six days of refrigerated storage. almost no tvac was recorded on scallops during the six days of storage; however, the number of marine bacteria decreased from 1.9 log cfu/g on day 0 to 1.8 log cfu/g on day 1 (p > 0.05). it increased to 2.0 log cfu/g on day 2 (p > 0.05) and to 3.4 log cfu/g on day 6 (p < 0.05). in this study, almost no general viable cell count was recorded during the six days of observation (figure 1). the viable bacterial count of scallops acclimatized overnight in artificial seawater at 10 °c after fishing increased from approximately 3.0 log cfu/ g on day 0 to 3.0–4.0 log cfu/g on day 6 when stored at 5 °c (yanagiya, shimada, yamabi, koizumi, & nagamine, 1999). meanwhile, the viable bacterial count of scallops stored in a refrigerator for 1–2 days has been reported to be 2.69–2.75 log cfu/g (narita et al., 2018). these values are higher than those observed in this study, where no bacteria were observed in standard agar. moreover, bacteria were not observed on the surface of the scallop adductor. bacteria do not exist in muscles when marine organisms are alive, and they move from the internal organs to the surface postmortem (seki et al., 2017). here, hygienic practices were employed during scallop preparation using all sterilized tools, and not all samples were rinsed with fresh water after separating the adductor muscles. it is possible that the adductor muscles were still in a sea water-like environment for six days, and bacteria that are not resistant to salt could not have grown on the seki, h. page 112 of 118 squalen bull. mar. fish. postharvest biotech. (2021) 16(3): 110-118 surface of the adductor muscle. therefore, the internal or gans of mar ine orga nisms should be removed immediately after post-mortem. in addition, compared with the findings of previous studies ( kimur a , na r ita , noma ta , fukushi, & takahashi, 1998; kimura, narita, imamura, ushiro, & yamanaka, 2000), the small number of bacteria might be due to immediate sample collection post mortem of the scallops. the abundance of general viable bacteria in scallops processed in a cleanroom is reportedly less than that in scallops not processed in a cleanroom (yanagiya et al., 1999). in the present study, almost all scallops were processed in hygienic environments, which could be a reason for the low general viable cell count. in contrast, the number of marine bacteria evaluated in this study was approximately 2.0 log cfu/g on day 0. the number was increased over the six days. in similar studies, the abundance of marine bacteria in scallops increased from approximately 3.0 log cfu/g on day 0 to approximately 4.0 log cfu/g on day 6 when stored at 5 °c (kimura et al., 1998, kimura et al., 2000). these results were considered lower than the changes in the tvc of marine bacteria observed in the current study. although the changes in the present study were relatively high, the tvc of marine bacteria after six days of storage was similar to that of previous studies (kimura et al., 1998, kimura et al., 2000). several microorganisms reported in marine products, such as scallops, are halophilic and mainly detected once cultivated in a salt-containing medium (onodera, miyamoto, ishikawa, & nakaya, 1997; yanagiya et al., 1999). nozawa et al. (2004) reported that vibrio is the predominant microbe in the scallop adductor muscle. in general, vibrio was not detected in a standard agar medium, as it requires salt for growth, but some vibrio species may grow well in 0% nacl medium (u. s. food & drug association, 2004). changes in atp-related compound levels and k value figure 2 shows the changes in the levels of atprelated compounds over time. the atp level was 4.5 µmol/g on day 0, and then sharply decreased to 0.0 µmol/g on day 3 (p < 0.05). the adp level was 3.0 µmol/g on day 0, and then slightly decreased to 2.7 µmol/g on day 2 (p > 0.05) and to 0.35 µmol/g on day 6 (p < 0.05). the amp level was 0.61 µmol/g on day 0, increased to 2.5 µmol/g on day 3 (p < 0.05) and decreased to 0.89 µmol/g on day 6 (p < 0.05). imp was not detected throughout the 6 days. the hxr level was 0.35 µmol/g on day 0, increased sharply to 3.7 µmol/g on day 4 (p < 0.05), and decreased to 2.0 µmol/g on day 6 (p < 0.05). the hx level was 0.38 µmol/g on day 0 and increased to 1.3 µmol/g on day 4 (p < 0.05); no change was observed during the remaining days (p > 0.05). the atp level in this study was 4.5 µmol/g on day 0, but it decreased with time and was undetectable on day 3 (figure 2). in similar studies, the atp level on day 0 has been reported to be 5.8 µmol/g (nagamine, yamabi, matsubara, & fukuda, 1988) and 8.0 µmol/g (kimura et al., 2000). seasonal variations in the atp level have been observed. the atp level in scallops caught in september (the same harvested month as in the current study) was approximately 9.9 µmol/g (shizukuishi, onishi, tanaka, & narita, 2004) and 7.5 µmol/g (kimura, 2003). the atp values were higher than those recorded in this study. further, the atp level of 5.4 µmol/g on day 3 (nagamine et al., 1988) and 5.0 µmol/g on day 4 (kimura et al., 2000) were also higher tha n those obser ved in this study. the degradation of atp-related compounds depends on temperature and time. the duration from harvest to sampling in the previous study was one day longer compared to that in current study. in addition, the atp level sharply decreased from approximately 45% on seki, h. page 113 of 118 figure 1. changes in the tvac and tvc of marine bacteria during storage. error bars denote the standard deviation of the mean (n = 6). the dashed line indicates the maximum (allowable) tvac at 5.0 log cfu/g (ichinohe et al., 2015). : tvac, : tvc of marine bacteria ■ figure 2. changes in the level of atp-related compounds in scallops during storage. error bars denote standard deviations of the mean (n = 5). atp, adp, amp, imp, hxr, hx. squalen bull. mar. fish. postharvest biotech. (2021) 16(3): 110-118 day 1 to 5.0% on day 3 in scallops stored at 5 °c in a previous study (matsumoto, 1996). according to this study, the atp level decreased from 50% to 0% from day 0 to day 3, showing the same trend as observed in a previous study (kimura, 2003). based on the study, the adp level was the highest on day 0; at the same time, the amp level was the highest on day 3. even in scallops stored at 5 °c, in which atp degradation was observed in this study, the adp level was the highest (30% of the total atprelated compounds) on day 0. on the contrary, the amp level was 20% of the total atp-related compounds on day 0 and 5% of the total atp-related compounds on day 1. this indicates a decrease after reaching approximately 20% of the total atp-related compounds on day 3 (matsumoto, 1996). in this study, the adp level on day 0 was approximately 34% of the total atp-related compounds, and the amp level on day 3 was around 32%, indicating the same trend. imp was not detected throughout the six days of storage since the amp deaminase was absent in scallops, and these results were similar to those of previous studies (nishi & nishida, 1976; kawashima & yamanaka, 1992; kimura, narita, nomata, kaneko, & yamanaka, 1997; kimura et al., 2000). it has been reported that amp is degraded via two pathways, imp and adenosine (kawashima & yamanaka, 1992). in consequence, a small amount of imp has been detected in scallops (nagamine et al., 1988, matsumoto, 1996). amp is decomposed by imp when divalent metal ions are lost (wei et al., 2020). however, as atp is generally bound to mg2+ in vivo ( kimur a , 2003) , the concentration of mg2+ in muscle increases with atp decomposition. typically, imp is not detected in that amp is decomposed into adenosine due to the presence of mg2+ the hxr level increased sharply from days 2 to 3. hardening of the scallop adductor muscle over time causes a reduction in the atp level and increases the hxr level (kimura et al., 1997). in this study, atp was not detected, and the hxr level increased sharply on day 3. this means hardening is related to a sharp incr ea se in the hxr level. hx a ccounted for approximately 28% of the total level on day 6. in scallops stored at 5 °c, it has been reported to increase to approximately 50% on day 6 (matsumoto, 1996), higher than the hx level observed in this study. however, a s the hxr level in this study was high, the decomposition of hxr to hx was slow. figure 3 shows the changes in the k value after six days of observation. the k value was 8.2% on day 0, increased to 15% on day 1 (p < 0.05), and then slightly increased to 18% on day 2 (p > 0.05). it increased rapidly to 66% on day 4 (p < 0.05) and gradually increased to 72% on day 6 (p < 0.05). since the determination of k value is species-dependent, the maximum k value of refrigerated scallop was calculated to evaluate the other parameters such as glu, glycogen, and atp-related components. the k value of scallops has been reported to increase sharply from 20% on day 4 to approximately 60% on day 7 when stored at 5 °c (kimura et al., 1997). although the day on which the k value increased in this study was different from that reported in a previous study (kimura et al., 1997), a rapid increase occurred in both studies. this rapid increase is associated with a decrease in ph because of hardening and the rapid decomposition of atp (nishi & nishida, 1976; kimura et al., 1997; yamanaka, 2002). in this study, the atp level decreased considerably from days 0 to 3 (figure 4), suggesting that these factors rapidly increased the k value. the recommended k values are as follows; fresh fish, <10 %; sashimi, 10%– 20%; moderately fresh fish, 20%–50%; raw material for processing, 35%–60%; and spoiled material, >60% (choi et al., 2020). based on the evaluation of k value and the findings of the current study, it might be figure 3. changes in the k value of scallops over six days of storage. error bars denote the standard deviations of the mean (n = 5). fresh fish should have a value of <10 %, sashimi should have a value of 10%–20%, moderately fresh fish should have a value of 20%–50%, raw material for processing should have a value of 35%–60%, and spoiled material has a value of >60% (choi et al., 2020). figure 4. changes in the ph of scallops during storage. error bars denote the standard deviations of the mean (n = 8). 5.5 6.0 6.5 7.0 7.5 seki, h. page 114 of 118 figure 5. changes in the color value of scallops during storage. error bars denote the standard deviation of the mean (n = 3). : :l*value, :a* value, :b* value. squalen bull. mar. fish. postharvest biotech. (2021) 16(3): 110-118 recommended that scallops can be consumed raw within two da ys a nd should be hea ted befor e consumption after three days of refrigerated storage. changes in ph and color values figure 4 shows the changes in ph over the six days. the ph was 7.0 on day 0; dropped to 6.0 on day 3 (p < 0.05), and continued slightly dropped to 5.9 on day 5 (p > 0.05). however, the value was insignificantly increased (p > 0.05) on day 6. the ph of marine or ga nisms dr ops shor tly post mor tem ( koseki, kitakami, kato, & arai, 2006). the ph of scallops stored at 5 °c gradually decreased over time, shown by the lessen to 6.9 on day 0 and 6.1 on day 6 (kimura et al., 1997). the ph of scallop has been reported to be 7.1, which dropped to 5.9 on day 6 (kawashima & yamanaka, 1994; kimura et al., 2000). these results are similar to those obtained in this study, whereas the ph of the scallop was 7.0 on day 0 and decreased to 5.9 on day 5. the ph of scallops eases due to octopine and lactic acid accumulation and the increase in hydrogen ions associated with atp decomposition (kimura et al., 1997; kimura et al., 2000). in this study, the atp level and ph decreased over three days (figs. 2 and 4), indicating the atp decomposition caused the reduction in ph. furthermore, the ph of scallops increased to 6.1 on day 6 of observation. the increase in ph has been reported to be caused by the accumulation of alkaline bio-transformers due to the accumulation of bacteria over time (ocano-higuera et al., 2006). figure 5 shows the changes in the l*a*b* value during the six days of storage. the l* value was 54 on day 0 and slightly decreased to 52 on day 6 (p > 0.05). the a* and b* values fluctuated between -0.70 and 0.27 and between 6.7 and 9.6, respectively, throughout the six days with minor changes (p > 0.05). the l*a*b* value showed almost no change during the six days. the l* value ranged from 52 to 54 and showed no significant changes (p>0.05) in this study. this observation is similar to a previous study, where the l* value of scallops stored at 0 °c remained unperturbed over 15 days (ocano-higuera, maedamartinez, lugo-sanchez, & pacheco-aguilar, 2006). in contrast, it has been reported that the l* value of oval squid (sepioteuthis lessoniana) and pacific ocean perch (sebastes alutus) increases after their shelf-life (okamoto et al., 2008; shamaila, skura, & nakai. 1995). this might be due to differences in flesh or tissue characteristics; when caught, these fish have transparent flesh but lose their transparency over time post mortem. moreover, their l* values increase over time due to the pr esence of cloudiness. this phenomenon is not observed in the scallop adductor muscle based on the findings of this study and previous studies. the a* value of scallops from hokkaido was between -2 and 4 (takeda, nomata, ohta, kaneko, & hashimoto, 1993), and the b* value was approximately 5 (kaneko, kimura, nomata, fukushi, & nishi, 1996). the a* value of scallop in this study ranged from 0.70 to 0.27, which was in the range of the previous study. in contrast, the b* value of the scallop in the current study was 6.7 to 9.6 during the six days of observation, and it was outside the range reported previously. the l*a*b* values represent the following: l* for lightness, a* for hue, and b* for chroma, and these indicate hue and saturation. an l* value of 100 represents white, whereas a value of 0 signifies black. the a* value represents red in the + direction, green in the direction, the b* value represents yellow in the + direction and blue in the direction. in the current study, the scallop was white with a slight yellow tinge. it has been reported that because of rigor, scallops turn black with time. still, the onset of rigor can be significantly delayed by wrapping the scallops with antibacterial sheets within five days (kimura, 2003). the onset of rigor may be due to an increase in bacteria. figure 6. changes in the glycogen content in scallops during storage. error bars denote the standard deviation of the mean (n = 9). seki, h. page 115 of 118 squalen bull. mar. fish. postharvest biotech. (2021) 16(3): 110-118 in this study, the onset of rigor was not confirmed as no significant increase in the bacterial count was observed (figure 1). in scallop, the color changes due to the maillard reaction on heating both saccharides and amino acids. since the scallops were not heated in this study, no significant difference in coloration was observed. changes in the glycogen content the changes in the glycogen content over the 6day storage period are shown in figure 6. the glycogen content was between 2.0% and 3.0%, and no significant difference was observed dur ing the six days of refrigerated storage. the glycogen content in scallops shows considerable seasonal fluctuations, decreasing below 0.5% from december to march, increasing from 3.0% to 4.0% from july to september, and decreasing to december (kimura, 2003; shizukuishi et al., 2004). this study was performed in september, and the glycogen content wa s almost the sa me a s tha t previously reported. the glycogen content is related to the amount of feed consumed by the scallop. generally, scallop feeding increases in the growing season from april and reaches the highest around september (kimura, 2003); however, it fluctuates every year. hereinafter, the amount of energy stored as glycogen also fluctuates, leading to the variation of glycogen content in the scallop. the difference in glycogen content in scallop up to 6% was observed between the same month of two different years (miyazono & kurata, 1995). in this study, although minor changes in the glycogen content were observed each day, they were not significantly different (p>0.05). this could be because glycogen, being an energy source, is not used after post-mortem; hence, no changes in the glycogen content were observed (figure 6). it has been reported that the taste level of hard clam soup correlates with the glycogen level (yamamoto & kitao 1993), indicating that the glycogen level is related to good flavors. although the reference glycogen level in raw scallops acceptable for consumption has not been defined clearly, glycogen is an important factor influencing the taste of scallops. in this study, glycogen metabolism in the organisms stopped post mortem; consequently, the glycogen level did not change significantly for six days. changes in the glutamic acid content figure 7 shows the changes in the glutamic acid content in the scallop during the 6-day refrigerated storage period. the glutamic acid (glu) level varied between 41 and 58 mg/100 g throughout the six days and with no significant increasing or decreasing trend (p > 0.05). the glu level in this study was generally in the range of the preferred concentration of above 46 mg/100 ml, as reported earlier (chamoun et al., 2019). the glu level varies with glycogen consumption during survival and atp production (kimura, 2003). after the post-mortem of an organism, glutamic acid metabolism stops. therefore, its level did not change significantly during the six days in this study. the glu level in scallops was as high as 132 mg/ 100 g in may but decreased sharply after june, and it ha s been r epor ted to be 35–40 mg/100 g fr om september to november (kimura, 2003). the high level of glu in may might be due to spawning and feeding effects. furthermore, a survey in another area reported that the level was 100 mg/100 g or more in may–july but decreased in august (shizukuishi et al., 2004). the current study was performed in september, and the results are similar to those reported previously (kimura, 2003). conclusions in this study, the quality of refrigerated ezo giant scallops adductor was evaluated by investigating the umami-related parameters (such as glycogen, imp, amp, and glu levels) and the changes of quality-related parameters (such as microorganisms abundance, k value, ph, and color). the quality-related parameters such as tvc of marine bacteria and the k value of scallops significantly increased during the refrigerated storage of 6 days. the k value of scallops has exceeded the acceptable level of 20% for sashimi consumption after two days of observation. meanwhile, the ph of scallops slightly decreased until five days of observation and then relatively increased on the sixth day of observation. although the umami-related parameters such as imp, amp, glycogen and glu level varied, there figure 7. changes in the glutamic acid level in scallop over storage time. error bars denote standard deviations of the mean (n = 9). seki, h. page 116 of 118 were no significa nt changes during r efr igerated storage, except in the amp level. based on the findings in this study, refrigerated scallops can be consumed as sashimi for only two days post mortem, and further processing on the scallops is recommended after two days of storage. the fourth-day refrigerated scallops are unsuitable for human consumption. in the future, it is recommended to investigate the abundance of pathogenic marine bacteria such as vibrio sp. for safety evaluation, analyze the changes in mg2+ ion over time, perform sensory scallops assessment, and determine the correlation among umami-related components, quality, and sensory characteristics. acknowledgments the author would like to thank konda, t., kudou, a., furihata, a., ebisawa, s., kikuchi, m., yui, a., murakoshi, a., nakanishi, y., taira, n., and harada, r. for assistance with the numerical simulations, and also the society for research supported this work on umami taste. references bennett, l. w., keirs, r. w., peebles e. d., & gerard p. d. 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(2016). effect of freezing and thawing on the quality of northern bluefin tuna thunnus thynnus (linnaeus 1758). asian fisheries science, 29(4), 232-244. doi: 10.33997/j.afs.2016.29.4.006 shamaila, m., skura, b. j., & nakai, s. (1995). monitoring quality of packaged pacific ocean perch (sebastes alutus) in storage. journal of aquatic food product technology, 4(3), 35-57. doi: 10.1300/j030v04n03_04 shizukuishi, s., onishi, c., tanaka, j., & narita, s. (2004). seasonal variation of components in scallop (patinopecten yessoensis) adductor muscle. report of aomori prefectural local food research center, 1, 19-24. takeda, t., nomata, h., ohta, t., kaneko, h., & hashimoto, k. (1993). sea area and monthly variation of color tone of patinopecten adductor muscle. scientific reports of abashiri fisheries research institute, 1991, 324-327. u. s. food & drug (2004). bacteriological analytical manual chapter 9: vibrio, https://www.fda.gov/food/laboratorymethods-food/bam-chapter-9-vibrio/ accessed on 11/8/ 2021. wei, h., tian, y., lin, y., maeda, h., yamashita, t., yu, k., takaki, k., & yuan, c. (2020). condition-dependent adenosine monophosphate decomposition pathways in striated adductor muscle from japanese scallop (patinopecten yessoensis). journal of food science, 85(5), 1462-1469. doi: 10.1111/1750-3841.15142. yamamoto, y. & kitao, n. (1993). effects of cooking time on the free amino acid contents and the taste of soup of hard clam (ushiojiru). science of cookery, 26(3), 214-217. doi: 10.11402/cookeryscience1968.26.3_214 yam an aka, h . (20 02 ). r elation between post m ortem biochemical changes and quality in the muscle of fish and shellfish. nippon suisan gakkai shi, 68(1), 5-14. doi: 10.2331/suisan.68.5 yamanaka, h. (2007). fish brands scallop. food and science, 49(9), 29-34. yanagiya, s., shimada, t., yamabi, t., koizumi, m., & nagamine, f. (1999). sanitary processing condition of scallop and freshness change under low temperature storage. report of aomori prefectural marine products processing research center, 1997, 38-46. https://www.maff.go.jp/primaff/ https://www.fda.gov/food/laboratory1 introduction wound healing is an active area of research on accounts of its importance in the treatment of burns, prevention of post surgical adhesions and cosmetics surgery (risbud et al., 2000). the purpose served by dressing  includes  protecting  wounds,  promoting healing, and providing, retaining or removing moisture ~ synthesis of polyvinyl alcohol-chitosan hydrogel and study .......... (t. wikanta, erizal, tjahyono, and sugiyono) synthesis of polyvinyl alcohol-chitosan hydrogel and study of its swelling and antibacterial properties sintesis hidrogel polivinil alkohol-kitosan dan studi sifat mengembang dan antibakterinya thamrin wikanta1), erizal2), tjahyono2) and sugiyono1) 1)research  and  development  center  for  marine  and  fisheries  product  processing  and  biotechnology 2)center  for  the application  isotopes  and  radiation  technology,  jl.  lebak  bulus  raya  no.49.  jakarta  selatan *correspondence author:  thamrin  w ikanta,  ks.  tubun  petamburan  vi  jakarta  pusat  10260,  e-mail:  thamrin_wikanta@yahoo.com abstract the aim of this research was to synthesize a hydrogel for wound dressing by mixing of polyvinyl alcohol  (pva)  and  chitosan  (cts)  and  processed  by  combination  technique  of  freezing-thawing and  irradiation  by  gamma ray, and to study  of  its  properties. pva aqueous  solution  10%  (w/v) was mixed  with  2%  (w/v)  chitosan  (cts)  solution  and  homogenized.  the  pva-cts  mixture  was processed  by  freezing-thawing  up  to  3  cycles,  and  then  irradiated  by  gamma  rays  at  the  dose ranged of 20-50 kgy  (dose rate was 10 kgy/hour). result showed that pva-cts hydrogel with the gel fraction of 83%, 87%, 90%, and 83% were obtained at the irradiation dose of 20 kgy, 30 kgy, 40 kgy, and  50  kgy, respectively. increasing of irradiation dose caused increasing  of water absorption of hydrogel, i.e. 1.700 %, 1.715  %, 1.913  %, and 2.036  %, respectively, and  the hydrogel reached the  equilibrium  in  25  hours.  the  hydrogel  showed  very  slow  water  evaporation  rate  (~  2%)  at  the initial time  (1  hour)  and  then  increased very  fast (up  ~50  %)  at 24  h,  i.e.  43%,  39.13%,  44%, and 53%,  respectively.  the  elongation  at  break  of  hydrogels  were  obtained  245%,  322%,  322%,  and 205% with the maximum value were obtained at irradiation dose ranged of 30-40 kgy. the presence of  chitosan  in  the  pva  hydrogel  made  it  having  higher  antibacterial  properties  with  the  inhibition zone  value  of  8  mm  at  irradiation  dose  of  30-40  kgy  compared  to  pva  hydrogel  as  a  negative control  (6  mm)  and  to  chloramphenicol  as  a  positive  control  (8  mm). keywords: hydrogel, polyvinyl alcohol (pva), chitosan (cts), freezing-thawing, gamma irradiation abstrak t uj u an   d ari  pen elitian   in i  adalah   men sintesis  h idro gel  u ntu k  p em balut  lu k a  den gan mencampurkan  polivinil  alkohol  (pva)  dan  kitosan  (cts)  dan  diproses  dengan  kombinasi  teknik beku-leleh  dan  irradiasi  sinar  gamma,  serta  mempelajari  sifat-sifatnya.  larutan  pva  10%  (b/v) dicampur dengan 2% (b/v) larutan kitosan (cts) dan dihomogenkan. campuran pva-cts diproses dengan  beku-leleh  hingga  3  siklus,  dan  kemudian  diirradiasi  dengan  sinar  gamma  pada  kisaran dosis  20-50  kgy    (laju  dosis  adalah  10  kgy/jam).  hasil  penelitian  menunjukkan  bahwa  hidrogel pva-cts  dengan  fraksi  gel  83%,  87%,  90%,  dan  83%  didapatkan  pada  dosis  irradiasi  masingmasing  20  kgy,  30  kgy,  40  kgy,  and  50  kgy.  meningkatnya  dosis  irradiasi  mengakibatkan meningkatnya  absorpsi  air  oleh  hidrogel,  yaitu  masing-masing  1.700  %,  1.715  %,  1.913  %,  dan 2.036  %,  dan  hidrogel  mencapai  kondisi  keseimbangan  dalam  25  jam.  hidrogel  menunjukkan laju  penguapan  air  sangat  lambat  (~  2%)  pada  awal  waktu  (1  jam)  dan  selanjutnya  meningkat dengan sangat cepat (hingga ~50 %) pada 24  jam, yaitu masing-masing 43%,  39.13%, 44%, dan 53%.  nilai  perpanjangan  putus  dari  hidrogel  didapatkan  245%,  322%,  322%,  dan  205%  dengan nilai  maksimum  didapatkan  pada  dosis  irradiasi  berkisar  30-40  kgy.  adanya  kitosan  dalam hidrogel  pva  menjadikannya  memiliki  sifat  antibakteri  lebih  tinggi  dengan  nilai  zona  inhibisi  8 mm  pada  dosis  irradiasi  30-40  kgy  dibandingkan  dengan  hidrogel  pva  sebagai  kontrol  negatif  (6 mm)  dan  dengan  kloramfenikol  sebagai  kontrol  positif  (8  mm). kata kunci: hidrogel, polivinil alkohol (pva), kitosan (cts), beku-leleh, irradiasi gamma ~ 2 (park &  barbul,  2004). hydrogels  are  cross-linked hydrophilic  polymer  networks  which  absorb  large amounts  of  water.  hydrogel  wound  dressing  has humectants properties such that the wound is kept hydrated to prevent any scabbing  or drying out so that the wound is allowed to heal from inside out. the absorption of secretion causes an expansion of the hydrogel making room for the inclusion of foreign bodies such as bacteria detritus and odor molecules that are irreversibly taken up along the liquid (pai et al., 2006). based on their similar physical properties to human tissues  and  their  excellent  tissue  compatibility, hydrogels have been studied extensively for biomedical applications. they can be used as soft contact lenses (dillehay & miller, 2007; cheng et al., 2004), tissue engineering scaffold (seng et al., 2012), controlled drug-release vehicles (barndl et al., 2010) and wound dressings (wang et al., 2012). hydrogels have many advantages as wound dressings, for instance, absorb excess of wound exudates, protect the wound from secondary infection and effectively promote the healing process by providing a moisturized wound healing environment.  they  also  can  be  removed  without causing trauma to the wound (thomas, 2007). polyv inyl  alcohol  (pva)  is  a  water-soluble polyhydroxy polymer. it has been used in practical applications because of its easy preparation, chemical resistance and physical properties, biodegradable, and cheap (anon., 2006; stasko et al., 2009). chitosan (cts), partially deacetylated of chitin, is a well known material in the wound dressing field. it has an excellent antibacterial activity, biodegradability, hemostatic, and biocompatibility (goosen, 1997; tombs & harding, 1998; liu et al., 2001; panos et al., 2008). the use of chitosan as an additive in hydrogels can improve its performance for wound dressing. chemical structure of pva and cts are presented in fig. 1. hydrogels can be prepared by irradiation, freezingthawing or chemical methods. irradiation is considered to be a suitable tool for the formation of hydrogel. the main disadvantage of hydrogel prepared by irradiation is  their  poor  mechanical  strength.  in  addition,  the technique is easy control of processing, no necessity to add any initiators or cross-linkers which may be harmful and difficult to remove, and possesses the possibility  of  combining  hydrogels  formation  and sterilization in one technological step. hydrogels of pva in aqueous solutions prepared by freezing-thawing have shown many interesting properties (stasko et al., 2009). the main disadvantages of this hydrogel are its opaque appearance and the limited swelling capacity  and  thermal  stability.  they  have  good mechanical strength, stable at room temperature, and with no initiators or cross-linkers. up  to  date,  much  work  hav e  been  done  on preparing hydrogels by irradiation (erizal & chosdu, 2009;  erizal  et al.,  2011)  or  by  freezing-thawing (lopergolo et al., 2002; nugent et al., 2005; zhao et al., 2003; bajpai & saini, 2005). however,  there is very little information on preparation of hydrogels by combining  the  two  processing  techniques  (nho  & park, 2002; erizal et al., 2011), especially by freezingthawing followed by irradiation. in  this  research,  pva  hydrogels  containing chitosan was prepared by combination technique of freezing-thawing and followed by γ-irradiation. the properties of the hydrogel including the gel fraction, the water absorption, the water evaporation rate, and the antibacterial properties were investigated. figure 1. repetitive units on the chemical structure of chitosan and polyvinyl alcohol.                 a= d-glucosamine; and b = n-acetyl-d-glucosamine; p = polyvinyl alcohol. a                                                                                      b a= d-glucosamine b = n-acetyl-d-glucosamine;  squalen vol. 7 no. 1, may 2012: 1-10. 3 material and method materials polivinyl alcohol (pva) with molecular weight of 72000 was purchased from merck and used without any pretreatment. chitosan (cts) was purchased from bi o tech  suri n do,  i ndone si a  wi th  d egree   of deacetylation of 92 %.  acetic acid was purchased from merck. all chemicals were of analytical grade and distilled water was obtained in our lab using aqua distilator. the 7 (seven) species of bacteria indicator namely staphylococcus aureus, salmonella typhimurium, escherichia coli, pseudomonas aerouginosa, streptococcus sp., bacillus stearothermophillus, and bacillus subtilis were  obtained  from  collection  of bacteri a  cul ture  l aboratory  at  research  and development center for marine and fisheries product processing and biotechnology. preparation of pva-cts hydrogel a  10%  (w/v)  of  pva  solution  was  prepared  by dissolving pva in distilled water and autoclaving at 121oc for 20 minutes. a 2% (w/v) of chitosan solution was prepared by dissolving chitosan in 1% acetic acid sol u ti on.  b oth  of   sol uti ons  wer e  m i x ed   and homogenized by stirring at room temperature to get the final volume of 500 ml. the mixture was poured into polyethylene plastic bags, dimension  of (20 cm x 10 cm x 0.5 cm), sealed and kept it in freezer at 80oc for 2 hours, then was thawed at room temperature for 1  h (  this is called  as 1  cycle).  the  process of freezing-thawing was repeated for 3 cycles. finally, all the samples were irradiated by gamma rays from 60co source at the doses of 20 kgy, 30 kgy, 40 kgy, and 50 kgy  (dose rate: 10 kgy/h) at room temperature. the hydrogels were washed directly for gel fraction determination  and  the  rest  were  used  for  swelling studies. each treatment was done in triplicate. gel fraction the hydrogel samples (2 cm x 2 cm x 0.5 cm) were put in the glass cup containing excess distilled water and were taken into shaker incubator at room temperature for 24 h, to remove the soluble fraction. the gels were dried under vacuum to constant weight. the insoluble fraction in the samples was determined gravimetrically and calculated as follow: gel fraction (%) = (w 2 /w 1 ) x 100% where w 1   is the initial weight of the gel and w 2  is the weight of dry gel after extraction. water absorption the water absorption of hydrogel was determined by  gravimetric  method. the  gel  samples  (dried to constant  weight)  were  immersed  in  a  glass  cup containing excess distilled water at room temperature. the hydrogel were periodically weighed after the water on the gel surface was removed with a filter paper. the water absorption was calculated as follow: water absorption = {(ws-wd)/wd} x 100% where ws   is the weight of the swollen gels at time t and wd is the initial weight of dried gels. elongation at break elongation  at  break  is  an  important  physical parameter of hydrogel representing its elasticity, and measured based on astm standard method by using instron tester instrument. the hydrogels were moulded with dumbbell for the preparation of the standard size measurement. both ends of the pieces were firmly clamped in the jaws of a testing machine. one jaw was fixed and the other was movable. the movable jaw  moved  at  the  rate  of  30  mm/min  at  room temperature. the resultant data was showed at the recorder. this procedure was repeated for three times for each result. the elongation at break was calculated as follow: elongation at break = l 1 /l o    x 100% where l o  is the initial length and l 1  is the final length. antibacterial assay antibacterial activity of pva-cts hydrogels were tested using kirby-bauer method (rollins & joseph, 2000) against seven species bacteria, escherichia coli (e. col i), s taph yloco ccus aure us, b acil lus stearothermophilus, salmonella typhimurium, streptococcus sp., pseudomonas aeroginosa, bacillus subtilis. as a negative control was disc blank (pva  hydrogel   disc),  a  positi v e  control   was chloramphenicol  (pva  hydrogel  disc  +  100  ppm chloramphenicol),  and  a  treatment  was  pva-cts hydrogel disc. at amount of 15 ml agar mueller hinton was poured into 90 mm petridish until solidification. at about 105 cfu/ml microbial culture suspension was spread over the surface of a sterile agar plate evenly by a sterile swab, then added another 15 ml agar  solution,  homogenized  and  waited  at  room temperature  until  the  plates  cooled  down.  the hydrogels treatment and control were applied on the centre of plates and the mixture was kept at 37°c for 48 hour. the antibacterial inhibition of hydrogel was synthesis of polyvinyl alcohol-chitosan hydrogel and study .......... (t. wikanta, erizal, tjahyono, and sugiyono) 4 irradiation h2o h*, ho*, h2o2 (1) p p* + h* (2) p + h* p* + h2 (3) p + oh* p* + h2o (4) p + p p – p (crosslinking) (5) when air is present, the expected reaction is assumed to be: h* + o2 ho2* (6) p* + o2 poo* (7) poo* + ho2* c + h2o2 + o2 (8) o c degradation (9) o o* p + ho2* po*( ch2 c ch2 ) + h2o (10) oh o* o ch2 c ch2 ch2 c + *ch2 (degradation) (11) oh oh figure 2. effect of gamma irradiation on polyvinyl alcohol polymer in aqueous solution, crosslinking and degradation reaction (sakurada & ikada, 1963). ch2 ch polyvinyl alcohol n oh + chitosan + irradiation ch2 ch ch 2 c* radical n n o* oh oh ch 2 ch ch 2 c crosslinking n-m m o o ch 2 c ch2 ch n-m m oh chitosan (cts)  polyvinyl alcohol (pva) figure 3. effect of gamma irradiation on polyvinyl alcohol polymer in aqueous solution (sakurada & ikada,             1963). interpenetrating polymer network (ipn) formed on pva-cts (hoarea & kohaneb, 2008). squalen vol. 7 no. 1, may 2012: 1-10. 5 determined by measuring the diameter of each clear zone  in  millimeter  at  around  of  gels  using  a  ruler provided. all of the operation procedures were done under aseptic condition. result and discussion when  irradiation  from  source  interacts  with  a polymer material, the polymer absorbs its energy and active species such as radicals are produced, thereby, initiating various chemical reaction. crosslinking and degradation are two competing process that always co-exist under radiation (mishra et al., 2007). in brief, there was a mechanism of crosslinking and degradation on this material when it is exposed to the irradiation. let say, p is a symbol of pva, and p*  is  a  radical  produced  as  a  result  of  hydrogen abstraction from the main-chain, and the radiolysis of pva aqueous solution can be described in fig. 2 and fig. 3 (sakurada & ikada, 1963). the amount of p* produced by reaction (2) is very small compared to that  produced  by reaction  (3) and  (4).  the  rate of reaction of (7) is generally consider to be much higher than that of (5). gel fraction the variation of gel fraction of pva-cts hydrogels versus irradiation dose on hydrogels is presented in fig. 4. it shows that the increase of the irradiation dose, the gel fraction of hydrogels gradually increased from 80% to 85% at the irradiation dose of 20 kgy and 30 kgy, respectively, and reached the maximum value of gel fraction of 90 % at the irradiation dose of 40 kgy. then, the gel fraction decreases until 80% at the  irradiation  dose  of  50  kgy.  thus,  there  is  a significant decrease in the gel fraction value as the irradiation dose increased in the range of this study. this result indicates that the pva-cts hydrogels with high  gel  fraction  of  90%  can  be  obtained  in  the presence of 2% chitosan at the irradiation dose of 40 kgy. rekso & sunarni (2009) used 10% pva and cts solutions with the ratio of pva-cts as 80% :  20% and irradiated the polymer at the dose of 20 kgy, 30 kgy, and 40kgy. he obtained the gel fraction of the polymer as 76.4%, 86.3%, and 87.4%, respectively. this gel fraction result is lower compared with our result  by  using  the  ratio  of  pva-cts  as  83.33% :17.67% and same irradiation dose, i.e. 83%, 87%, and 90%. hydrogel of pva-cts blend has the higher gel fraction then pva alone. based on those data, the higher the cts content in the hydrogel, the lower the gel fraction. chitosan  is  a  natural  polysaccharide,  which  is degraded on  irradiation by breakdown of  the main chains (hien et al., 2012). however, in this study pva is the main component that is known as a polymer and crosslinked in aqueous medium (zheng et al., 2008).  when  the  mixture  of  pva-cts  is  freezedthawed and then it is irradiated, the interpenetrating polymer  network  (ipn)  is  formed  (fig.  3)  with  the chemical crosslink of pva and physical crosslink of cts (herman et al., 2009). soerens et al. (2005, usa patent 6967261) and yang et.al (2008, usa patent 20090297587) reported that the hydrogels with the gel fraction of ~80 % are suitable to be applied as wound dressings. it means that based on gel fraction, all  treatments  used  in  this  research  are  met  the requirement for wound dressing, and the best one is resulted from irradiation dose of 40 kgy. water absorption the water absorption of pva-cts hydrogel with variation of the irradiation dose is presented in fig.5. it shows that the absorption of water increases along 6040200 100 80 60 40 20 0 dose (kgy) g el f ra ct io n ( % ) figure 4. relationship of the irradiation dose (kgy) and the gel fraction (%) of pva-cts hydrogels. synthesis of polyvinyl alcohol-chitosan hydrogel and study .......... (t. wikanta, erizal, tjahyono, and sugiyono) 6 2826242220181614121086420 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 time (hrs) w at er a bs or pt io n (% ) 20 kgy 30 kgy 40 kgy 50 kgy figure 5. relationship of time (hours) and water absorption (%) of pva-cts hydrogel that was irradiated with               different dose. on the applications of hydrogel as a wound dressing it is needed to change wound dressing very often at about every 2-3 days. based on those statements, the hydrogel prepared by irradiation dose of 50 kgy is the  most ideal  one,  and  that  of  by  40  kgy is  also ideally.  in contrast, rekso & sunarni using pva-cts ratio  as  80%  :  20%  reported  that  the  higher  the irradiation  dose,  the lower the water absorption  of hydrogel, i.e. at the irradiation dose of 20 kgy, 30 gy, and  40  kgy,  got  the  water  absorption  as  166.1%, 104.6%,  and  90.9%,  respectiv ely.  this  water absorption result is lower compared with our result by using the ratio of pva-cts as 83.33% : 17.67% and same irradiation dose, i.e. 1700%, 1715%, and 1913 %, respectively. rekso & sunarni the found the water absorption of hydrogel with the irradiation dose of 20 kgy, 30 gy, and 40 kgy were decreased, i.e. 92.2%, 57.8%, and 54.5% at 2 hours and 104%, 72.5%, and 70.3% at 4 hours dipping in aquadest, respectively. whilst, we got much higher result and were increased on the water absorption of hydrogel with the irradiation dose  of  20  kgy,  30  gy,  and  40  kgy,  i.e.  1.244%, 1.316%, and 1.475% at 2 hours and 1.332%, 1.384%, and  1.563%  at  4  hours  dippi ng  in  aquadest, respectively. the difference result probably because of mainly they used a pva with lower molecular weight and also the chitosan with a lower molecular weight and degree of deacetylation, that it was not specified. water evaporation to examine the possibility of the hydrogel to be used as  wound dressing, we investigate the water evaporation rate from hydrogel at the temperature of 30°c with 40% humidity. result is presented in fig. squalen vol. 7 no. 1, may 2012: 1-10. ~ with the times howevers after 26 hours, it reached a limiting value which is an equilibrium condition. it was found  that  the  water  absorption  of  hydrogels  was increasing in accordance with the increasing of the irradiation  dose.  all  hydrogels  were  reached  the equilibrium  condition  in  25  hours,  i.e.  1.700%, 1.715%, 1.913%, and 2.036%, respectively, with the highest water absorption of ~ 2.000% at the irradiation of 40 kgy and 50 kgy. the  water  absorption  of  hydrogels  may  be supported by hydroxyl (oh) groups of pva and amino (-nh 2 ) groups of cts which is interacted with water molecules  through  hydrogen  bonding,  and  by  the presence  of  porous  network  in  the  hydrogel.  by increasing the irradiation dose, the crosslink density of pva hydrogel increase, therefore the porosity of pva hydrogel increase and the water diffusion into hydrogel increase. on the other hand, the increasing the irradiation dose the degradation of ipn cts in the hydrogel increase, and the degradation product of its cts was dissolved out of the hydrogel, and in turn due to the porosity of pva hydrogel increase, the water absorption of hydrogel or water diffusion into hydrogel increase. the  pva-cts  hydrogel  prepared  by  gamma irradiation at the irradiation dose range of 40 kgy to 50  kgy  has  water  absorption  of  ~  2.000%,  it  is equivalent to swelling ratio of 20 g/g. thus, it indicates that  this  hydrogel  absorb  all  the  effusive  wound exudates if it is used as wound dressing. soeren et al. (2005) reported that the swelling ratio of hydrogel at value of 20 g/g is an ideal value to absorb an excess of wound exudates. according to yang et al. (2008, usa patent 20090297587) if the hydrogel has a water absorption value of 1/2 times lower of the ideal value, ~ 7 6. the water evaporation rate from hydrogel irradiated at the dose of 50 kgy at the initial 1 hour was very slow (~2%) and then increased very fast up to ~50 % at 24 hour. the water evaporation rate of hydrogel irradiated at the dose of 20 kgy, 30 kgy, 40 kgy, and 50  kgy  were  43%,  39.13%,  44%,  and  53%, respectively. the water evaporation rate of hydrogel irradiated by 20 kgy, 30 kgy, and 40 kgy are relatively 26242220181614121086420 60 50 40 30 20 10 0 time (hrs) w at er e va p o ra ti o n ( % ) 20 kgy 30 kgy 40 kgy 50 kgy have same value, at about 40%, except irradiated by 50kgy is much higher. it is known that swelling capacity can prevent the accumulation  of  wound  exudates,  and  a  smaller evaporation rate can avoid very often changing the wound  dressing.  therefore,  hydrogel  prepared  by irradiation dose of 20 kgy, 30 kgy, 40 kgy are suitable for wound dressing, and among of those, hydrogel irradiated by the dose of 30 kgy is the most suitable one. whilst, hydrogel prepared by 50 kgy is the worse. yang et al. (2008) reported that irradiation dose of 30 kgy is suitable irradiation dose for preparation of pvacts hydrogel. they obtained that water evaporation rate of the pva-cts hydrogel was very fast (~100 %) for 6 hour standing at room temperature. elongation at break elongation at break is an important physical factor of hydrogel representing its flexibility when it is used for wound dressings. the aims of measurement on the elongation at break of hydrogel in this experiment was  to get  a  supporting  data for  hydrogel  product specification. until now, there is no available reference related with the standard values of elongation at break for wound dressing. generally, the more flexible of hydrogel the easier to follow the skin surface contour (soerens  &  malik,  2005;  yang  et al.,  2008).  the relationship  of  elongation  at  break  of  pva-cts hydrogel with the irradiation dose is presented in fig 7.  the  initial  elongation  at  break  of  hydrogel  with irradiation dose of 20 kgy is 245%. the elongation at break  of  hydrogel  increased  with  increasing  the irradiation dose from 20 kgy to 30 kgy, become 322%, and  kept  constant  at  40  kgy  (322%),  and  then decreased at 50 kgy, become 205%. it indicates that irradiation dose at range of 30-40 kgy is the optimum dose to get maximum value of the elongation at break of pva-cts hydrogel. decreasing of elongation at break of hydrogel at the irradiation dose more than of 40 kgy may be due to  degradation  of  chitosan  in  the  hydrogel  matrix, distributed unhomogeneously and uncrosslinked with resulting a harder and less extensible hydrogel. in vitro antibacterial assay in the biomedical field, the product used must be free of bacteria or has an antibacterial activity. hydrogel prepared for wound dressing or biomedical application has to be sterilized or free of any microorganisms. here, the pva hydrogel with and without chitosan have been made by irradiation and were tested against many bacteria to confirm whether chitosan addition on pva can  improve  the  antibacterial  activity  of  the  pva hydrogel  or  the  product  meet  the  requirement  for biomedical  application.  the  clear  zones  diameter formed as representing the inhibition activity of pvacts hydrogel tested against 7 bacteria is presented in table 1. the results showed that all the hydrogel containing chitosan (treatment) inhibited the growth of all the bacteria tested with bigger inhibition zone (7-8  mm)  than  pva  hydrogel  without  chitosan  or neg ati v e   con trol   (6-7  m m ).  com p ari ng   to figure 6. relationship of time and water evaporation rate of pva-cts hydrogel that was irradiated with different   dose. synthesis of polyvinyl alcohol-chitosan hydrogel and study .......... (t. wikanta, erizal, tjahyono, and sugiyono) 8 6040200 400 300 200 100 0 dose (kgy) e lo n g a ti o n a t b re a k ( % ) figure  7. relationship of irradiation dose (kgy) and elongation at break of pva-cts hydrogel. ba cte ria 1 2 3 4 5 6 7 8 9 e c . 6 6 6 7 7 8 8 7 8 s a 6 6 6 7 7 7 8 7 8 b s t 6 6 6 7 7 7 8 7 8 s ty 6 6 6 7 7 8 8 7 8 s h 6 6 6 7 7 8 8 7 8 p a 6 6 6 7 7 8 8 7 8 b s 6 6 6 7 8 8 8 7 8 inhibition zone dia m e te r (m m ) of hydroge l table 1. antimicrobial activities of irradiated pva-cts hydrogel notes: negative control were 1). pva ( 20 kgy); 2). pva (30 kgy); 3). (40 kgy); 4). pva (50kgy); treatments were  5). pva-cts  (20 kgy);  6). pva-cts (30 kgy);  7). pva-cts  (40 kgy);   8). pva-cts (50 kgy); positive control was 9). chloroamphenicol; disc diameter was 5 mm; bacteria tester : ec = e. coli; sa = staphylococcus aureus; bst = bacillus stearothermophilus, sty = salmonella typhimurium; sh = streptococcus sp.; pa = pseudomonas aeroginosa; bs = bacillus subtilis; chloramphenicol  as a positive  control (8  mm),  the antibacterial activity of pva-cts hydrogel (30 kgy and  40  kgy)  hav e  same  potency  (8  mm)  with chloramphenicol.  the  antibacterial  properties  of hydrogel containing chitosan is due to a positive charge of  the amino-group  in chitosan  that and  it has  the ability to bind to a negative charge in bacteria (tsai & su, 1999). conclusion a series of pva hydrogel containing chitosan were synthesized by combination of freeze-thawing and irradiation  technique  and  their  properties  were compared with pva hydrogel without chitosan. results showed that pva-cts hydrogels have high gel fraction (83-90%),  high  water  absorption  capacity  (1.2011.441% in 1  hour; 1.700-2.036%  in 25 hours),  low water evaporation rate (43-53%), high elongation at break (205-322%),  good  antibacterial  activity,  and translucent appearance. increasing the irradiation dose of 20 kgy until 50 kgy, will result in increased the gel fraction, increased the water absorption, relatively low and constant of water evaporation rate except at the dose of 50 kgy, increased elongation at break except at the dose of 50 kgy, and increased the antibacterial activity except at the dose of 50 kgy. squalen vol. 7 no. 1, may 2012: 1-10. 9 all the pva-cts hydrogels showed an antibacterial activity  against  e. coli, staphylococcus aureus, bacillus stearothermophilus, salmonella typhimurium, streptococcus sp., pseudomonas aeroginosa, and bacillus subtilis. based on all parameters measured, pva-chitosan hydrogels produced by irradiation dose of 40 kgy is the best one. in fact, the product of 30 kgy and 40 kgy has similar properties, hence from economical view point (time of irradiation, dose rate was  10  kgy/hour),  the  product  of  30  kgy  is  more promising. acknowledgment the  authors  were  grateful  to  colleagues  at irradiation facilities patir-batan which  has been providing  the  samples  irradiation  and  to  prof.  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hydrogels  treated  with  radiation  used  as  artificial cartilage.  applied surface science.  p.  568–570. squalen vol. 7 no. 1, may 2012: 1-10. qualen bulletin of marine and fisheries postharvest and biotechnology review article published online: 24 may 2021 page 41 of 55 * department of chemistry, kulliyyah of science, international islamic university malaysia, 25200 kuantan, pahang, malaysia * corresponding author: deny@iium.edu.my received: 11 september 2020 accepted: 22 april 2021 published: 24 may 2021 ©squalen bulletin of marine and fisheries pos th a rves t a nd b i ot echn o lo gy, 2 0 21 . accreditation number:148/m/kpt/2020. is sn: 2089 -569 0, e -is sn: 2406 -927 2. doi: 10.15578/squalen.514 o pe n acces s squalen bulletin introduction sea weeds, a lso known a s ma cr oa lga e, a r e composed of large, diverse macroscopic, eukaryotic, photosynthetic and non-vascular marine organisms. garcia-vaquero, lopez-alonso, and hayes (2017) had listed 10,000 different species of seaweeds, which occupy the littoral zone. the structure of seaweeds is varied, as some are filamentous with a few millimeters in height, while others have huge fronds up to 60meter long. also, the seaweeds’ chemical structure and bioactive compounds are varied based on their habitats, which are either in the harsh marine or terrestrial environment (garcia-vaquero et al., 2017). seaweeds are classified into three higher taxa, namely chlorophyta (green seaweed), phaeophyta (brown seaweed) and rhodophy ta ( r ed sea weed) , ba sed on their pigmentation. bioactive compounds, cosmeceutical and nutraceutical applications of green seaweed species (chlorophyta) fatin shazwani ruslan, deny susanti*, normawaty mohammad noor, nurul iman aminudin, and muhammad taher abstract seaweeds are valuable marine plants that have garnered much attention from the public due to their high bioactive, nutrients and minerals content. seaweeds have been used in multiple applications, including in cosmeceutical, nutraceutical and pharmaceutical industries. nevertheless, this review will focus on the bioactive compounds of chlorophyta and their potential application in nutraceutical and cosmeceutical industries. chlorophyta are believed to possess a significant amount of nutrients and minerals, sufficient to meet the daily requirements of nutrients and minerals in the human body. considering the nutritional aspect, deficiency in nutrients may lead to severe ailments, including heart disease, neurological disorder and cancer. the main compounds studied in this review are polysaccharides, proteins, amino acids, lipids, fatty acids, pigments, minerals, vitamins and secondary metabolites. among all, polysaccharides are the most exploited compounds and used in many advanced applications in the nutraceutical and cosmeceutical industries. this review also offers insights into the beneficial biological properties of chlorophyta, highlighting their potential in cosmeceutical and nutraceutical applications. further research is required to highlight the chlorophyta sp. aquaculture, its extraction method, and the most targeted bioactive compounds from the species. therefore, the challenge is to increase public awareness of the promising application of this species in the nutraceutical and cosmeceutical industries. keywords: chlorophyta, cosmeceutical, nutraceutical seaweeds have been consumed as a type of sea vegetable in most countries such as japan, china and korea. they have become a source of hydrocolloids (alginate, carrageenan, and agar), thickening and gelling agents and had been utilized in industrial and foods across western countries (pereira, 2018). at present, there is a high demand for seaweeds as many quarters have begun consuming healthy and ‘natural foodstuffs’, mainly because seaweeds are rich in minerals, vitamins, and proteins. additionally, the french consumed several types of macroalgae and microalgae in their meals as vegetables or condiments. they have also been widely known and used as a source of fertilizer and thickening agents. meanwhile, in japan, seaweeds make up to 10-25 % of food intake as they used seaweeds as sushi wrappings, seasonings, condiments and vegetables (miyamoto, yabuta, kwak, enomoto, & watanabe, 2009). seaweeds species have been in high demand in mailto:deny@iium.edu.my squalen bull. mar. fish. postharvest biotech. (2021) 16(1): 41-55 ruslan, s. f., susanti, d., noor, m. n., aminudin, i. n., and taher, m. page 42 of 55 most industr ies including nutr a ceutica l a nd cosmeceutical as they contain high carbohydrates, proteins, fiber, vitamins, minerals and low-fat content. also, seaweeds are an excellent source of b group vitamins (b1, b2, and b12), vitamins with antioxidant a ctivities, vitamins c and e, provita min a and carotenoids (škrovánková, 2011). hence, seaweeds can be an alternative food or supplement for those following a special diet and strict vegetarians. yaich et al. (2011) asserted that several seaweed species, such a s ulv a sp., ha ve been a uthor iz ed for huma n consumption. therefor e, it can be deduced that seaweeds have become valuable vegetables and an essential food ingredient in the human diet. figure 1 shows seaweed (u. lactuca) in chlorophyta family. according to pati, sharma, nayak, and panda (2016), the bioactive component such as carbohydrate content in the chlorophyta family is higher than that in rhodophyta and phaeophyta, which also depending on their species and habitats. for instance, higher carbohydrate content was recorded in u. lactuca (35.27 %) from chlorophyta, compared to that in dictyota dichtoma (10.63 %) from phaeophyta family. priyan, kim, kim, and jeon (2019) also discussed that carbohydrates from chlorophyta had been proven to exhibit ma ny cosmeceutica l pr operties such a s antioxidant, anti-wrinkle and moisturizing properties. in a study conducted by roleda et al. (2021), they discussed the chlorophyta sp. for its nutritional content that will benefit the public, anchored on the presence of polysaccharides, pigments, fatty acids, polyphenols and peptides. these compounds may as well contribute to the development of nutraceutical and cosmeceutical. sever a l studies highlighted the utiliza tion of monostroma latissimun, c. racemosa, u. lactuca and, u. australis in nutraceutical applications. most asian countries such as china, japan and korea have consumed these seaweed species as medicinal foods, dietary supplements and fortified products for human consumption through their diets. this is due to the biologically active compounds present in these species, such a s carbohydrates, dietar y fiber s, vitamins, minerals and others that provide great human health benefits and deputized for an inexhaustible source of materials for the nutraceutical and cosmeceutical applications (cotas, leandro, pacheco, gonçalves, & pereira, 2020). however, seaweeds from chlorophyta sp. appear to be unexploited, especia lly when compared to rhodophyta and phaeophyta. this species has not been utilized optimally by the community due to limited research work concerning this species, despite their beneficial application for human diet. therefore, the focus of this review is nar rowed down to green seaweed, chlorophyta sp., by emphasizing the bioactive compounds in this species that had been explored and utiliz ed in the nutr a ceutical a nd cosmeceutica l industries. generally, a nutraceutical compound is defined as a compound that will intensify the food pr oducts’ benefits when a dded. meanwhile, cosmeceutical compounds will add a therapeutic value on cosmetic products (cotas et al., 2020). a few typical examples of chlorophyta sp. were successfully discovered for their bioa ctive compounds a nd applications, namely ulva, monostroma, enteromorpha and caulerpa (kumar, ganesan, suresh, & bhaskar, 2008). nevertheless, only a handful of studies have assessed biological and nutritional contents in chlorophyta sp. this is beca use less r esea r ch wa s conducted concerning the extraction of bioactive compounds from this species than those from other species. this review probed into these seaweed species to identify their nutritional values and their potential benefits in the nutraceutical and cosmeceutical applications. figure 1. ulva lactuca, from chlorophyta collected from merambong island, johor bahru, malaysia. squalen bull. mar. fish. postharvest biotech. (2021) 16(1): 41-55 bioactive compounds in chlorophyta in malaysia, plenty of seaweed species can be found in mer a mbong isla nd, loca ted in j ohor sta te. merambong island offers a vast range of habitats, apart from providing a stable environment that is adequate to support the growth of these seaweeds (zainee, ismail, taip, ibrahim, & ismail, 2019). according to zainee et al. (2019), there are 25 genera, including 46 species of seaweeds found in merambong island. chlorophyta was found to be the highest inhabitant in mer ambong isla nd, a ccommoda ting 11 gener a , followed by rhodophyta (10 genera) and phaeophyta (4 genera). therefore, this review focused on the highest inhabitant in merambong island, which is the green seaweed, chlorophyta sp. chlorophyta sp. are richer in nutritional contents than plants that live on land because they utilize only a small amount of energy to form circulatory systems, leaves, roots, stem and reproductive organs. thus, more phytonutrients, protein and lipids can be stored (hasan, 2017) . there ar e sever al bioa ctive a nd nutritional compounds reported in the chlorophyta sp., e.g., natural pigments (nps), polyunsaturated fatty acids (pufas), lipids, proteins and polysaccharides (khalid, abbas, saeed, bader-ul-ain, & ansar rasul suleria, 2018; kumar et al., 2008). these commercial bioactive compounds are believed to possess many potential health benefits that later may be utilized in nutraceutical and cosmeceutical industries. however, environmental changes such as changes in light, nutrients, contaminants, salinity, ph, temperature and co 2 availability may cause variation of bioactive compounds present in seaweeds (khalid et al., 2018). polysaccharides marine plants such as seaweed contain ma ny polysaccharides, which can be found in their cell wall structure (kumar et al., 2008). polysaccharides are complex biological macromolecules that constitute monosaccharides polymers connected by glycosidic (ether) links. the extracted polysaccharides are usually found in sulfated and non-sulfated forms (hentati et al., 2020). generally, the polysaccharides (matrix and storage) present in the three different seaweed species (chlorophyta, phaeophyta, and rhodophyta) are macroalgae species-specific. for instance, ulvans and xylans were found abundantly in chlorophyta (hentati et al., 2020; kumar et al., 2008). in general, ulvan is differentiated from other seaweed polysaccharides because of the remarkable rare sugars rhamnose and iduronic acid, which are identical to the mammalian glycosaminoglycans (figueira, da silva, enrich-prast, yoneshigue-valentin, & de oliveira, 2020). meanwhile, xylans have a molecular structure of 1,4--d-xylans like higher plants (aizatul, abdul, rahim, yusof, & atikah, 2021). ulvans the cell walls of chlorophyta sp. are made up of ulvan. it is a sulfated polysaccharide that consists of a centr al fr amework of disacchar ide modules, lr ha mnose 3sulpha te, which is linked to ( i) ulvabiouronic acid unit a; (ii) ulvabiuronic acid unit b; (iii) ulvabiose unit a; or (iv) ulvabiose unit b (shah et al., 2020). moreover, ulvan is mainly composed of va riable amounts of rha mnose, glucuronic acid, iduronic acid, xylose and high level of charged sulfated polyelectrolytes (figueira et al, 2020; aizatul et al., 2021). ulva (family ulvacea) from chlorophyta sp. offer ed a wide r a nge of nutr a ceutica l a nd pharmacological applications since they have the capacity to manufacture ulvans moieties of different sugar units (cunha & grenha, 2016). for instance, a low molecular weight (28.2 kda) ulvans of u. pertusa exhibited high inhibitory activity against hydroxyl and superoxide radicals, also showed a strong reduction capacity and metal chelating properties (shah et al., 2020). thus, these properties can be further exploited in the industries for their powerful antioxidant agents. in addition, studies also showed that ulvans from chlorophyta sp. manifested the potentiality as an antiviral, anticancer and anti-aging (figueira et al., 2020). although many studies have discussed the exploitation of polysaccharides from other seaweeds species in the industries, ulvans from chlorophyta sp. are largely untapped. xylans xylans can be found in the cell wall of chlorophyta sp. such as in the caulerpa, which is composed of 1,3--d-xylans (hsieh & harris, 2019). in a study performed by aizatul et al. (2021), they highlighted the utilization of xylan. xylan was highly used in coatings, binding and packaging, and also works well a s a n oxygen ba r r ier. even so, xylans fr om chlorophyta sp. have not been well characterized as less research is concerning on this species. proteins and amino acids proteins and amino acid content in seaweeds differ from one another. according to the previous studies, chlorophyta sp. recorded higher protein contents, which were 10 – 47 % of the dry weight (notowidjojo, 2021). a study highlighted u. lactuca exhibited a relatively high protein content (6–32 % of the dry weight) compared to that of other species (pangestuti & kim, 2015). the protein contents in c. racemosa and u. fasciata were 8.8–19.9 % and 8.0–11.1 %, ruslan, s. f., susanti, d., noor, m. n., aminudin, i. n., and taher, m. page 43 of 55 squalen bull. mar. fish. postharvest biotech. (2021) 16(1): 41-55 respectively (magdugo et al., 2020). the protein content variation in chlorophyta sp. has been influenced by the habitat and depth of the seaweed area. similarly, u. lactuca recorded a higher protein content (19 % of the dry weight) than that in wheat (14.2 % of the dry weight) (van der heide, stødkilde, nørgaard & studnitz, 2021). hence, this species may become a suitable substitute for wheat in extracting a remarkable amount of protein content. furthermore, chlorophyta sp. do not compete with higher plants for space and resources; thus it may become the most significant alternative protein source in the future. moreover, various protein extraction methods concerning this species ha ve a lr ea dy been per for med, such a s enz yma tic hydr olysis and ultr a sonic-a ssisted extraction, which will benefit the public in consuming this species. on top of that, chlorophyta sp. was reported to be rich in essential (arginine) and non-essential amino acids (glycine, alanine and glutamic acid) (ganesan et al., 2020). caulerpa was also recorded to afford the highest amounts of essential and non-essential amino acids among the other green seaweeds. the essential amino acids, namely leucine, lysine, methionine, phenylalanine and valine, content were 0.2, 0.5, 0.4, 0.2 a nd 1.3 mg/g pr otein, r espectively ( tanna , brahmbhatt, & mishra, 2019). not to mention, the histidine and taurine content in ulva and caulerpa also play a vital role in fetus development (ganesan et al., 2020). in contrast, u. pertusa accommodated a remarkable amount of both essential and non-essential amino acids, which were arginine and glycine (ganesan et al., 2020). arginine and glycine are involved in the metabolic pathways, regulation of intestinal function, and protein synthesis and performance (barekatain et al., 2019). a known food source for essential amino acids such as lysine and histidine is usually acquired from meat, eggs, dairy cheese, soy, fish, and fish products. ca nned sa r dines, macker el, tuna and marinated anchovies showed an exceptional content of biogenic amines (histamine, tyramine, tryptamine, putrescine and cadaverine), ranged from 26.58 to 406.55 mg/kg. these biogenic amines were formed from the free amino acids histidine, tyrosine, tryptophan and lysine (bilgin & gençcelep, 2015). attia, al-harthi, kor ish, and shiboob (2020) discussed the total essential and non-essential amino acid contents in eggs from jeddah, saudi arabia. the eggs contained different percentage of essential (arginine, histidine, isoleucine, leucine, lysine, methionine, methionine + cysteine, phenylalanine, threonine, tryptophan, valine) and non-essential amino acids (alanine, aspartic acid, glutamic acid, glycine, proline and serine), which were 56.34 and 70.33 mg/g, respectively (attia et al., 2020). although total amino acid contents were higher in eggs and others than those in green seaweeds, several strict vegetarians do not consume eggs, dairy cheese and any other product derived from animals. hence, this species can be exploited in the future to become an alternative or a potential source of food proteins and amino acids. lipids and fatty acids seaweeds were reported to contain low lipids content (pati et al., 2016). nonetheless, it is a good sour ce of polyunsatur ated fa tty a cids ( pufas) compared to other foods derived from plant and animal sources. pufas manifested a significant part in regulating blood clotting, blood pressure, brain and nervous system (notowidjojo, 2021). nevertheless, a high level of pufas was observed from a cold-water geographical region where the chlorophyta sp. lies; meanwhile a high level of saturated fatty acids and oleic acid wa s r ecorded fr om a wa rm wa ter chlorophyta sp. (notowidjojo, 2021). this can be deduced that lipids and fatty acids composition vary between geographic regions. according to pati et al. (2016), e. clathrata from chlorophyta exhibited the highest lipid content of 4.6 % , followed by c. tomentosum (2.53 %). meanwhile, low lipid content has also been noted in e. intestinalis (1.33 %) and u. lactuca (1.6 %) (pati et al., 2016). besides, lipid content recorded in u. rigida collected from chillka lake, india, was 12 %, which was the highest compar ed to previously mentioned lipid content in other seaweeds species (satpati & pal, 2011). on the contrary, the lipid content in fish displayed a varied range, usually from 0.2 to 25 % content and exhibited a less amount of lipid and fatty acid composition than those in red meat (pal, shukla, maurya, & verma, 2018). in addition, the lipid and fatty acids composition in fish is highly dependent on the species and seasons, which might be a disadvantage in acquiring sufficient lipid and fatty acid in the future. in the recent research, a study on the fatty acid compositions in quinoa was investigated. the main fatty acids detected were linoleic acid, oleic acid, palmitic acid, and -linoleic acid (pellegrini et al., 2018). overall, the fatty acid content in quinoa comprised 4.87 to 6.48 g/100 g, which also showed a higher content when compared to that of chlorophyta sp (pellegrini et al., 2018). despite that, chlorophyta also contains -linolenic acid, also known as an omega3 fatty acid that plays a vital role in human physiology (hasan, 2017). therefore, it can be manifested that different seaweed species and habitats accommodated a great variation of total lipid and fatty acid contents, which might be advantageous for human consumption rather than foods that are derived from other plant and animal sources. ruslan, s. f., susanti, d., noor, m. n., aminudin, i. n., and taher, m. page 44 of 55 squalen bull. mar. fish. postharvest biotech. (2021) 16(1): 41-55 pigments chlorophyta carried a photosynthetic pigment pattern that is almost analogous to the higher plants. sever al photosynthetic pigments ar e pr esent in chlorophyta, namely chlorophyll a, chlorophyll b and, carotenoids. chlorophylls the most common types of chlorophylls present in chlorophyta are chlorophyll a and chlorophyll b. chlorophyta utilized these photosynthetic pigments to absorb light needed for the growth, with the presence of carbon dioxide and carbohydrates. chlorophylls contained reduced porphyrin rings, with a central magnesium atom and a long hydrophobic tail, making them less soluble in water (martins et al., 2021a). in most industries, chlorophylls have been used as natural colorants in foods and beverages and been reported to portray antioxidant, antitumor and antimicrobial activities (martins et al., 2021a). martins et al. (2021b) revealed that a maximum yield of chlorophyll (5.96 mg/g dw) was successfully extracted from u. rigida, using a cost-effective extraction method, using 250 mm tributyl(tetradecyl)phosphonium chloride as the solvent. carotenoids the ma in ca r otenoid compounds found in chlorophyta were -carotene, lutein, neoxanthin, antheraxanthin, zeaxanthin and violaxanthin. the latter was only present in chlorophyta sp. (othman, amin, sani, fadzillah, & jamaludin, 2018). these carotenoids present absorbed light at different wavelengths than chlorophyll, allowing the species to acquire some light to survive in the low sunlight conditions (martins et al., 2021). present studies recorded that c. lentilifera had the highest zeaxanthin and -carotene contents 21.3 and 10.7 µg/g dw, respectively, compared with other seaweed species (othman et al., 2018). similarly, aditi, jaishini, raisa, aamna, and rupali (2020) reported that carotenoids were identified for green microalgae; spirogyra neglecta, pithophora oe dogonia and m ic rospora indic a, ba sed on thinla yer chromatography (tlc) and high-performance liquid chromatography (hplc) analysis. u. lactuca also presented a significant content of carotenoids in which -carotene recognized as major compounds (11.44 – 11.47 %) (abd el hafez, elkomy, saleh, and aboulela, 2020). this promising total amount of pigment contents in chlorophyta make it a perfect target for use as a colorant, antioxidant, antimicrobial and active pharmaceutical ingredient (api) in the future. however, only a handful of studies had reported efficient purification processes of the pigments at a high purity level required by most industries. minerals seafood, including seaweed species, are usually known to hold an abundance of minerals such as iodine, magnesium, calcium, phosphorus, iron, potassium, copper and fluoride (gokulkrishnan, anantharaman, manivannan, thirumaran, & balasubramanian, 2011). these minerals play a significant role as a cofactor of the enzyme in the human body. for instance, calcium and magnesium help maintain the bone and teeth strength, while sodium and potassium involve in the transfer of nutrients (gokulkrishnan et al., 2011). hence, it is believed that seaweeds are valuable for human consumption, as studies showed that seaweeds have great minerals content. u. reticulata presented the maximum contents of chromium, copper and magnesium. at the same time, halimeda tuna only exhibited low miner al contents of cobalt, ir on, ma gnesium, ma nga nese, nickel, lea d a nd z inc (gokulkrishnan et al., 2011). besides, c. lentillifera from the kei islands of indonesia contained magnesium, potassium and zinc, whereas c. lentillifera from the seribu islands contained calcium (119.20 g/kg), sodium (34.18 g/kg) and iron (0.34 g/kg), respectively (tapotubun et al., 2020). also, a study on c. taxifolia from the shores of kanyakumari, india yielded the mineral contents of copper (9.1 ± 0.017 µg g-1 dry weight), zinc (19.96 ± 0.115 µg g-1 dry weight), manganese (53.05 ± 0.058 µg g-1 dry weight) and chromium (5.15 ± 0.087 µg g-1 dry weight). however, the variation of total mineral content was observed when the seaweed was extracted for their mineral contents during two differ ent months, april and december (sethi & karmegam, 2020). secondary metabolites chlorophyta is also known to become a main source of secondary metabolites, which benefits humans in combating various pathogens (levasseur, patrick, & victor, 2020). according to recent research, ulva and caulerpa have shown multiple bioactivities such as antibacterial, antitumor, antiviral and anti-inflammatory due to the presence of secondary metabolites (shah et al., 2020). among the secondary metabolites available in chlorophyta are phenols, alkaloids and terpenes. caulerpa was one of the genera reported to contain phenolic compounds (tannins and flavonoids), terpenes and steroids (shah et al., 2020). alkaloids alkaloids is a cyclic organic compound with a nitrogen-containing base, which also exhibited a diverse ruslan, s. f., susanti, d., noor, m. n., aminudin, i. n., and taher, m. page 45 of 55 squalen bull. mar. fish. postharvest biotech. (2021) 16(1): 41-55 a nd vital physiological effect on huma ns. the physiological effects exerted were owing to the nitrogen found in the molecular structure of alkaloids. alkaloid compounds, namely bisindole alkaloids and caulerpine, were mainly isolated from caule rpa. the novel bisindole a lka loids, ra cemosins a a nd b, wer e successfully extracted from caulerpa collected from zhanjiang coastline in the east china sea, china. this a lka loid compound was believed to show a neuroprotective action (shah et al., 2020). in contrast, ca uler pine a nd ca uler pic a cid fr om caule rpa demonstrated anti-inflammatory and antinociceptive (shah et al., 2020). cantarino, coutinho, soares, duarte, and martinez (2020) varied the extraction method of caulerpin from c. racemosa. the highest caulerpin content (32.06 %) was recorded from the soxhlet extraction method, followed by the maceration technique (19.50 %), microwave-assisted extraction (17.70 %) and ultrasonic-assisted extraction (11.84 %), respectively. therefore, it can be said that the alkaloid contents may be varied depending on the type of extraction techniques used. terpenes other than alkaloids, terpenoids are one of the pr imar y secondar y meta bolites widely found in chlorophyta. terpenoids can be categorized as mono, sesqui-, di-, sester-, triand tetraterpenoids based on the number of isoprene units. c. taxifolia contained sesquiterpenes (three units of isoprene, the backbone of the c15 carbon) with an unusual aromatic carbon skeleton of valerenance type (caulerpals a and b) (shah et al., 2020). these metabolites, caulerpals a and b, wer e r esponsible for inhibiting huma n pr otein tyrosinase phosphatase. meanwhile, chakraborty & santra (2008) studied the diterpenoids (four units of isoprene, the backbone of c20) in u. fasciata, which has been assumed to exhibit an antibacterial effect (shah et al., 2002). phenols a phenolic compound is defined as a group of small molecules that bearing at least one phenol unit. it can be divided into different subgroups based on its chemical structure, namely phenolic acids, flavonoids, tannins, coumarins and a few more. chlorophyta exhibited a unique content of phenolics and flavonoids compounds. al-malki, barbour, al-zahrani, and moselhy (2018) studied the different total phenolic and flavonoid contents in u. lactuca. the phenolic content ranged between a minimum mean of 38.9 gae/g when extracted using chloroform. the highest total phenolic content was observed at a maximum mean of 77.3 gae/g when treated with ethyl acetate solvent. similarly, the total flavonoid content was recorded at 31.2 and 60 mgqe/g when recovered by chloroform and ethyl acetate solvents, respectively (al-malki et al., 2018). for this reason, it can be disclosed that the ethyl acetate solvent will significantly increase the yield of phenolic and flavonoid contents in other seaweed species in the comparative research. a wide range of antioxidant byproducts may be well established from the tabulation of the total phenolic compounds in chlorophyta. vitamins sufficient and good nutrition is the key to a healthy lifestyle. intake of nutritious food keeps diseases at bay, apart from enhancing the well-being of a human. nutritional deficiency often occurs among the elderly, which may cause them to suffer from nutritional anemia, such as iron, folate and vitamin b12 (shahar, budin, bakar, umar, & halim, 2005). besides, the world has reported multiple issues related to health, such as vitamin deficiencies that affect all ages, including pregnant and lactating women. strict vegetarians are more susceptible to vitamin b12 deficiency since they limit meat consumption (bo et al., 2019). as stated by ganesan, tiwari, and rajauria (2019), algae contain the highest amount of both essential and non-essential vitamins. for example, vitamin b12 content is higher in microalgae chlorella, which was 33.3 µg/kg fresh weight than macroalgae nori (1 µg/kg fresh weight). however, nearly 60 % of active vitamin b12 aggregated coenzymes from the macroalgae nori would cover the daily need of biologically active vitamin b12 if it is for tified in a smoothie (ganesa n et a l., 2019) . subramanian, manivannan, sona, ravi, and sasikala (2015) highlighted the vitamins a, b1 and b2 contents in u. rigida (vitamin a: > 0.6 ± 0.12 mg/kg, vitamin b1: 5.85 ± 0.04 mg/kg, vitamin b2: 1.22 ± 0.01 mg/ kg) and u. lactuca (vitamin a: > 0.5 ± 0.11 mg/kg, vitamin b1: 5.22 ± 0.06 mg/kg, vitamin b2: 0.97 ± 0.01 mg/kg), respectively. despite the lack of studies on vitamin content in green seaweed, the outcomes revealed that the seaweeds can become a source of nutrients and as an alternative source of several vitamins. biological activities of chlorophyta in terms of biological activities, green seaweeds ha ve been r epor ted to exhibit a ntimicr obia ls, antioxidants, antiviral, anti-obesity, anti-inflammatory and immunostimulatory properties. these properties are essential to suppress the effects of various diseases. the antimicrobial activity in u. lactuca has been found to be effective in controlling human pathogenic microorganisms (yu-qing, mahmood, shehzadi, & ruslan, s. f., susanti, d., noor, m. n., aminudin, i. n., and taher, m. page 46 of 55 squalen bull. mar. fish. postharvest biotech. (2021) 16(1): 41-55 ashraf, 2016). the u. lactuca extract was tested against some human pathogenic bacteria, such as salmonella paratyphi, pseudomonas aeruginosa, vibrio cholera, staphylococcus aureus, shigella dysentriae and klebsiella pneumonia, yet the active compound responsible for the antimicrobial activity was not mentioned. this antimicrobial activity was tested using the inhibition zone method. as a result, 11.2 mm of inhibition zone was noted for the extract against p. aeruginosa (vallinayaga m, arumugam, kannan, thirumaran, & anantharaman, 2009). also, a review done by zamimi, halim, mustafa, darnis, and musa (2020) discussed the antimicrobial activity of the c. lentillifera and c. racemosa extracts against s. aureus, which the minimum inhibition concentration (mic) values recorded were about 125.25 to 375.75 mg/ml, respectively. the same inhibitory activities against s. aureus also were observed when c. cuppressoides extract was introduced, which the inhibition zone was 6 mm. this chlorophy ta sp. possesses these antimicrobial properties. mainly due to the presence of fatty acids, polysaccharides, and pigments (zamimi et al., 2020). on top of that, polysaccharides from seaweeds can also become a potentia l sour ce of antioxidants. antioxidants scavenging free radicals exhibited a significant role in preventing reactive oxygen species (ros)-induced diseases such a s carcinogenesis, cardiovascular disease, alzheimer ’s disease, aging and neurological disorders (li et al., 2020). as reported in their studies, the high sulfate content purified ulvan, extracted from u. pertusa, successfully improved the antioxidant activity in mice. moreover, oligosaccharides from u. lactuca demonstrated an anti-aging effect when applied to senescence accelerated mouse-prone 8 (samp8) mice. it was observed that the total antioxidant capacity of the mice increased. hence, it can be deduced that oligosaccharides from u. lactuca can be utilized in the making of nutraceutical products and in cosmeceutical to prevent aging problems (liu et al., 2019). in addition, abd el hafez et al. (2020) reviewed the antioxidant activity possessed by u. prolifera by the total antioxidant capacity method. from their studies, the total antioxidant capacity exhibited by different solvent extracts, namely hexane, ethyl acetate and methanol extracts, were 0.97 ± 0.09, 1.23 ± 0.04 and 1.63 ± 0.09 mg equivalent ascorbic acid/g dw, respectively, which was due to the polysaccharide, lipid, and protein contents in u. prolifera (abd el hafez et al., 2020). additionally, the pigment contents in chlorophyta can be beneficial in acting as antioxidants as pigment-like carotenoids can inhibit active radicals (hidayati, yudiati, pringgenies, oktaviyanti, & kusuma, 2020). seaweed species, particularly chlorophyta, are also known for their ability against viral infections; hence this species can be exploited to formulate some antiviral drugs. as reported by mattos, romanos, de souza, sassaki, and barreto-bergter (2011), the incorporation of seaweed species in the antiviral drug formulation now aims to investigate their activities against herpes simplex virus (hsv) and human immunodeficiency virus (hiv). few reports described several antiviral activities against hsv1, japanese encephalitis virus (je) and white spot syndrome virus (wssv) of the polysaccharides from ulva sp. (sun et al., 2018). moreover, there is also a review done by riccio et al. (2020), which analyzed the antiviral activity showed by sulfated polysaccharides extracted from u. pertusa, by targeting avian influenza virus (aiv) particle attachment to the cells. aguilar-briseño et al. (2015) reported the ulvan compounds from u. clathrata possessed the ability to target the newcastle disease virus (ndv) by inhibiting the cell-cell fusion via a direct effect on the f0 protein. furthermore, ulvan extracted from enteromorpha sp. targeted wssv, yet the action mechanism was not reported (riccio et al., 2020). a study was conducted on the antiviral treatment of zika virus (zikv), which presented the inhibition of c. racemosa towards zikv replication in a dose-dependent manner. the study found that c. racemosa extract exhibited the highest cytotoxicity (cc 50 ) effect, 732 µg/ml against zikv (cirne-santos et al., 2017). it can be said that chlorophyta extracts can be a promising species for further studies for the development of new antiviral agents. not only that, chlorophyta, specifica lly c. okamurae are rich in active compounds such as minerals, fiber, vitamin a, vitamin c, alkaloids, sitosterol and essential unsaturated fatty acids that was reckoned to possess an anti-obesity effect. a study done on 6-week-old male c57bl/6j mice exhibited a significantly lower body weight when supplemented with 250 mg/kg body weight of c. okamurae group compared to a mouse fed with high fat diet (hfd) alone. it was observed that the weight of epididymal and perirenal adipose and the total fat of the mouse were decreased when fed with c. okamurae diet (gómez-zorita et al., 2020). given these studies, it can be deduced that c. okamurae extract has the potential to prevent obesity. this is because c. okamurae supplementa tion r educed the pr otein expressions of peroxisome proliferator-activated receptor-gamma (ppar) and ccaat/enhancerbinding protein alpha (c/ebp) that increased due to the hfd (gómez-zorita et al., 2020). similarly, sharma, kim, kim, park, and rhyu (2017) also highlighted the anti-obesity effect demonstrated by c. okamurae extracts. it was found that the ethanolic ruslan, s. f., susanti, d., noor, m. n., aminudin, i. n., and taher, m. page 47 of 55 squalen bull. mar. fish. postharvest biotech. (2021) 16(1): 41-55 extract from c. okamurae was successfully inhibited the lipid accumulation, reduced the expression of ppar, sterol regulatory element-binding protein-1c (srebp1-c) and c/ebp in 3t3-l1 adipocytes of a hfd-fed mice (sharma et al., 2017). extract from ulva sp. also exhibited a promising anti-inflammatory activity. mccauley, winberg, meyer, and skropeta (2018) discussed the lipid-rich extract of ulva, which had been cultivated under high nutrient conditions, possessed anti-inflammatory activity. this study resulted that ulva sp. exhibited a strong antiinflammatory activity of > 94 %, as well as no cell toxicity (mccauley et al., 2018). other than that, the low dose of sulfated polysaccharides (1 mg/kg) from u. lactuca also presented an anti-inflammatory effect. the sulfated polysaccharides content inhibited osmotic edema, that is characterised by bradykinin action (de araújo et al., 2016). it is indicated that the structural compound from sulfated polysaccharides from u. lac tuc a pla ys a r ole in contr olling these a ntiinflammatory pathways. another study was done on the u. lactuca hydroethanolic extract, which worked a s a n a ntiinfla mma tory a gent in suppr essing r heuma toid a r thr itis in ma le wista r ra ts. the improvement was observed on the arthritis rats after three weeks of orally treated with a dose level of 100 mg/kg weight of u. lactuca hydroethanolic extract (ahmed, soliman, mahmoud, & gheryany, 2017). the administration of hydroethanolic extract of u. lactuca improved the elevated level of the rheumatoid factor (rf), prostaglandin e2 (pge2), tumor necrosis factoralpha (tnf-), interleukin-17 (il-17), and interleukin1-beta (il-1b), also the lowered interleukin-4 (il-4) level (ahmed et al., 2017). moreover, bioactive compounds from chlorophyta sp. also exhibited an immunostimulatory activity. berri et al. (2017) discussed an immunostimulatory activity of sulfated polysa cchar ides extr a cted fr om u. armoricana. the capacity of extracted ulvan from u. armoricana to act as immunostimulatory was examined on the gut using an in vitro system of porcine intestinal epithelial (ipec-1) cells. it showed that the mrna and protein expression of cytokines such as ccl20, il8, and tnf- was significantly increased with the concomitant of ulvan extract (berri et al., 2017). besides, the galactans compound extracted from c. c upre ssoide s wa s a lso r eported to possess immunostimulatory activity. it was reported that the production of nitric oxide, reactive oxygen species and proinflammatory cytokines tnf- and il-6 were increased with the presence of c. cupressoides extracts containing galactans (da silva barbosa et al., 2020). overall, it can be deduced that various species from chlorophyta sp. may exhibit immunostimulatory activity with potential therapeutic applications. table 1 summa r iz es the bioa ctive compounds of some chlorophyta species with their important biological activities. application of chlorophyta in industries chlorophyta has gained keen interest by two primary industries, cosmeceutical and nutraceutical industries. in general terms, cosmeceutical is referring to a cosmetic product with medicinal or drug-like advantages. cosmetics are routinely used by most individuals today since many outdoor activities are being practiced throughout the day. thus, extra care needs to be practiced to protect the skin from external stimuli. the external stimuli may be direct exposure from the ultraviolet coming from the sun, the dust from the sur r ounding, a nd ma ny mor e. despite tha t, chlorophyta has also been widely incorporated as dietary supplements in the nutraceutical industries. the purpose of adding a dietary supplement into the regular diet is to boost and enhance the diet with a nutritional and physiological effect. in general, the nutraceutical is defined as any food or part of food that caters to medical or health benefits and acts as a tool in preventing diseases. examples of nutraceuticals are natural foods, which include antioxidants, dietary supplements, fortified dairy products, vitamins, minerals, cereals, herbals and milk. incorporation of chlorophyta in cosmeceutical industries people have recognized a traditional cosmetic with a more natural composition in this age, as the value of a pplying a hea lthy and safe cosmetic ha s been highlighted. a healthy cosmetic must be used to avoid any side effects that can cause toxicity to the user. several studies have reported adverse side effects such as contact dermatitis and allergic reactions when a pplying cer ta in cosmetics tha t conta in high concentra tion, exceeding the optimum limits of bioactive substances (panico et al., 2019). in this context, it is crucial to invent a new whole cosmetic product that is consumer-friendly to prevent these unwanted reactions. thus, seaweed species such as chlorophyta were exploited in the cosmeceutical industries in manufacturing a new line cosmetic product which will act as a thickening, gelling agent and many more. generally, the incorporation of seaweed species stimulates the extracellular tissue matrix (etm) production by increasing the neocollagenesis, thus enhancing the consumer youth and well-being and the skin replacement (pimentel, alves, rodrigues, & oliveira, 2018). the most common cosmeceutical products containing seaweed extracts included creams, moisturizers, serums and lotions. ruslan, s. f., susanti, d., noor, m. n., aminudin, i. n., and taher, m. page 48 of 55 squalen bull. mar. fish. postharvest biotech. (2021) 16(1): 41-55 table 1. bioactive compound of some chlorophyta species with their promising biological activities polysaccharides from chlorophyta, particularly ulvan and its highly sulfated derivatives, have been reported to possess radical scavenging activity, reducing power and metal chelating ability, which are useful in synthesizing certain chemicals (priyan et al., 2019). ulvan, which contains polyaldobiuronan has been used to synthesize aromas, producing strawberry odor and flavoring agents such as furaneol. this was due to the polyaldobiuronan moieties rich in the 6-deoxyhexose sugar rhamnose (priyan et al., 2019). in addition, lahaye and robic (2007) discussed the synergistic skin protective and bioactive effects exhibited by rhamnose and fucose in combating skin aging problems. similarly, mannans and xylans possess desirable properties to be used in the manufacturing industries. for instance, glucomannan has been incorporated in cosmeceutical as an emulsifier (srivastava & kapoor, 2005). chlorophyta is also known to contain a sustainable sour ce of amino acids and peptides, wher e the cholorophyta peptides have a potential to protect collagen stores and enhance collagen synthesis (palareti et al., 2016). for instance, u. lactuca has been reported to revive collagen synthesis in human fibroblasts due to tripeptide containing an arginine-glycine-aspartic acid sequence (palareti et al., 2016). similarly, the peptides from c. vulgaris have shown the ability to reduce the matrix metalloproteinase-1 (mmp-1) expression in human skin cell fibroblasts responsible for the collagen breakdown (chen, liou, chen, & shih, 2011). in this case, the fibroblast is vital in human skin as its role is to repair and remodel the dermis during the skin antiaging process (de araújo, lôbo, trindade, silva, & per eir a , 2019) . ther efor e, the peptides fr om chlorophyta may be capitalized in manufacturing a cosmeceutical product that will help prevent skin aging problems. apart from that, a study conducted by nurjanah, nurilmala, hidayat, and sudirdjo (2016) discussed the amino acid contents in caulerpa could be utilized as cosmetics material. the concentration of amino acid contents such as glutamate, histidine, arginine, aspartate, tyrosine, alanine and valine was high, above 100 mg/100 g. this amino acid content is vital in skin regeneration and in maintaining healthy skin. ruslan, s. f., susanti, d., noor, m. n., aminudin, i. n., and taher, m. page 49 of 55 biological activities s pecies bioactive compounds references immunostimulatory u. lactuca,c. lentilifera,c. racemosa,c. cuppressoides, u. pertusa, u. prolifera fatty acids, p oly saccharides, p igments, galactans yu-qing et al. (2016); vallinay agam et al. (2009); zamimi et al. (2020) antioxidants u. pertusa, u. lactuca, u. prolifera, u. armoricana, u. rigida poly saccharides; mainly ulvans, oligosaccharides, lip ids, p roteins, carotenoids, chlorop hyll a, chlorop hy ll b shah et al. (2020); figueira et al. (2020) anti-inflammatory u.lactuca, u. rigida, c. racemosa poly saccharides; mainly ulvans shah et al. (2020) antimicrobial u. rigida, c. racemosa fatty acids, p oly saccharides, p igments, galactans, chlorophy ll a, chlorop hy ll b, terp enes yu-qing et al. (2016); vallinay agam et al. (2009); zamimi et al. (2020), m artins et al. (2021), shah et al. (2020) antiviral ulva sp ., u. pertusa, u. clathrata, c. racemosa poly saccharides; mainly ulvans figueira et al. (2020) anti-obesity c. okamurae m inerals, fibers, vitamin a, vitamin c, alkaloids, fatty acids, gómez-zorita et al. (2020); sharma et al. (2017) sy nthesis of p rotein, involved in metabolic p athway s ulva sp ., caulerpa sp. proteins; arginine, gly cine ganesan et al. (2020); barekatain et al. (2019) fetus develop ment ulva sp., caulerpa sp . proteins; histidine, taurine ganesan et al. (2020) neurop rotective action caulerpa sp ., c. racemosa alkaloids; bisindole alkaloids, caulerp ine, racemosin a and b, terp enes; diterp enoid shah et al. (2020); squalen bull. mar. fish. postharvest biotech. (2021) 16(1): 41-55 moreover, mycosporine-like amino acids (maa) also was found abundant in green macroalgae and seemed to be potential cosmetic agents. the example of maa family included mycosporine-glycine (mgly), palythine, palythinol, asterina-330, porphyra-334 and shinorine (berthon et al., 2017). these compounds ca r r ied significa nt chemopr otective effects in preventing photoinduced skin aging. other than that, carotenoids of green seaweeds can also become sources of ingredient in the cosmetic industry anti-inflammatory, anti-aging, antiphotoaging, colorants and radical scavengers’ properties (christaki, bonos, giannenasa, & florou-paneria, 2013). carotene, for instance, can revitalize and enable the skin to fight against skin aging and reduce the risk of skin cancer a mong the users (joshi, kumari, & upasani, 2018). furthermore, -carotene from u. lactuca acidic extract (155 x 10 g/l) exhibited antioxidant properties analogous to the commercial antioxidants and had a skin-irritant effect of 0.1 % (balboa et al., 2014). thus, it was safe for topical use in cosmetics. consequently, u. reticulata crude extract ha d been for mula ted into a n a ntia ging ser um for mula tion ( septiya nti, lia na , sutr iningsih, kumayanjati, & meliana, 2019). joshi et al. (2018) also reported that the industry incorporated astaxanthin obtained from haematococcus pluvialis in cosmetics, food and beverages. other than polysaccharides and pigment, u. lactuca has been reported to be abundantly incorporated in the cosmetic industry as an anti-wrinkle agent since this species contained a remarkable amount of several vitamins and minerals such as vitamin a, b, c and e, magnesium, iron and amino acids (£êska, messyasz, & schroeder, 2018). therefore, it can be inferred that chlorophyta sp. ca n be a pr omising sour ce in cosmeceutical industries. several other species are believed to be a promising source in cosmeceutical industries, as presented in table 2. incorporation of chlorophyta in nutraceutical industries chlorophyta species presented different nutritional values based upon their natural characteristics. the biochemical compounds originated from chlorophyta offered many nutraceutical benefits. for instance, u. fasciata and c. racemosa contain a high concentration of polysaccharides, lipids and amino acids (magdugo et al., 2020). in addition, magdugo et al. (2020) reviewed that ulva and caulerpa were consumed directly by humans in the philippines and have long been listed as nutraceutical products by the food and agricultural organization of the united nation (fao) for its various health-promoting benefits. moreover, carotenoids from chlorophyta have also been utiliz ed in nutr aceutica ls, a s well a s in pharmaceuticals. carotenoids may become an effective antioxidant; thus, it will be beneficial for human health. in ter ms of nutr aceuticals, ca rotenoids such a s tocopherol were used as a food preservative in some food products (shah et al., 2020). astaxanthin from h. pluv ialis ha s a lso been r epor ted to lessen inflammation, oxidative stress and enhance the immune system of pa tients who wer e suffered fr om cardiovascular disease (shah et al., 2020). apart from the nutra ceutica l mentioned a bove benefit of chlorophyta species, chlorophyta species, dried green seaweed (enteromorpha) has been expected to be an alternative for vitamin b12, especially to those who are on a special diet. u. lactuca species is also a vital source of vitamin b (macartain, gill, brooks, campbell, & rowland, 2007). they revealed that a daily intake of 1.4 g/day of u. lactuca is sufficient to meet the daily requirement of vitamin b12. therefore, the algal species is a promising species that acts as an alternative source of vitamins in the future, especially to older people and strict vegetarians, which later can be exploited in the nutraceutical industries. ta nna a nd mishr a ( 2018) ha d r epor ted few chlorophyta species were commercially available as nutraceutical products in a company located in vietnam. they reported that the commercialized ulva was rich in docosahexaenoic acid (dha), with an omega ratio in the range of 0.61 to 5.15:1. moreover, world health organization (who) recommended an 6/3 ratio of < 10 for nutraceutical to exhibit an ability to suppress neurologica l, infla mma tor y, a nd car diova scula r disorders (tanna & mishra, 2018). u. fasciata also exhibited an anticoagulant effect due to the presence of ga la cta ns a nd fuca ns ( ruocco, costa ntini, guariniello, & costantini, 2016). overall, the bioactive compounds in seaweed, especially in green seaweed species, offered many nutraceutical benefits to human health. table 3 summarizes the application of several species from chlorophyta in terms of their current and future nutraceutical properties and products. conclusion in short, seaweeds offered numerous nutrients to benefit huma n beings. sea weeds, pa r ticula r ly chlorophyta are rich in beneficial compounds such as polysaccharides, proteins, amino acids, fatty acids, minerals, and vitamins with medicinal and healthpromoting effects. therefore, this nutritional value makes chlorophyta a valuable future food supplement and a precious component as cosmetic ingredients. chlorophyta also benefits those on a strict diet or vegetarians due to excluding certain nutrients or ruslan, s. f., susanti, d., noor, m. n., aminudin, i. n., and taher, m. page 50 of 55 squalen bull. mar. fish. postharvest biotech. (2021) 16(1): 41-55 table 2. applications and functions of extracts extracted from chlorophyta sp. in the cosmetic industry minerals from their daily dietary intake, specifically vitamin b12. this problem is problematic as it can lead to nutrient deficiency that causes diseases. moreover, table 3. applications and functions of extracts from chlorophyta sp. in the nutraceutical industry recognition of seaweeds species as a source of nutrients is crucial to suppress some illnesses related to nutrient deficiency among the elderly and others. ruslan, s. f., susanti, d., noor, m. n., aminudin, i. n., and taher, m. page 51 of 55 cosmetic properties/products s pecies extracts/compounds references antioxidant e. linza, bryopsis plumose, u. rigida poly saccharides pereira (2018); thiy agarasaiy ar, goh, jeon & yow (2020) antioxidant – ddph inhibitions c. antennia fucoxanthin thiy agarasaiy ar et al. (2020) algotherm – soothing p ower, hy drating p rop erties, exfoliating gel u. compressa, codium tomentosum m agnesium, p oly saccharides m ichalak, dmy try k, & chojnacka (2020) m oisturizing agents u. rigida, u. lactuca poly saccharide ulvan, extracts priy an et al. (2019); pereira (2018); lahay e & robic (2007); pimentel et al. (2018)exfoliating gels, body masks, body scrubs, face p eelings, face masks, cleansing gels u. lactuca, u. compressa extracts, micronized algae pereira (2018) anti-stretch mark creams, body lotions, ey e creams, face masks c. vulgaris extracts pereira (2018) anti-inflammatory agent u. lactuca poly saccharide ulvan, carotenoids (astaxanthin, βcarotene, fucoxanthin, pimentel et al. (2018); pereira (2018) stimulation of collagen p roduction, increase the collagen sy nthesis u. lactuca tripep tide: arginine, gly cine, asp artic acid wang, paul, & luesch (2013) anti-aging, antioxidant, ty rosinase inhibitors, antiphotoaging agents, radical scavengers u. lactuca carotenoids (astaxanthin, βcarotene, fucoxanthin, lutein) pereira (2018) antibacterial u. lactuca chlorop hy lls pereira (2018) antiadhesive agents ulva sp . lectins pereira (2018) ty rosinase inhibitor to inhibit melanin p igment caulerpa sp . steroids, flavonoids, p henols pereira (2018) nutraceutical properties/products s pecies extracts/compounds references lettuce extracts, vegan alternatives to beef-derived gelatins ulva and enteromorpha sp. u. lactuca, u. armoricana poly saccharide ulvan tanna & m ishra, (2018); shannon & abu-ghannam, 2019 nutraceutical p roduct by vietnam comp any ulva docosahexaenoic acid (dha) tanna & m ishra, (2018) semi-sweet biscuit with antioxidant c. racemosa p oly saccharides kumar et al. (2008) food stabilizer and p reservatives u. lactuca , u. pertusa , u. clatharata , u. intestinalis , u. linza bromop henols and flavonoids leandro et al. (2020) dietary supp lements enteremorpha , u. lactuca vitamin b12 (cy anocobalamin) m acartain et al. (2007) healthy snacks in thailand u. rigida dietary fiber, p roteins, minerals thunyawanichnondh et al. (2020) squalen bull. mar. fish. postharvest biotech. (2021) 16(1): 41-55 seaweeds are becoming one of the most desirable natural sources for obtaining biological compounds due to their high potential for producing novel nutraceutical a nd cosmeceutica l pr oducts. therefore, fur ther r esea r ch is r equir ed to highlight the sea weed (chlorophyta) aquaculture, its extraction method, and the most targeted bioactive compounds from the species. in addition, the preservation of the bioactive compounds during extraction, which is mostly unstable upon contact with oxygen, high temperature, and light, must a lso be consider ed so the extr a ction a nd aquaculture methods can be easily applied in the industries. in the current research, the focus is more on the cultivation method of seaweed that will give a simila r or higher concentr a tion of bioa ctive compounds, which correspond with the bioactive compounds fr om wild sea weed. likewise, the optimization of the extraction method needs also to be further studied to yield better quality and quantity of the extract. also, it is crucial to develop an optimized extraction method that will help industries obtain more than one compound to create value added to the products. this review has emphasized the biological compounds, biological activities, and application in the industries that might also benefit future researchers in improving chlorophyta sp. cultivating methodology and optimizing extraction methods. acknowledgement the authors are thankful to the international islamic university malaysia for funding this work via grant no. p-rigs18-028-0028 and ministry of higher education (mohe) malaysia through fundamental grant scheme (frgs19-129-0738). references abd el hafez, m. s. m., elkomy, r. g., saleh, h., & aboul-ela, h. m. 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