03 Rahmadi.cdr Vol.12, No.3, September 2018, p 83-91 DOI: 10.5454/mi.12.3.3 Bacterial Population and Chemical Characteristics of Fermented Mandai Cempedak with Starter Induction * ANTON RAHMADI , KARTIKA SARI, NIKMATUL KHAIRIYAH, FRIO HANDAYANI, SITOHANG SATRIO, YULIANI, AND ASWITA EMMAWATI Department of Agricultural Products Technology, Faculty of Agriculture, Universitas Mulawarman, Jalan Kuaro, Samarinda Ulu, Samarinda, East Kalimantan 75119, Indonesia Traditionally fermented foods can be improved by introducing starter and hygienic production. The study observes the changes in population of lactic acid bacteria (LAB), pH, polyphenolic levels, and antioxidant activity of spontaneous and Lactobacillus casei induced mandai cempedak fermentation at 37 °C for seven days. The hygienic process included two steps boiling of inner-skin of cempedak at 80-90 °C for 15 minutes. LAB and non- LAB growth were quantified with plate count. Phenolic substances were spectrophotometrically quantified. Gallic acid (GAE), tannic acid (TAE), and catechin (CE) were used as standards. DPPH method was employed to measure antioxidant activity. As the result, the LAB dominated bacterial population during the course of -1 fermentation. The LAB grew from 3.3±0.5 to 8.8±0.6 log cfu mL for spontaneous fermentation and from 3.3±0.4 -1 to 9.0±0.5 log cfu mL for starter induced fermentation. The population of LAB in spontaneous and L. casei induced fermentation grew in almost similar pattern and can be approached by linear regression. The degree of acidity increased during the fermentation process and achieving pH 3.5 at the sixth day of fermentation. The fermentation process increased the phenolic contents both in spontaneous and L. casei induced fermentation, and resulting in enhancing the antioxidant activity. The phenolic contents, except total tannins, were higher in starter induced fermentation, thus lowering IC of inhibitions of DPPH reduction. Hence, L. casei produced fermented 50 products with better antioxidant activity in comparison to spontaneously fermented products. From these parameters, L. casei was successfully used as starter for mandai cempedak and optimum fermentation at 37 °C was 6 days. Key words: antioxidant, bacterial population, mandai cempedak, phytochemicals Makanan fermentasi tradisional dapat ditingkatkan kualitasnya dengan menggunakan biakan pemula dan produksi yang higienis. Penelitian ini bertujuan untuk mengamati perubahan populasi bakteri asam laktat (LAB), pH, kadar polifenol, dan aktivitas antioksidan dari fermentasi spontan dan biakan pemula L. casei dari mandai cempedak pada suhu 37 °C selama tujuh hari. Proses persiapan higienis dilakukan dengan dua tahapan perebusan kulit cempedak bagian dalam pada suhu 80-90 °C selama 15 menit. Pertumbuhan populasi LAB dan non-LAB dikuantifikasi dengan angka lempeng total. Kadar fenolik dikuantifikasi secara spektrofotometri. Asam galat (GAE), tanat (TAE), dan katekin (CE) digunakan sebagai standar. DPPH digunakan untuk mengukur aktivitas antioksidan. Bakteri asam laktat mendominasi populasi bakteri selama waktu fermentasi. Dari studi ini didapat -1 hasil sebagai berikut, populasi LAB tumbuh dari 3,3±0,5 ke 8,8±0,6 log cfu mL untuk fermentasi spontan dan dari -1 3,3±0,4 ke 9,0±0,5 log cfu mL untuk fermentasi dengan biakan pemula. Populasi LAB dalam fermentasi spontan dan yang diinduksi L. casei berkembang dengan profil yang serupa dan dapat diprediksi dengan regresi linier. Tingkat keasaman meningkat seiring dengan proses fermentasi dengan pH terendah adalah 3,5 pada hari ke-6. Fermentasi meningkatkan kadar fenolik baik untuk fermentasi spontan maupun yang diinduksi dengan L. casei, dan hal ini meningkatkan aktivitas antioksidan. Semua fenolik, kecuali total tanin, terdapat dalam konsentrasi yang lebih tinggi pada fermentasi dengan biakan pemula, sehingga fermentasi cempedak meningkatkan kapasitas pereduksi DPPH menjadi lebih baik. Fermentasi dengan induksi L. casei memiliki aktivitas antioksidan yang lebih baik dibandingkan pada fermentasi spontan. Sebagai kesimpulan, L. casei dapat digunakan sebagai biakan pemula pada fermentasi mandai dengan waktu optimum fermentasi pada suhu 37 °C adalah enam hari. Kata kunci: antioksidan, mandai cempedak, populasi bakteri, phytochemicals MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 *Corresponding author: Phone: 62-541-741118; Fax: 62- 541-747479/732870 ; Email: arahmadi@unmul.ac.id traditional fermented products, spontaneous fermentation is commonly performed at room temperature (Nuraida 2015). Mandai cempedak is made from the inner-skin of cempedak (or jackfruit) by utilizing salt to promote LAB growth (Nur 2009; Rahmadi et al. 2013; Emmawati 2015). Lactobacillus plantarum and Leuconostoc sp. are predominant bacteria that can be isolated from these products. In Kalimantan, in addition to be consumed as fresh fruit and seed, the inner-skin of Cempedak or Tiwadak (Artocarpus champeden) fruit can be processed into a fermented food called mandai or dami (Emmawati et al. 2015). To produce mandai cempedak and other Stages of processing including peeling the outer skin of the fruit, removing the epidermis, and immersing in salt water in order to preserve and soften the texture. The length of fermentation is observed from several hours to one month. The disadvantage of the traditional fermentation process includes the use of salt in high concentrations (15-25% w/v) that leads to an increase in the body's salt intake (NaCl). High salt diet has health implications (Song et al. 2017). The use of high salt content also provides off and bitter taste in the traditionally fermented products as a result of mineral accumulation in the product (Panagou et al. 2011). In community, hygienic practice during fermentation is often neglected, resulting in dissimilar quality between batches and potential pathogenic microorganism contamination in the mandai cempedak product (Blana et al. 2014). There should be an effort to improve the process of traditional food fermentation. Lactobacillus plantarum is traditionally found in plant-based fermented products. This is due to the versatility of L. plantarum enzymes in comparison to other LABs (Siezen et al. 2012). Lactobacillus plantarum produces amylase, b-glucosidase, decarboxylase, lactate dehydrogenases, peptidases, phenolic acid decarboxylases, phenol reductase, proteinase, and tannase enzymes. On the other hand, regardless more commonly found in dairy-based products, L. casei may be employed for plant-based fermentation, as it is recorded to produce amylase, lactate dehydrogenases, peptidases, and proteinase enzymes (Hur et al. 2014). The LAB starter utilized in this research was prepared from Lactobacillus casei Shirota strain. This study aims to compare changes in bacterial populations, pH, phenolic (total phenolics, total tannins, and flavonoids) contents, and total antioxidants without addition of salt by spontaneous and starter induced fermentation of mandai cempedak at temperature of 37 °C. MATERIALS AND METHODS Mandai Cempedak Fermentation. The part of cempedak fruit used was the inner-skin that has been sorted and cleaned. The inner-skin of cempedak was 3 diced to the size of 3-5 cm . The pieces of the inner-skin of cempedak were boiled at temperature of 80-90 °C for 15 minutes to remove the sap and then the water was drained. The inner-skin of cempedak was separated into sealed containers, each was weighted at 100 g. Potable water was poured into the container until the entire dices were submerged. The samples were boiled once more at temperature of 80-90 °C for 15 minutes. Half of the samples were inoculated with L. casei at a 6 8 concentration of 4x10 to 4x10 cfu, or approximately 4 mL of the concentrated L. casei inoculum for each container. The samples were further fermented for seven days at 37 °C. Observations on total bacteria, total LAB, pH, phenolic contents, and antioxidant activity potency were performed daily until the seventh day. Total Plate Count. Total Plate Count (TPC) was performed as described by Fardiaz (1993). Nutrient Agar (NA) medium (Accumedia, USA) was used for total bacteria count, while De Mann Rogosa Sharpe Agar (Himedia, India) medium used for LAB count. The media was sterilized at 121 °C for 15 minutes, then the media was cooled to 60 °C. The warm media was poured aseptically in a sterile Petri dish which was then homogenized to spread evenly across the surface. Petri dishes containing media were allowed to solidify. As many as 0.1 mL of samples were poured into a Petri dish and flattened. The samples were incubated in the incubator in reversed position at 37 °C for 24 hours. The calculation based on the number of colonies ranges -1 from 25 to 250 cfu mL , considering dilution factor. The calculation of non-LAB population was performed by subtracting the log of total bacterial population obtained from the NA medium with log of LAB population of the MRSA medium. pH. The degree of acidity was measured using Sudarmadji et al. (2007) method. About ± 50 mL of sample product was placed into a small jar and the pH was measured in duplicate for each product. Prior to analysis, the pH meter had been checked and calibrated in buffer pH 4 and 9. Total Phenolics. Phenol analysis was performed by spectrophotometric method employing Folin- Ciocalteu (Mu'nisa et al. 2012; Nurhayati et al. 2012). Gallic acid (Sigma-Aldrich, USA) was used as the standard. The total content of phenolics in the extract was expressed in gallic acid equivalent (GAE). Each sample was weighed as much as 5 mg, then dissolved in 2 mL of 95% ethanol. Further, about 5 mL of aquades and 0.5 mL of Folin-Ciocalteau (Sigma-Aldrich, USA) 50% (v/v) were added. Samples were allowed to stand for 5 minutes and a 5% (w/v) of Na CO (Sigma-2 3 Aldrich, USA) solution was added to make 10 mL of total volume. The solution was homogenized in dark room for one hour. The solution was measured at a wavelength of 752 nm (Genesys 20, Thermo-Fischer, 84 RAHMADI ET AL. Microbiol Indones Volume 12, 2018 Microbiol Indones 85 USA). Total Tannins. Total tannins were tested according to Malangngi et al. (2012). A total of 0.5 g of the product was extracted with 10 mL of diethyl ether (Merck, USA) for 20 hours, then filtered. The residue obtained was boiled in 100 mL of aquadest (Soil Science Laboratory, Mulawarman University, Indonesia) for two hours, then cooled and filtered. The extract obtained was added with distilled water until the extract volume reached 100 mL. A total of 0.1 mL of the extract was added with 0.1 mL of Folin Ciocalteu reagent (Sigma Aldrich, USA) and was vortexed. About 2 mL of Na CO (Sigma Aldrich, USA) was 2 3 added and the sample was vortexed once more. The absorbance was read at 760 nm of wavelength (Genysis 20, Thermo Fischer, USA) after incubation for 30 min at room temperature (28±2 °C). The results obtained were plotted against the standard curve of tannic acid (Sigma Aldrich, USA) that was prepared in the same procedure. The total content of tannins was expressed as tannic acid equivalent (TAE). Total Flavonoids. Total Flavonoids were measured by the method of Zou et al. (2004). About 1 mg of extract was dissolved in 10 mL of 95% of ethanol (Kimia Farma, Indonesia) and 0.7 mL of distilled water was added. About 0.1 mL of 5% NaNO (Sigma 2 Aldrich, USA) was added into the mixture. After 5 minutes, 0.1 mL of AlCl 10% (Sigma Aldrich, USA) 3 was added. After 6 minutes, 0.5 mL of 1 M NaOH (Merck, USA) was added. All ingredients were mixed evenly and the samples were incubated for 10 minutes. The absorbance was measured at a wavelength of 510 nm, after 1 mL of sample was replaced with 1 mL of 95% ethanol solvent. The results obtained were plotted against standard curve of catechin (Sigma Aldrich, USA) that was prepared in the same way. Total flavonoids were expressed as catechin equivalent (CE). Antioxidant Activity. Total antioxidants were performed by spectrophotometric method using 2,2- diphenyl-1-picrylhydrazyl (DPPH) (Sigma-Aldrich, USA) reduction principle (Farhan et al. 2012). A total of 1 mL of diluted extract in ethanol (Kimia Farma, Indonesia) was added to 1 mL DPPH (prepared at 0.15 mM in ethanol) and at the same time, a control consisting of 1 mL of DPPH with 1 mL of ethanol was prepared. The reaction mixture was vortexed and then incubated in the dark at room temperature for 30 minutes. The absorbance was measured at 519±2 nm (Genesys 20, Thermo-Fischer, USA). Vitamin C (Sigma Aldrich, USA) was used as a positive control and ethanol was used as the substract. The ability to inhibit DPPH reduction of the extract was calculated by comparing the absorbance of the reduced control of sample divided by the absorbance of the control. The total antioxidant value was then plotted in a linear regression equation: [antioxidant potential] = a [ingredient in ppm] + b to obtain its IC value. 50 RESULTS Total Bacteria and LAB. The growths of microbes in mandai cempedak without addition of salt were quantified in seven days of fermentation course with -1 initial microbial concentration of 3,3±0,3 log cfu mL -1 for spontaneous fermentation and 3,5±0,5 log cfu mL for starter induced fermentation. The dominance of the LAB in mandai cempedak fermentation was estimated in linear equations presented in Figure 1 and Table 1. The LAB grew to achieve the density of 8,8±0,6 log cfu -1 mL for spontaneous fermentation and 9,0±0,5 log cfu -1 mL for starter induced fermentation at the end of the observation period (Fig 2). Until the seventh day, LAB continued to grow and dominate in both spontaneous and starter induced fermentation (Fig 2). Confirmation of LAB growth was performed with MRSA medium and partial biochemical tests including Gram positive confirmation, microscope observation, the absence of spore, non-motility, and positive catalase. LAB and non-LAB growths were observed from day one to day seven at 37 °C (Fig 1). In the spontaneous mandai cempedak fermentation, L. plantarum was dominated microbial growth, while in the starter induced fermentation, there were at least two distinct LAB isolates observed under microscope. However, we are yet to determine the species. This data proved that LAB was able to survive in the heat process applied in the initial processing of mandai cempedak. LAB is known to produce lactic acid in an amount that is sufficient to increase the acidity of the fermented product. The increase of acidity of mandai cempedak started from the second Table 1 The linear equation of bacterial growth in mandai cempedak fermentation with and without starter Linear equation Spontaenous Fermentation L. casei induced Fermentation Total Bacteria Y = 0.9029*X + 2.687 Y = 0.8717*X + 2.681 Total LAB Y = 0.9008*X + 2.149 Y = 0.9128*X + 2.081 Fig 1 Linear regression of bacterial growth in mandai cempedak fermentation with and without starter. A: The linear regression of growth of bacteria and LAB in spontaneous fermentation B: The linear regression of growth of bacteria and LAB in L. casei induced fermentation. * Significantly different in the multiple T- test. SEM is symbolized with (┬) located above and below the observation point. 86 RAHMADI ET AL. Microbiol Indones A B Fig 2 The LAB and non-LAB population in mandai cempedak fermentation with and without starter. A: The LAB and non-LAB population in spontaneous fermentation B: The LAB and non-LAB population in L. casei induced fermentation. * Significantly different in the multiple T-test. SEM is symbolized with (┬) located above and below the observation point. A B Volume 12, 2018 Microbiol Indones 87 day of the fermentation course to the last observed day. The lowest pH (3.5) was observed on the sixth day of fermentation. Overall, there was no significant difference of the lowest pH of spontaneous and starter induced mandai cempedak, although slight variations of pH on day to day observation were measured (Fig 3). Fermentation is proven to increase the phenolic contents in certain food products, and as resulting in enhancing the antioxidant activity. These phenomena were highlighted in mandai cempedak fermentation (Fig 4). The difference of total phenolic and flavonoid contents between spontaneous and starter induced fermentation products was observed, in which the use of L. casei induced fermentation resulting in higher concentrations of total phenolic and flavonoid contents. In addition, there was no difference of total tannins for both products in any day of observation (Fig 4). Hur et al. (2014) stated that antioxidative activity may be increased in fermented plant-based food products as a result of microbial enzymatic hydrolysis of phenolic compounds. DISCUSSION The result is in line with the research of De Angelis et al. (2004) and Fiocco et al. (2007) which stated that several strains of LAB were able to survive after mild heat processing because they produced proteins that were protective to heat. The inner-skin of cempedak contained fibrous materials that provide an extent of supports for cell immobilization, giving protection to the viability of lactobacilli (Lye et al. 2012). This also explained the survivability of the LAB during mild heat treatment. It is noted that cempedak medium promoted LAB growth and domination. Heat treatment of mandai cempedak may resulted in the release of nutritious compounds such as free amino, nitrogen, and sugars into the fermentation media, leading to their increased availability to be utilized by LAB. Teh et al. (2010) postulated that the sugars from the skin of durian (Durio zibethinus), cempedak, and mangosteen were assimilated by lactobacilli and utilized for growth. Acidity. From the lowest achieved pH, both optimum fermentations at 37 °C were 6 days. The pH is used as general indication of optimum LAB fermentation as stated by Mousavi et al. (2010). Rhee et al. (2011) reported a decrease in pH as a result of lactic acid production, which was also an indicator of the success of fermentation of traditional food products by LAB. The L. plantarum and L. casei are regarded to be facultatively heterofermentative bacteria (Ashraf et al. 2011; Zago et al. 2011), therefore produces a variety of organic acids to reduce the pH. Cueva et al. (2013) stated that during the in vitro fermentation of up to 48 h of grape seeds by Lactobacilli, organic acids such as 4- hydroxyphenylacetic acid, phenylpropionic acid, 3- hydroxyphenylacetic acid, phenylacetic acid, 3-(4- hydroxyphenyl)-propionic acid, and 4-hydroxy-5- (phenyl)-valeric acid were significantly increased by the fermentation process. These compounds, along with lactic acid, caused the increasing acidity of the fermented mandai cempedak. Phytochemistry and Antioxidant Activity. Lactobacillus casei and L. plantarum are known to Fig 3 pH of mandai cempedak fermentation with and without starter. * Significantly different in the multiple T- test. SEM is symbolized with (┬) located above and below the observation point. 88 RAHMADI ET AL. Microbiol Indones Fig 4 The total phenolic, tannins, flavonoid contents and antioxidant activity in fermentation of mandai cempedak with and without starter. A: The total phenolic contents in spontaneous and L. casei induced fermentation. B: The total tannin contents in spontaneous and L. casei induced fermentation. C: The total flavonoid contents in spontaneous and L. casei induced fermentation. D: The IC of inhibition of DPPH 50 reduction in spontaneous and L. casei induced fermentation. * Significantly different in the multiple T- test. SEM is symbolized with (┬) located above and below the observation point. A B C D have -glucosidase and -galactosidase activities that increase flavonoids in fermented food products, i.e. in soybean fermentation (Marazza et al. 2009). In this regard, Hur et al. (2014) proposed that LAB fermentation causes formation of organic acids that influences pH, Maillard reaction, and pentose phosphate pathway which subsequently attenuate redox balance and radical scavenging activity. The fermentation also causes activations of -glucosidase, - galactosidase, tannase, and phosphoketolase that assist hydrolysis and de-polymerization of phenolics. The downstream processes have an implication of liberation of phenolic substances which in turn provides hydrogen or electron donor and positively modulates metal ion chelation activity. As a result, radical scavenging activity may increase. Quercetin and gallic acid were significantly higher during LAB fermentation of Graptopetalum paraguayense in any stage of fruit maturity (Wu et al. 2011). Hole et al. (2012) concluded that fermentation of cereal products with specific probiotics exhibited significant increase of free phenolic acids, i.e. caffeic acid, p-coumaric acid, ferulic acid, sinapic acid, 5,5-diferulic acid, 8-o-4-diferulic acid, and 8,5-diferulic acid. Selected LABs were able to increase free phenolic acids due to high feruloyl esterase activity (FAE). In solid state fermentation, the increase of antioxidant activity with regard to modulation of polyphenolic content is also proven. Lee et al. (2008) reported that an increase of antioxidant activity is observed when employing different starter to ferment koji bean, which was attributed to the increase of phenol and anthocyanin contents. Further, the increase in phenolic substances were measured during fermentation of cooked grass pea seed, wheat grains, and soybean products (Starzynska-Janiszewska et al. 2008; Bhanja et al. 2009; Singh et al. 2010; Dajanta et al. 2013). However, the changes of phytochemical contents are medium and strain specific (Martins et al. 2011). In other condition, i.e. tea fermentation, it is stated that the monomeric flavonoids were transformed to polymeric derivatives as the tea leaves were further fermented (Kim et al. 2011). As a result, polyphenolic contents were lessened as the higher degree of fermentation occurred. Further, external factors affecting the changes of phytochemical contents are duration of fermentation course, temperature, pH, inhibitors, stimulators, and the composition of atmosphere in the fermentation chamber (Hur et al. 2014). The change in IC values of DPPH reduction 50 inhibition is limitedly influenced by pH if the difference of the pH is less than 1. However, if the difference of pH at range of greater than 2, the IC 50 value of DPPH reduction inhibition between products will differ significantly (Pekal and Pyrzynska 2015). In this study, the comparison of IC inhibition of DPPH 50 reduction was performed only between the same day fermented of mandai cempedak products, where the difference of pH was less than 1 between spontaneous and starter induced fermented products. It was concluded that IC inhibitions of DPPH reduction of 50 starter induced fermentation were lower than the values p r o d u c e d f r o m s p o n t a n e o u s f e r m e n t a t i o n . Lactobacillus casei produced fermented products with better antioxidant activity in comparison to L. plantarum fermented products. As conclusion, the growths of bacteria in mandai -1 cempedak initially started from 3,3±0,3 log cfu mL -1 and 3,5±0,5 log cfu mL for spontaneous fermentation and starter induced fermentation, respectively. The growth curves of the LAB in spontaneous and L. casei induced mandai cempedak fermentation in the seven day of fermentation at 37 °C were in log-linear pattern. 9 -1 The LAB grew to achieve the density of 10 cfu mL for both types of fermentation at the end of the observation period. There was no significant difference of the lowest pH of spontaneous and starter induced mandai cempedak, although slight variations of pH on day to day observation were measured. The starter induced fermentation products contained higher total phenolic and flavonoid in comparison to spontaneous fermentation products, while there was no difference of total tannins for both products in any day of observation. Lactobacillus casei produced fermented products with better antioxidant activity in comparison to L. plantarum fermented products, indicated by IC 50 inhibitions of DPPH reduction. From these parameters, L. casei was successfully used as starter for mandai cempedak and optimum fermentation at 37 °C was 6 days. ACKNOWLEDGMENT The authors would like to thank the Indonesian Directorate General of Research and Development Strengthening (DRPM) Ministry of Research, Technology and Higher Education, Republic of Indonesia for financing this research through Penelitian Strtegis Nasional Institusi (PSNI) scheme. Microbial population, flavonoid levels, and total antioxidants in spontaneous fermentation have been Volume 12, 2018 Microbiol Indones 89 90 RAHMADI ET AL. Microbiol Indones presented in Indonesian Association of Food Technologists (PATPI) National Seminar on 10-11 October 2017 in University of Lampung. REFERENCES Ashraf R, Shah NP. 2011. 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