CONTACT : ANDI NUR FAIZIAH PALLA andinurfaiziah15@gmail.com 222 ISSN : 2580-2410 eISSN : 2580-2119 Protein Hydrolyzate of Grouper Viscera : Effects of Crude Bromelain Extract Concentration and Hydrolysis Time on Yield and Degree of Hydrolysis Andi Nur Faiziah Palla1*, Metusalach2, Nursinah Amir2 1 Fisheries Science Study Program, Faculty of Marine Science and Fisheries, Hasanuddin University, Makassar, Indonesia. 2 Department of Fishery, Faculty of Marine Science and Fisheries, Hasanuddin University, Makassar, Indonesia Introduction In fish processing industry, demersal fish like grouper is usually processed into fish fillet which produces high amount of by-product such as head, scales, bones, frames, skin, fins, and viscera. This by-product may account up to 45-65% of the total weight of the fish. Fish viscera is one of the fish processing by-product that is still unutilized or underutilized despite of its high protein content (Mardina et al., 2018). The fish viscera contains high level of protein (Atma, 2016), reaching up to 31.20% of protein on a dry basis (Aditya, 2018), and making it a highly potential raw material for the production of certain or specified fish produc t such as fish protein hydrolyzate. Fish protein is highly digestible and has an excellent essential OPEN ACCESS International Journal of Applied Biology Keyword By-product; degree of hydrolysis; fish viscera; protein hydrolyzate; proteolytic enzyme Article History Received November 19, 2022 Accepted December 14, 2022 I nternational Journal of A pplied Biology is lic ensed under a C reative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. Abstract Grouper is a fishery commodity that has high demand for both domestic and export markets. For export market, the grouper is commonly sold in a fillet form. However, production of the grouper fillet generates substantial amount of by-products, such as skin, scales, bones and viscera. The viscera contains high amount of protein and can be converted into protein hydrolyzate. The enzymatic processing of the protein hydrolyzate requires proteolytic enzymes, such as bromelain. This study aimed to analyze the effect of concentration of the bromelain crude extract and hydrolysis time on yield and degree of hydrolysis of grouper viscera protein hydrolyzate. The experimental design used was a completely randomized factorial design with different enzyme concentrations (4, 6, 8, 10 and 12%) and different length of hydrolysis time (4, 6, 8 and 10 h). The results showed that the concentration of the bromelain enzyme with the length of time in the waterbath had an effect on the yield and degree of hydrolysis of the grouper viscera (P<0.05). The highest yield was 18.17%, while the highest degree of hydrolysis was 89.29%, indicating the potential use of the grouper viscera and the crude extract of bromelain in the production of the fish protein hydrolyzate. International Journal of Applied Biology, 6(2), 2022 223 amino acid (EAA) profile that suits the needs of the human diet as defined by the World Health Organization (WHO) (Egerton et al., 2017). Fish protein hydrolyzate can be produced using chemical or enzymatic hydrolysis. For the enzymatic hydrolysis, a proteolytic enzyme is into the fish or fish parts raw materials to accelerate the hydrolysis process under controlled conditions with the end result being a mixture of protein components (Piggot and Tucker 1990). Proteolytic enzymes that are often used in the food industry include bromelain and papain. Of the two enzymes, the papain is more commonly used in the production of the protein hydrolyzates. However, previous research has shown that the papain enzyme leaves a bitter taste (Somanjaya, 2013). So the bromelain enzyme is used which is a protease that has considerable potential for application in the food industry. The bromelain is relatively inexpensive (Wijayanti et al., 2016), but it causes no bitter taste to the protein hydrolyzate. Bromelain can be obtained from the pineapple plant (Ananas comosus) including the stalks, skin, leaves, fruit and stems in varying amounts (Masniar et al., 2016). Research on fish protein hydrolyzate from grouper viscera with bromelain enzymes has not been carried out. This research utilized the grouper viscera for the production of the fish protein hydrolyzate and examined the effects of the concentration of the bromelain crude extract on the yield and degree of hydrolysis of the grouper viscera protein. It is hoped that this research may offer help to produce an added value product for the grouper processing industry. Materials and Methods This research was carried out from July to November 2022 at the Animal Microbiology Laboratory, Faculty of Veterinary Science, Hasanuddin University. Materials used in this study were the viscera of grouper, pineapple, K2SO4, HgO, H2SO4, H3BO3, HCl, CH3COOH, NaOH, aquadest. The equipment used included commercial blender, calico cloth, measuring cup, erlenmeyer, hotplate with temperature control, centrifuge and oven, a set of laboratory equipment for chemical analysis of fish hydrolyzate such as Kjeldahl flasks, digestion and distillation units, soxhlet, oven, desiccator, porcelain cup, ashing furnace, filter paper, pH meter, stirrer. Methods This research used an experimental method, employing the experimental design used was a completely randomized factorial design. The factors used were concentration of the bromelain (crude extract of the pineapple) and hydrolysis time. The bromelain concentration consisted of five levels (4, 6, 8, 10, and 12% of the homogenized viscera weight) while hydrolysis time consisted of three levels (4, 6, 8, and 10 h). All experimental treatments were carried out in three replicates. A certain amount of pre-homogenized grouper viscera was thoroughly mixed with the crude extract of bromelain at a proposed concentration. The mixture was the hydrolyzed in a waterbath at 55°C for a period of time according to the proposed hydrolysis time. At the end of the hydrolysis, the hydrolyzate was recovered by filtration, enzyme-inactivated at 90°C, centrifuged at 3500 rpm, and then the solid hydrolyzate was dried in an oven. After drying, the yield of the protein hydrolyzate was calculated and the degree of hydrolysis was measured. International Journal of Applied Biology, 6(2), 2022 224 Preparation of Pineapple Crude Extract The pineapple fruit used was a ripe one, peeled, the peeled fruit was splitted and chopped into small pieces and then homogenized using a commercial blender. To recover the crude extract of the bromelain, the homogenized pineapple was then squeezed and filtered to separate suspended fine particles. The filtrate was then transferred into a tight-capped plastic bottle until used for the experiment. Production of Fish Protein Hydrolyzate An approximately 250 g of the pre-homogenized grouper viscera were transferred into a 500 ml glass beaker, added with either 4, 6, 8, 10, or 12% of the bromelain extract and then mixed thoroughly. The beakers containing experimental samples were arranged properly in a waterbath, and then the hydrolysis process was carried out at 55°C for either 4, 6, 8, or 10 hours. The pH of the samples was maintained at 7 during the hydrolysis process, using either CH3COOH or NaOH solutions. At the end of each of the proposed hydrolysis time, each sample was filtered through a 160 mesh filter cloth then inactivated the enzyme at 90°C for 20 minutes, and centrifuged at 3500 rpm for 15 minutes. The solid (protein hydrolyzate) was recovered, spresded evenly in a drying pan and dried in an oven at a temperature of 65°C for 24 hours. The dried protein hydrolyzate was then finely grinded to obtain the protein hydrolyzate flour. The parameters tested in this study were the yield and degree of hydrolysis. a. Yield The yield of the protein hydrolyzate was calculated by dividing the weight of the hydrolyzate powder with the initial sample weight, and multiplied by 100%. 𝑌𝑖𝑒𝑙𝑑 (%) = ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑧𝑎𝑡𝑒 𝑝𝑜𝑤𝑑𝑒𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔) 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔) b. Hydrolysis Degree The degree of hydrolysis was analyzed following Amiza et al. (2012) and Nurilmala et al. (2018). As much as 20 mL of protein hydrolyzate was thoroughly dissolved in 20 mL of 20% TCA (v/v). The mixture was then allowed to stand for 30 minutes for pre cipitation to occur, then centrifuged (7,800 x g) for 15 minutes. The supernatant was analyzed for nitrogen content using the Kjeldahl method (AOAC 2005). The degree of hydrolysis is calculated using the following formula (Hoyle and Merritt, 1994). 𝐷𝑒𝑔𝑟𝑒𝑒 𝑜𝑓 ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑏𝑙𝑒 𝑛𝑖𝑡𝑟𝑜𝑔𝑒𝑛 𝑖𝑛 20% 𝑇𝐶𝐴 𝑇𝑜𝑡𝑎𝑙 𝑛𝑖𝑡𝑟𝑜𝑔𝑒𝑛 𝑖𝑛 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑋 100 Data Analysis The experimental design used in this study was a completely randomized factorial design with different concentrations of the bromelain enzyme (4, 6, 8,10 and 12%) and different lengths of hydrolysis (4, 6, 8 and 10 hours). The parameters measured in this study were the yield and degree of hydrolysis. Data were analyzed using analysis of variance International Journal of Applied Biology, 6(2), 2022 225 (ANOVA) and if the treatment showed a significant effect, a Tukey test was performed. Significant difference was determined at 95% level of probability (α = 0.05). Results and Discussion Yield The yield of the protein hydrolyzate of the grouper viscera in this study ranged from 8.00 to 18.17%, where the lowest yield was at 4% bromelain enzyme concentration with 4 hours lengths of hydrolysis time, whereas the highest yield was obtained at 10% bromelain enzyme concentration with 8 hours lengths of hydrolysis time (Table 1). Table 1. The yield of the grouper viscera protein hydrolyzate produced at various concentration of bromelain and length of hydrolysis time. Bromelain enzyme concentration (%) Yield (%) 4 Hours 6 Hours 8 Hours 10 Hours 4% 8.00 ± 0.24ay 15.22 ± 0.82abw 11.39 ± 0.50ax 9.97 ± 0.50az 6% 11.93 ± 0.54bz 14.86 ± 0.01ay 15.86 ± 012bx 11.40 ± 0.47bz 8% 14.80 ± 1.27cy 16.60 ± 1.33bcy 14.78 ± 0.09cy 13.59 ± 0.22cz 10% 14.92 ± 0.34cy 15.29 ± 0.14by 18.17 ± 0.54dx 10.68 ± 0.23az 12% 14.16 ± 0.78cy 17.97 ± 0.18cx 14.19 ± 0.19ey 10.76 ± 0.40abz Val ues fol l owed by the s ame l etter i n the s ame row (x,y,z..) or i n the s ame col umn (a,b…) i ndi cati ng no di fference at 95% confi dence l evel (p>0.05) The results of a two-way Anova showed that the concentration of bromelain enzyme and hydrolysis time had a significant (p<0.01) effect on the yield. Based on the hydrolysis time, Tuckey test showed that at 4 hours hydrolysis time, the yield of the 8, 10, and 12% bromelain concentration was similar (p>0.05) but was significantly higher (p<0.05) as compared those of the 4 and 6% bromelain concentrations. At 6 hours hydrolysis time, the 12% bromelain concentration produced a significantly higher yield than those of other concentration, except with the 8% bromelain concentration. However, significant difference (p<0.05) of the yield existed between all the concentration treatments for the 8 hours hydrolysis time, being highest at 10% and lowest at 4% bromelain concentrations. For the 10 hours hydrolysis time, only the concentration of 8% bromelain produced a significantly higher yield (p<0.05), while the other concentration were relatively similar in their yields (p>0.05). For any concentration tested, the highest yield produced was obta in at either 6 or 8 hours of hydrolysis time. The highest yield of the combination of 10% bromelain enzyme concentration and 8 hours lengths of hydrolysis time may indicate the optimum combination for the production of the protein hydrolyzate from the grouper viscera. Wijayanti et al (2016) showed that the increase in the yield protein hydrolyzate of the milkfish meat was primarily due to the increase in the concentration of the bromelain enzyme. Similarly, Harahap (2022) reported that the increase in the yield was due to the contribution and activity of the bromelain enzyme in the process of protein hydrolysis. The yield of the protein hydrolyzate of using the bromelain enzyme has been reported by several authors, such as Wijayanti et al (2016) for International Journal of Applied Biology, 6(2), 2022 226 milkfish meat (11.41%); Purbasari (2008) for golden clams (17%); Amelia et al (2021) for the viscera of bombay duck fish (12.55%), and Widadi (2011) using papain enzyme for African catfish (25%). The yield in this study was much higher compared to the results of Anissa et al (2017) for tilapia (5.64%), for milkfish (2.73%) and for sharks (2.83%), and Kamini et al. (2016) for Siamese catfish viscera (2.77%). However, it may worth to note that these researchers used different concentrations of enzymes and hydrolysis times. Degree of Hydrolysis The degree of hydrolysis of the grouper viscera in this study ranged from 55.18 to 89.29%, being lowest at 8% bromelain concentration with 10 hours hydrolysis time and highest at 10% bromelain concentration with 8 hours hydrolysis time (Table 2). Table 2. The degree of hydrolysis of the grouper viscera at different concentration of bromelain and length of hydrolysis time Bromelain enzyme concentration (%) Hydrolysis Degree (%) 4 Hours 6 Hours 8 Hours 10 Hours 4% 70.19 ± 4.50ay 80.50 ± 5.75ax 80.72 ± 6.84ax 84.39 ± 1.98acx 6% 63.56 ± 4.03abz 83.80 ± 3.87ay 87.18 ± 2.13abx 79.76 ± 3.57ay 8% 67.73 ± 3.55aby 79.18 ± 3.00ax 83.74 ± 4.19ax 55.18 ± 9.54bz 10% 58.08 ± 9.38bz 74.03 ± 7.48ay 89.29 ± 1.12bx 77.31 ± 5.25ay 12% 67.23 ± 7.65abz 64.39 ± 2.69by 88.02 ± 4.26abx 87.44 ± 2.02cx Val ues fol l owed by the s ame l etter i n the s ame row (x,y,z..) or i n the s ame col umn (a,b…) i ndi cating no di fference at 95% confi dence l evel (p>0.05) The results of a two-way Anova showed that the concentration of the bromelain and length of hydrolysis time significant affected (p<0.01) the degree of protein hydrolysis of the grouper viscera. The Tuckey test showed that some significant differences (p<0.05) existed among the bromelain concentration as well as the hydrolysis time treatments regarding the degree of protein hydrolysis of the grouper viscera. In line with the yield, the highest degree of hydrolysis was also observed for the 10% of the bromelain at 8 hours hydrolysis time . Also, the 8 hours hydrolysis time tended to produce higher degree of hydrolysis at any concentration of the bromelain enzyme. The highest degree of protein hydrolysis of the grouper viscera at 10% bromelain with 8 hours hydrolysis time may indicate the optimum conditions for the hydrolysis process. When considering the whole concentration of the bromelain used, the hydrolysis time of 8 hours seems to be the best to obtain a maximum degree of hydrolysis the high degree of hydrolysis in this study may confirm Harahap (2022) that the bromelain enzyme is more active in hydrolyzing protein in animals. The high degree of hydrolysis in this study indicated that the protein hydrolysis process of the protein took place efficiently. The degree of hydrolysis obtained in this study was far greater than that of Fakhrija (2021) for black sea cucumbers (10.11%), and Harahap (2022) for eel (6.46%). International Journal of Applied Biology, 6(2), 2022 227 The availability of the enzyme in sufficient quantity can increase the rate of the hydrolysis. According to Hasnaliza et al. (2010), enzyme concentration greatly determines the degree of protein hydrolysis. However, too much of the enzyme does not help in maximizing not only the yield but also the degree of the protein hydrolysis as shown by this study. The concentration of the enzyme required is proportional to the number of peptide bonds that need to be hydrolyzed. The increase in protein content requires an increasing concentration of the added enzyme, but to a certain extent the addition of excess enzyme will produce a constant or even decrease amount of hydrolyzate due to the short of substrate to act on. Where the substrate is abundant, the higher the concentration of the enzyme used the greater the amount of amino acid protein hydrolyzate produced as reported by Wij aya and Yunianta (2015). Conclusions The yield and degree of hydrolysis of the grouper viscera protein hydrolyzate were affected by the concentration the bromelain enzyme and the length of the hydrolysis time. The best condition for producing the protein hydrolyzate using viscera of the grouper fish is the bromelain concentrations of 10% with a length of hydrolysis time of 8 hours. International Journal of Applied Biology, 6(2), 2022 228 References Aditya, R.P. 2018. Aktivitas antioksidan dan antibakteri hidrolisat protein hasil fermentasi telur ikan cakalang [tesis]. 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