JOURNAL OF INTERNATIONAL ACADEMIC RESEARCH FOR MULTIDISCIPLINARY Impact Factor 1.625, ISSN: , Volume 2, Issue 10, November 2014

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1 ISOLATION AND CHARACTERIZATION OF ENZYMES PRODUCING MICROBIAL FLORA FROM GASTRO INTESTINAL TRACT OF FRESHWATER FISH CIRRHINUS MRIGALA IN RESPONSE TO DIFFERENT DIETS DR SUDESH RANI* *Dept. of Zoology, Maharshi Dayanand University, Rohtak, Haryana, India ABSTRACT The present study was undertaken to isolate and characterize the enzymes producing microbial flora from gastro intestinal tract of freshwater Cirrhinus mrigala (mrigal) in response to eight different diets having 40% protein (1 4 raw soybean based and 5 8 processed full-fat soybean based). Fishmeal based diet was taken as control diet. Proteolytic amylolytic, cellulolytic and lipolytic microfloras were identified from the culture plate using selective media. The isolates were qualitatively screened on the basis of their extracellular enzyme producing ability. The selected strains were further quantitatively assayed for protease, amylase, cellulase and lipase activities. All activities were exhibited by almost all the bacterial isolates, while strain isolated from fish feed on diet-5 (contain 25% processed soybean meal and 75% fish meal as protein source), showed considerable proteolytic, amylolytic and cellulolytic activities. Further, strain isolated from fish feed on fishmeal based reference diet showed high lipolytic activity. The study indicates that there is a distinct microbial source of the digestive enzymes protease, amylase, cellulase and lipase apart from endogenous sources in fish gut. KEYWORDS: Fishmeal, raw soybean, processed full-fat soybean, microflora, mrigal, gastro- intestinal tract INTRODUCTION Fish get bacteria in the digestive tract from aquatic environment through water and food which are populated with bacteria. Being rich in nutrient, the environment of the digestive tract of fish confers a favorable culture environment for the microorganisms. There are only a few probiotics in the gut as it is highly acidic. The number of these beneficial bacteria increases dramatically in the intestine (Fukami et al 2002). Enzymes are produced by a variety of microorganisms and played an important role during fish sauce fermentation. Several protease-producing bacteria found in fish sauce fermentation, including halophilics, halotolerants and lactic acid bacteria. These bacteria hydrolyze fish protein to peptides and amino acids (Lopetcharat et al 2001). Protease-producing bacteria found in fish sauce are 411

2 Pseudomonas sp. (Vermelho et al 1996), Bacillus sp. Micrococcus sp. Staphylococcus sp. Streptococcus sp. Pediococcus sp. Coryneforms (Thongthai and Suntinanalert, 1991, Noguchi et al 2004), Halobacillus thailandensis sp. (Chaiyanan et al 1999) Tetragenococcus halophililus and T. muriaticus (Trust and Sparrow 1974). A few reports concerning microbial cellulase (Stickney and Shumway 1974; Prejs and Blaszcyk 1977; Lindsay and Harris 1980; Lesel and Lesel, 1986; Das and Tripathi 1991; Saha and Ray 1998) amylase and lipase production in the gastrointestinal tract of fish are however, available. For different experimental purposes, the microbial gut flora of fish has been studied by several workers. This includes relation between environment and fish micro flora (Horsely 1973), microbial flora as food of fish (Kamjunke et al. 2002) and microbial flora help in production of enzymes (Bairagi et al. 2002). For all these reasons, study of bacterial flora of the gut is important. In this study we have analyzed the enzyme producing microflora in the gastrointestinal tract of freshwater culturable teleosts, Cirrhinus mrigala. MATERIALS AND METHODS Diet Formulation: Eight diets were formulated for different levels of raw and processed soybean replacing by fishmeal. Soybean was hydrothermically processed (for 15 min at 121 C at 15 lb) before incorporating in diet after then diets (1-4 raw soybean based, 5-8 processed soybean based) with 40% protein level were formulated by blending raw and processed soybean at four different inclusion levels viz. 65, 130, 195 and 260g kg 1 with fish meal. A diet with fish meal as the protein source was used as reference diet. A mechanical pelletizer was used and 0.5mm thicker pellets were obtained which then dried in oven (60-62 C) before using in feeding trials. Diet Formulation was done by following the procedure of Jana et al. (2012). Fish examined: Freshwater teleost, Cirrhinus mrigala provide eight diets were taken for the present study. Fishes fed on Fishmeal based diet was taken as control group. Different diets and average weight of the fishes studied are presented in Table 1. After 90 days the fishes were sampled and were starved for 24 h in order to clear their alimentary tracts before being dissected. 412

3 Post-mortem examination: The ventral surface of the fish was thoroughly scrubbed with 1% iodine solution (Trust and Sparrow 1974) immediately after being pithed. The fish were dissected on ice and their alimentary tracts were removed and cleaned with sterile physiological saline solution. The alimentary tracts were homogenized with 10 parts of chilled 0.89% sodium chloride solution (Das and Tripathi 1991). Microbial culture: Microbial culture of the intestinal mucosa collected from the test fish was carried out for bacterial isolation. For this purpose, the homogenate of the intestinal mucosa of each of the test fish was used after five serial 1:10 dilutions (Beveridge et al. 1991). Samples (0.1 ml) were taken from each dilution and poured aseptically within a laminar flow on sterilized Tryptone Soya Agar (TSA) plates, in duplicate. These culture plates were incubated at 34 C for 24 h. They were then examined for the development of bacterial colonies. The wellseparated colonies with apparently different morphological appearance were streaked separately on TSA plates to obtain pure cultures. Single, isolated colonies from the streaked plates were transferred to TSA slants. To isolate and enumerate protease, amylase, cellulase and lipase producing bacteria, diluted gut homogenate was poured on peptone-gelatin-agar, starch-agar, carboxymethylcellulose( CMC)-agar, and tributyrin-agar media containing plates, respectively. These culture plates were incubated at 34 C for 24 h. It was assumed that the microflora, which had formed colonies on the peptone-gelatin-agar, had proteolytic activity. Similarly, it was assumed that the microflora grown on starch plate, CMC plate and tributyrin plate had amylolytic cellulolytic and lipolytic activities respectively. By multiplying the number of colonies formed on each plate by the reciprocal of dilution, colony numbers per unit sample volume of gut homogenate were determined (Rahmatullah and Beveridge 1993). Screening of isolates for extra-cellular qualitative enzyme production: Isolates were screened for the production of extra-cellular protease, amylase, cellulase and lipase. For extra-cellular protease production, the isolates were streaked on peptone-gelatin enriched nutrient agar (4% gelatin) plates and incubated at 32 C for 15 h. The appearance of a clear zone around the colony after flooding the plate with 15% HgCl2 indicated the presence of proteolytic activity (Jacob and Gerstein 1960). For screening of amylase 413

4 producing strains, isolates were streaked on starch (1%) supplemented nutrient agar plates and incubated at 32 C for 48 h. The culture plates were then flooded with 1% Lugol s iodine solution (Jacob and Gerstein 1960) to identify amylase activity. For screening of cellulase producers, isolates were grown in carboxymethlycellulose (1%) nutrient broth and incubated at 32 C for 72 h. The amount of reducing sugar produced per microgram protein in the culture broth was determined using dinitrosalisylic acid reagent (Sadasivam and Manickam 1996).Lipase producers showed a clear zone surrounding their colony in tributyrin plates (1%) (Sangiliyandi and Gunasekeran 1996). Quantitative enzyme assay: Liquid media were used for quantitative assay of enzyme production from different strains. For protease, amylase and cellulase, selective media (without agar) were used. For lipase production the medium contained (mg/ml): Olive oil, 1% (v/v); Sucrose, 5; MnSO4.H2O, 0.01; ZnSO4.7H2O, 0.01; Na2HPO4, 3; KH2PO4, 1; KCl, 0.5; MgSO4.7H2O, 0.5; NaNO3, 5. The culture flasks were incubated for 48 h at 32 C. The contents were centrifuged (10,000 g, 10 min, 4 C) and the cell-free supernatant was used for enzyme assay. The protein content of the enzyme extract was estimated spectrophotometrically at 660 nm according to Lowry et al. (1951). Protease assay: Protease activity was detected by caseinase assay method (Walter 1984). Amylase assay: Amylase was assayed by the dinitrosalicylic acid method based on the estimation of reducing sugars at 560 nm using maltose as the standard (Bernfield 1955). Cellulase assay: The production of reducing sugars due to cellulolytic activity was measured following dinitrosalicylic acid method at 540 nm using glucose as the standard (Sadasivam and Manickam 1996). Lipase assay: Lipase activity was detected by the method of Colowick and Kaplan (1955). Emulsion of olive oil and 2% polyvinyl alcohol solution was used as the substrate. The liberated free fatty acids in the enzyme-substrate complex were titrated with 0.02 N NaOH. RESULTS AND DISCUSSION The aerobic bacterial population in the gastrointestinal tract of fish mrigal fed different diets on TSA plate are presented in (Table-2). Analyses show that the aerobic bacterial population on TSA plate is maximum in Cirrhinus mrigala fed on diet-5 (1.1x10 6 ) followed by fish fed fishmeal based reference diet ( bacterial cells/g digestive tract) and minimum in C. 414

5 mrigala, fed on raw soybean based diets ( bacterial cells/g digestive tract). When enumeration of specific enzyme producing bacterial flora was done and it was observed that proteolytic bacterial flora present in all the fish studied and the maximum count was observed in the gut of mrigal fish fed on diet-5 th (5.2x10 5 ) followed by fish fed on fishmeal based reference diet (4.8x10 5 ). Densities of amylolytic bacterial flora were observed high in mrigal fed on processed soybean based diets 5 th and 6 th. The cellulolytic population exhibited maximum densities in mrigal fish fed on processed soybean based diets. Lipolytic bacterial flora were detected in all the fish studied and the maximum population density was however recorded in fish fed on reference diet ( 10 3 bacterial cells/g digestive tract). The intensity of extracellular enzyme production by the bacterial strains isolated from the mrigal fed different diets was assayed qualitatively (Table-3). Maximum protease activity was observed in PSV-1 and PSV-2 strains isolated from mrigal fed on processed soybean based diets (Table-4). Peak specific amylase and cellulase activities were exhibited by bacterial strains PSV-1, PSVI-1, PSVIII-2 and PSV-2 respectively (Table-5&6). Specific lipase activity was found to be maximum in FM-1, a strain isolated from fish fed fishmeal based reference diet (Table-7). In the present investigation, the presence of a considerable population of bacterial flora has been found in the gastrointestinal tracts of the Cirrhinus mrigala and certain strains exhibit proteolytic, amylolytic, cellulolytic and lipolytic activities. Proteolytic bacteria were detected in the gut of all the fish examined in our study. However, assay of extracellular protease activity of the bacterial isolates showed highest value in PSV- 2, a strain isolated from mrigal fed on diet-5. The occurrence of proteolytic bacteria in the gut of fish seems to support the presence of a diet dependent microbial population in fish. The results of the present study indicate that the microorganisms isolated from the fish digestive tract are capable of producing proteolytic, amylolytic, cellulolytic and lipolytic enzymes. Kawai and Ikeda (1972) and Shcherbina et al. (1976) reported adaptive changes in the activity of proteolytic enzymes in common carp (Cyprinus carpio) in relation to the type of diet. Das and Tripathi (1991) observed optimum protease activity between ph 7.6 and 8.4 inboth the fingerling and adult grass carp, and among fingerlings the activity increased proportionately with higher amount of protein in diet up to a limit. Das and Tripathi (1991) reported high amylase activity in the gastrointestinal tract of grass carp, which appeared to be the result of its omnivorous feeding habit. Sarbahi (1951), Dhage (1968) and Phillips (1969) suggested that amylase activity in the intestine of herbivorous carp 415

6 is much more intense than in carnivorous fishes, this is in agreement with our study in which we observed very low amylase activity in mrigal fish fed on fishmeal based diet. Amylase is secreted by the entire intestine in the Indian major carps, Catla catla, Labeo rohita and Cirrhinus mrigala, and its activity is high toward the proximal end (Dhage 1968). Though reports on microbial amylase activity in fish gut are scanty, endogenous amylase activity in fish is evident. However, the amylolytic bacteria have been detected in fish guts after24 h of starvation in our study, and it seems that some of the flora forms a persistent population. Reports on existence of cellulase activity in the digestive system of fish are also scant, and moreover, conflicting, with contradictory results. Microbial intestinal cellulase activity was observed by Das and Tripathi (1991) in grass carp and Saha and Ray (1998) in rohu fingerlings. In the present investigation presence of a considerable population of cellulolytic bacteria and their active role in extracellular cellulase production in the C. mrigala fed on processed soybean based diets has been confirmed. Stickney and Shumway (1974) earlier concluded that cellulase activity within fish has resulted from a stable microflora maintained within the digestive tract, irrespective of feeding habit. Our observation is in agreement with this. The results of the present investigation also suggest a possible positive correlation between the food habits and intestinal microbial cellulase activity. Cellulolytic microbial flora was not detected in the gastrointestinal tract of the C. mrigala fed on fishmeal based diets showed carnivorous feeding of fish. All the information available about fish intestinal lipase is about its endogenous source. In the present context, microbial lipolytic activity was studied in the gut of the selected C. mrigala fish and maximum lipase activity was shown by a strain named FM-2, isolated from fish fed on fishmeal based diets. Carp normally ingest lipid in their diet and this is known to be a source of energy, essential fatty acids and lipid-soluble vitamins. Al-Hussaini (1949) observed the occurrence of lipase in cyprinids and the activity is more concentrated in the anterior intestine than in the posterior intestine. Lipase activity was concentrated in the anterior one-fifth of the intestine in Cirrhinus mrigala and Labeo rohita but was found to be totally absent in the entire intestine of Catla catla (Dhage 1968). In commercial aquaculture beneficial bacteria could be introduced by incorporating them into artificial fish diets. Most of the experiments conducted in utilizing probiotics in aquaculture is mainly for alternative methods of disease control. In animal husbandry, probiotics are used to increase nutrient utilization and growth rate. Such experiments are not very common in aquaculture. The enzyme producing microorganisms isolated from the fish digestive tracts in 416

7 response to different diets in the present study can be beneficially used as a probiotic while formulating the diet for fish, especially in the larval stages. However, much more research should be conducted to determine if the addition of such isolates to fish feeds do, in fact, provide some kind of benefit to the fish involved before advocating their use. Conclusion Present study indicates that there is a distinct microbial source of digestive enzymes (protease amylase, cellulase and lipase) apart from the endogenous sources in fish gastrointestinal tracts. References 1. Al-Hussaini A.H. (1949). On the functional morphology of the alimentary tract of some fish in relation to differences in their feeding habits: cytology and morphology. Quartarly Journal of Microscopical Science 90: Bairagi A., K.S. Ghosh, S.K. Sen, A.K. Ray (2002). Enzyme producing bacterial flora isolated from fish digestive tracts. Aquacul. Int. 10: Bernfield P. (1955). In: Colowick S.P. and Kaplan N.O (eds), Methods of Enzymology. Vol. I. Academic Press, New York, p Beveridge M.C.M., P.K. Sikdar, Frerichs G.N. and S. Millar (1991). The ingestion of bacteria in suspension by the common carp Cyprinus carpio L. Journal of Fish Biology 39: Chaiyanan S., S. Chaiyanan, T. Maugel, A. Huq, F.T. Robb, R.R. Colwell (1999). Polyphasic taxonomy of a novel Halobacillus, Halobacillus Thailandensis sp. nov. Isolated from fish Sauce. J. Appl. Microbiol. 22: Colowick S.P. and N.O. Kaplan (1955). In: Colowick S.P. and Kaplan N.O (eds), Methods of Enzymology. Vol. 1. Academic Press, New York, p Das K.M. and S.D. Tripathi (1991). Studies on the digestive enzymes of grass carp, Ctenopharyngodon idella (Val.). Aquaculture 92: Dhage K.P. (1968). Studies of the digestive enzymes in the three species of the major carps of India. Journal of Biological Sciences 11: Fukami K., S. Ishiyama, H. Yaguramaki, T. Masuzawa, Y. Nabeta, K. Endo, M. Shimoda, (2002). Identification of distinctive volatile compounds in fish sauce. J. Agric. Food Chem. 50(19): Horsley R.W. (1973). A review of the bacterial flora of teleosts and elasmobranchs, including methods for its analysis. J. Fish Biology 10: Jacob M.B. and M.J. Gerstein (1960). Handbook of microbiology. D Van Nostrand Co. Inc. Princeton, New Jersey. 12. Jana S. N., Sudesh, S. K. Garg, V. P. Sabhlok and A. Bhatnagar (2012). Nutritive Evaluation of Lysine- and Methionine-Supplemented Raw Vs Heat-Processed Soybean to Replace Fishmealas a Dietary Protein Source for Grey Mullet, Mugil cephalus, and Milkfish, Chanos chanos. Journal of Applied Aquaculture. 24:1, Kamjunke, N., R. Mendonca, I. Hardewing and T. Mehner (2002). Assimilation of different cyanobacteria as food and the consequences for internal energy stores of juvenile roach. J. Fish Biology 60(3): Kawai S. and S. Ikeda (1972). Studies on digestive enzymes of fishes. II. Effect of dietary change on the activities of digestive enzymes in carp intestine. Bulletin of the Japanese Society for Scientific Fisheries 38: Lesel R., C. Fromageot and M. Lesel (1986). Cellulose digestibility in grass carp, Ctenopharyngodon idella and in goldfish, Carassius auratus. Aquaculture 54: Lindsay G.J.H. and J.E. Harris (1980). Carboxymethylcellulase activity in the digestive tracts of fish. Journal of fish Biology 16: Lopetcharat K., Y.J. Choi, J. W. Park, M. A. Daeschel (2001). Fish sauce products and manufacturing: review. Food Rev. Int. 17(1):

8 18. Lowry O.H., N.J. Rosebrough A.L. Farr and R.J. Randall (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193: Noguchi H., M. Uchino, O. Shida, K. Takano, L.K. Nakamura, and K. Komagata (2004). Bacillus vietnamensis sp. a moderately halotolerant, aerobic, endospore-forming bacterium isolated from Vietnamese fish sauce. Int. J. Syst. Evol. Microbiol. 54: Phillips A.M. (1969). Nutrition, digestion and energy utilization. In: Hoar W.S. and Randall D.J. (eds), Fish Physiology. Vol. 1. Academic Press, New York, pp Prejs A. and M. Blaszcyk (1977). Relationships between food and cellulase activity in freshwater fishes. Journal of Fish Biology 11: Rahmatullah S.M. and M.C.M Beveridge (1993). Ingestion of bacteria in suspension by Indian major carps (Catla catla, Labeo rohita) and Chinese carps (Hypophthalmichthys molitrix, Aristichthys nobilis). Hydrobiologia 264: Sadasivam S. and A. Manickam (1996). Biochemical Methods. New Age International (P) Ltd. Publishers, pp Saha A.K. and A.K. Ray (1998). Cellulase activity in rohu fingerlings. Aquaculture International 6: Sangiliyandi G. and P. Gunasekeran (1996). Extracellular lipase producing Bacillus licheniformis from an oil mill refinery effluent. Indian Journal of Microbiology 36: Sarbahi D.S. (1951). Studies on the digestive enzymes of goldfish Carassius auratus (Linn.) and large mouth black bass, Micropterus salmoides (Lacepede). Biological Bulletin 100: Shcherbina M.A., L.N. Trofimova and O.P. Kazlaskene (1976). The activity of protease and the intensity of protein absorption with the introduction of different quantities of fat into the food of the carp Cyprinus carpio. Journal of Ichthyology 16: Stickney R.R. and S.E. Shumway (1974). Occurrence of cellulase activity in the stomachs of fish. Journal of Fish Biology 6: Thongthai C., and P. Suntinanalert (1991). Halophiles in thai fish sauce (Nam Pla). Rodriguez-Valera, F. (ed.). General and applied aspects of halophilic microorganisms. pp New York : Plenum Press. 30. Trust T.J. and R.A.H. Sparrow (1974). The bacterial flora in the alimentary tract of freshwater salmonid fishes. Canadian Journal of Microbiology 20: Vermelho A.B., M.N.L. Meirelles, A. Lopes, S.D.G. Petinate, A.A. Chaia and Branquinha. (1996). Detection of extracellular proteases from microorganisms on agar plates. Mem. Inst. Oswaldo. Cruz. 91(6): Walter H.E. (1984). Methods of Enzymatic Analysis. Verlag Chemie, Weinheim, p Table 1. Average weight of the fishes examined Fish species Diets given Average weight (g) Cirrihinus mrigala Reference diet 2.83 Diet Diet Diet 3 7 Diet Diet Diet Diet Diet

9 Table 2. Bacterial strains isolated from fish gut and qualitative extracellular enzyme activity Fish species Cirrhinus mrigala Diets given Bacterial strains Enzyme Activity Protease Amylase Cellulase Lipase Reference diet Diet I Diet II Diet III Diet IV Diet V Diet VI Diet VII Diet VIII FM1 FM2 RSI-1 RSI-2 RSII-1 RSII-2 RSIII-1 RSIII-2 RSIV-1 RSIV-2 PSV-1 PSV-2 PSVI-1 PSVI-2 PSVII-1 PSVII-2 PSVIII-1 PSVIII-2, high,, moderate;, low;, nil. FM- Fishmeal based, RS-Raw soybean based, PS- Processed soybean based Table 3. Aerobic bacterial count in fish digestive tract Fish species Cirrhinus mrigala Diets given Bacterial population per g digestive tract Bacterial Proteolytic Amylolytic Cellulolytic count ( 10 5 ) ( 10 3 ) ( 10 3 ) in TSA plate ( 10 6 ) Reference diet Diet Diet Diet Diet Diet Diet Diet Diet Values in same vertical column are not significantly different (p < 0.05). Lipolytic ( 10 3 ) 419

10 Table 4. Profile of protease activity in the selected strains Bacterial strains Protease (U) a Protein (mg/ml) b Specific enzyme activity c FM1 FM RSI-1 RSI RSII-1 RSII RSIII-1 RSIII RSIV-1 RSIV PSV-1 PSV PSVI-1 PSVI PSVII PSVII PSVIII PSVIII a µg tyrosine liberated per ml of culture filtrate/min b culture filtrate c U/mg protein Table 5. Profile of Amylase activity in the selected strains Bacterial strains Amylase (U) a Protein (mg/ml) b Specific enzyme activity c FM1 FM RSI-1 RSI RSII-1 RSII RSIII-1 RSIII RSIV-1 RSIV PSV-1 PSV PSVI-1 PSVI PSVII PSVII PSVIII PSVIII a µg maltose liberated per ml of culture filtrate/min b culture filtrate c U/mg protein

11 Table 6. Profile of cellulase activity in the selected strains Bacterial strains Cellulase (U) a Protein (mg/ml) b Specific enzyme activity c RSI-1 RSI RSII-1 RSII RSIII-1 RSIII RSIV-1 RSIV PSV-1 PSV PSVI-1 PSVI PSVII PSVII PSVIII PSVIII a µg glucose liberated per ml of culture filtrate/min b culture filtrate c U/mg protein Table 7. Profile of lipase activity in the selected strains Bacterial strains Lipase (U) a Protein (mg/ml) b Specific enzyme activity c FM1 FM RSI-1 RSI RSII-1 RSII RSIII PSV-1 PSV PSVI-1 PSVI PSVII-1 PSVII PSVIII-1 PSVIII a micromole fatty acid liberated per min b culture filtrate c U/mg protein 421