alimentary substrate, as produced in course of digestive transit. In grass carp. Ctenopharyngodon idella, tryptic and amylolytic activities decreased sharply from ...
Advances in Fish Research, Pages 55–70 Edited by : U.C. Goswami Copyright © 2012, Narendra Publishing House
DIGESTIVE ENZYME PROFILEOF CARPS: AN OVERVIEW 1
Rina Chakrabarti and Jai Gopal Sharma 1
2 2
Aqua Research Lab, Department of Zoology, University of Delhi, Delhi-110007, India, Ministry of Earth Sciences, C.G.O. Complex, Lodi Road, New Delhi-110003, India
ABSTRACT The presence and level of activity of digestive enzymes are good indicators of the physiological status of fish. The ratio of membrane and cavity digestion actually changes in fishes with age. Development and regulation of the digestive enzymes depend on the progressive changes in the digestive tract. The present study reviews the digestive enzyme profile of carps during ontogenic development. Quality and quantity of diets play significant role in the secretion of digestive enzymes in fish. Intestinal microflora play important role in digestion.
Keywords: Carps, Digestive enzyme profile, Ontogenic development, Intestinal microflora
INTRODUCTION Digestion is the biodynamics of enzymes. Enzymes play major role in digestion of ingested food of fish. More than 60% of production cost of fish in intensive aquaculture is due to feed. Therefore, proper digestion, assimilation, absorption and utilization of food are crucial for the aquaculture industry. The ability of any fish to digest a given diet and absorption of its nutrients depends on the presence and the quality of digestive enzymes. Digestive processes in fish are less known than in mammals, although the data available in fish so far show that the digestive enzymes studied are qualitatively similar to those observed in other vertebrates. Many workers have described that the digestive enzyme activity in fish is influenced by age and/or stage of development (Kuz’mina, 1980, Lauff and Hofer, 1984, Cousin et al., 1987). The knowledge of temporal appearance of key enzymes in the gut of cultivable species is essential to understand age-specific formulation of feed that contributes to rapid and efficient growth rate. The assessment of the presence and level of activity of certain enzymes may be used as a comparative indicator of the rate of development of the larvae, as well as their further survival rate (Ueberschar, 1993). The ratio of membrane and cavity digestion actually changes in fishes with age. Development and regulation of the digestive enzymes depend on the progressive changes in the digestive tract and the subsequent response to composition and amount of the available food. This is either genetically programmed or induced by the corresponding substrate in the food (Gruzdkov et al., 1986).
India is popularly known as carp country. More than 87% of our inland aquacultural production is contributed by carps. Carps show diversity in feeding habits viz. some fishes are phytoplankton feeders, others are omnivores or zooplankton feeders; some are surface feeders, whereas others are mid column feeders or bottom feeders. Moreover, carps are passing through various developmental stages like spawn, fry, fingerling and adult. Each developmental stage is characterized by special morphological structures, which results into age-specific and species-specific feeding behaviour. Although several on-farm feeds are in use in aquaculture, commercial fish feeds manufactured on the basis of proper understanding of digestive physiology of fish are yet to be established for most of the cultured species (Seenappa and Devaraj, 1995). Selection of appropriate ingredients for the preparation of commercial diet for each developmental stage of individual species of carp is beneficial for the aquaculture industry. Digestibility of ingredients results into the bioavailability of essential nutrients to the organism. Proteins, lipids and carbohydrates are the source of energy to the fish. The nutritional value of carbohydrates varies among fish with warm water fish being able to utilize much higher levels of dietary carbohydrate than cold water and marine fish. No dietary requirement for carbohydrate has been demonstrated in fish. However, if carbohydrates are not provided in the diet, other nutrients such as protein and lipids are catabolized for energy and to provide metabolic intermediates for the synthesis of other biologically important compounds. Therefore, the estimation of enzymes involved in carbohydrates digestion is most essential for each developmental stage of individual species. As protein utilization is fundamental to growth, proteases have an important role to play in larval and adult fish. The digestive proteases of different species showed variations (Chakrabarti and Sharma, 2005), which may influence their digestive capability and feeding habits.
Constrains Associated with the Study of Enzyme Profile of Fish The difficulty in studying digestive secretions in fish involves the collection of these secretions rather than the methodology used in the analyses, since similar techniques to those developed in other vertebrates are followed. In addition, other factors cause variability in the final data: (1) there is no uniformity in the tissue used for enzymatic activity determinations; consequently, the procedure sometimes includes the homogenization of the attached glands and/or the whole digestive tract, whereas in other cases the extraction is performed by scraping the mucous layer from the digestive tract; (2) the nutritional status of the animals used in the experiments is not consistent, as the animals are sacrificed either after starvation or at different post-feeding times; and (3) in some cases, the digestive tract is washed before homogenization, whereas others have used the tract and its contents for extraction. These factors, as well as the broad variety of techniques used to determine the different enzymatic activities (different substrates, temperature of incubation, pH, etc.) make it difficult to obtain absolute values for the enzymatic activities from different species under specific physiological conditions and to compare these results to those obtained in other laboratories. The study of the digestive secretions in fish might clarify some aspects of their nutritive
physiology and, therefore, could also help to solve some nutritional problems in fish feeding (Hidalgo et al., 1999).
Distribution Pattern of Digestive Enzymes The most important part of the digestion occurs in close contact with the intestinal epithelium where the brush-border bounded enzymes realize the splitting and uptake of the main food components. The nature, distribution pattern and specific activities of these intestinal enzymes are related to the structure of the digestive tract and the nature of diets. Many of these enzymes are inducible by the presence of any new component in the diet. In common carp Cyprinus carpio, amyloglucosidase and leucine-aminopeptidase activities were found to be widespread among enterocytes from all part of the digestive tract (Fraisse, 1981). Alkaline-phosphatase and γ-glutamyl-transferase activities were predominant in the anterior and distal parts of intestine, respectively. Brush border enzymes are not completely lacking in one special part of the intestine, indicating that everywhere the enterocytes populations possesses similar digestive abilities with a trend to some regional specialization. It is interesting to note that amyloglucosidase which is found in renal tubule exhibits same immunological, electrophoretical and molecular properties as the intestinal one. The distribution pattern of digestive enzymes coincides with the availability of the concerned alimentary substrate, as produced in course of digestive transit. In grass carp Ctenopharyngodon idella, tryptic and amylolytic activities decreased sharply from fore-gut to hind-gut indicating an efficient reabsorption mechanism (Bitterlich, 1985).
Digestive Enzyme Activities Observed During Ontogenic Development Fishes experience evolutionary adaptations in the morphogenesis of their digestive system during early developmental stages due to the changing nutritional requirement and shifting from endogenous to exogenous feeding. This fact is reflected in the ontogeny of digestive enzyme patterns. The rate of development is likely to determine the survival of fish with physiological and environmental changing status. A comprehensive analysis of the ontogenic changes occurring during the early life stages of fish is essential for the design of adequate larval rearing and feeding strategies and for formulation of dry diets (Verreth and Segner, 1995). Detailed knowledge of digestive physiology during the developmental stages of such fish is essential as the maximum growth rate of fish is partly contributed by digestive capacity, oxygen availability and their metabolic capacity required to support tissue protein synthesis (Blier et al., 1997). Fish must be competent at procuring and assimilating food before yolk sac absorption (Green and McCormick, 2001). It is well documented that at the early developmental stage fishes experience a crucial metabolic phase during the shifting of feeding. Considerable attention should be given to evaluate the functional characteristics of the digestive enzymes that play important role in the ontogenesis of larvae.
Case Study The yolk sac of carp is resorbed within 3-4 days after hatching depending on water
temperature, whereas the activities of digestive enzymes are very low during this time. In grass carp, the ratio between amylase activity in hepatopancreas to its activity in mucosa increases with age, younger the fish, more significant role of amylase associated with the mucosa (Buzinova, 1973). The process of embryonic and pre-larval development is associated with enhancement of two alternatives - synthesis and catabolism of proteins, which is accompanied by increase in the activities of trypsin and chymotrypsin-like proteases in common carp Cyprinus carpio (Konovalov and Mestechkin, 1975). Kumar et al. (2000) studied the specific proteolytic enzyme activity and ultrastructure of the anterior part of the intestine of catla Catla catla larvae cultured under three different feeding schemes of live-food, refrigerated-plankton food and starvation during ontogenesis. Specific proteolytic activity of larvae increased with the age regardless of feeding conditions. There was no significant difference in enzyme activity between larvae fed live-plankton and refrigerated-plankton during the initial 9 days, but it became significantly (P < 0.001) higher in the former than the latter from day-10 onwards. Highest activity was observed in the -1 -1 26-day-old larvae of live-food treatment (0.987 ± 0.02 mg tyrosine mg protein h ). In the starvation treatment, activity was significantly lower throughout the culture period. Ultrastructure study of the digestive system also supported the results of quantitative estimation. Microvilli, microfilament bundles and secretory granules showed progressive changes with age. Age-related changes in the diversity and quantity of digestive enzymes appear to represent evolutionary adaptations to the different natural diets and nutritional requirements of distinct life-history stages. -1
-1
In common carp, amylase activity was recorded (505 ± 22 µg maltose mg protein h ) on day-4 after hatching. The enzyme activity showed a decreasing trend up to day-10. Then enhanced activity was recorded between days-12 and 18; again decreased up to day-26. Highest specific amylase activity was recorded on day-30 after hatching (Rathore et al., 2005a). Specific protease activity was minimum on day-4 after hatching and gradually increased up to day-12 and then showed a decreasing trend up to day-26. Highest protease activity was found on day-30 after hatching. Highest trypsin activity was recorded on day-28 after hatching. Comprehensive study of digestive enzyme profiles of three Indian major carps, catla Catla catla, rohu Labeo rohita and mrigal Cirrhinus mrigala during ontogenic development have documented some important facts which are relevant for feed formulation (Chakrabarti and Sharma, 1997; Rathore et al., 2005b; Chakrabarti et al., 2006a; Chakrabati and Rathore, 2009). In all these three fishes, amylase, protease, trypsin, chymotrypsin and lipase activities were detected on day-4 before first feeding. But there were significant difference in the quantity of each enzyme in individual species on day-4. Highest specific amylase activity -1 -1 was found in mrigal (0.722 ± 0.05 mg maltose mg protein h ), followed by rohu (0.59 ± -1 -1 -1 0.01 mg maltose mg protein h ) and minimum in catla (0.12 ± 0.01 mg maltose mg protein -1 h ). In mrigal and catla, highest activities were found on day-34, whereas in rohu highest activity was found on day-26 after hatching. The highest amylase activity of mrigal was significantly (P < 0.05) higher compared to rohu and catla.
Total protease activity was significantly (P < 0.05) higher in catla (286.96 ± 52 mU mg -1 -1 -1 -1 protein min ) compared to rohu (41.99 + 14.11 mU mg protein min ) and mrigal (28.61 ± -1 -1 8.90 mU mg protein min ) on day-4. But in mrigal significantly (P < 0.05) higher protease -1 -1 activity was found on day-34 (4337 ± 388 mU mg protein min ) compared to the maximum -1 -1 activity found in catla (2713 ± 147.2 mU mg protein min ) on day-32 and in rohu (1588 ± -1 -1 mU mg protein min ) on day-30. -1
Trypsin activities were 53.55 ± 4.57, 31.86 ± 1.12 and 19.37 ± 3.11 mU mg protein min in catla, mrigal and rohu, respectively before first feeding (day-4). In catla (118.1 ± -1 -1 7.09 mU mg protein min ) and rohu, highest trypsin activity was recorded on day-34, whereas in mrigal highest activity was found on day-26 after hatching. This was significantly (P < 0.05) higher in mrigal compared to the activities found in other two Indian major carps. In catla the enzyme activity showed constantly increasing trend from day-16 onwards and -1 -1 was maximum on day-34 (118.1 ± 7.09 mU mg protein min ). In rohu, trypsin activity exhibited exponential trend with the age of fish. -1 -1 In 4-day-old catla, chymotrypsin activity (57.63 ± 5.20 mU mg protein min ) was more than 3-fold higher compared to the chymotrypsin activities found in rohu and mrigal. Like trypsin, chymotrypsin activity was also significantly (P < 0.05) higher in mrigal (7140 ± 215 -1 -1 mU mg protein min ) on day-26 compared to the highest activities found in catla on day-32 -1 -1 (1789.0 ± 111.7 mU mg protein min ) and in mrigal on day-34 (1807.9 ± 124.58 mU mg -1 -1 protein min ). Among these three carps, highest lipase activity was found in rohu (183.69 ± 54.8 mUnits) compared to mrigal (61 ± 16 mUnits) and catla (28.11 ± 77 mUnits) on day-4. In catla, lipase activity increased steadily from day-22 onwards; in rohu lipase activity showed a polynomial relationship with age. In mrigal, significant increase in lipase activity was observed between days-24 and 34 after hatching. -1
Study of digestive enzyme and partial characterization of proteases of silver carp Hypophthalmichthys molitrix (male) and bighead carp Aristichthys nobilis (female) hybrid during early ontogeny was performed (Chakrabarti et al., 2006b). Specific amylase activity -1 -1 was observed in 4-day-old hybrid carp (0.07 ± 0.01 mg maltose mg protein min ). Specific amylase activity showed polynomial relationship with the age of fish. Total protease, trypsin and chymotrypsin activities were 14.37 ± 2.21, 11.38 ± 1.67 and 2.83 ± 0.50 mUnits mg -1 -1 protein min in 4-day-old fish, respectively. Total protease activity showed exponential trend, whereas trypsin and chymotrypsin activities showed polynomial relationships with the increasing age of the fish. Lipase activity was 2.33 ± 0.18 mUnits in 4-day-old hybrid carp. Lipase activity showed polynomial trend with the increasing age of fish. The enzyme activities found in 4-day-old larvae documented the presence of important digestive enzymes before the onset of exogenous feeding as the samplings were made prior to the first feeding. These results also have thrown some light on the digestion capability of these commercially important carps during early developmental stages. Higher specific amylase activity found in mrigal and rohu compared to catla and hybrid carp indicates the
advantage of incorporation of carbohydrate sources in the diets of these first two species at early stage. These results indicated the carbohydrate utilization capacity of these fishes during first feeding. This also showed that carbohydrate sources should be either minimized or totally avoided in the diets of catla and hybrid carp as these two species had lower specific amylase activities at early stage. Tanaka (1973) suggested that dietary carbohydrates might fill the energy gap between endogenous and exogenous protein demand of the fish. Silver carp, bighead carp and grass carp were reported to increase in their amylase activities from day-21 when they actively consume large phyto and zooplankton (Volkova, 1999). Cahu et al. (2004) suggested that regulation of amylase is post-transcriptional in early larval stages of sea bass Dicentrarchus labrax (till day-25) and become transcriptional towards the end of larval period. Total protease, trypsin and chymotrypsin activities were significantly (P < 0.05) higher in catla before the starting of exogenous feeding compared to the other carps. Pancreatic secretions like trypsin and chymotrypsin play a key role in digestive physiology of the carps as the stomach is absent. In all these species, enzyme activities increased during ontogenic development. It was demonstrated that the exocrine part of the pancreas just begin to accumulate secretary granules at the early developmental stages of carp, i.e. the pancreas function at a lower level, and secretion was activated after two weeks of development, whereas, the activity of alkaline proteases increased in the intestine (Ostroumova and Dement’eva, 1981). This is clear from this study that zooplanktivore catla has better capacity to digest proteins compared to omnivore rohu and detritivore mrigal at first feeding. Lipase activity was maximum in rohu at first feeding and the remaining study period compared to catla and mrigal. This also indicated that incorporation of lipid substances might be useful to this species during the time of preparation of commercial diet for early larval stage. Major lipase in fishes appeared to be non-specific and bile salt dependant (Gjellesvik et al., 1992).
EFFECTS OF FOOD Quality and quantity of diets play significant role in the secretion of digestive enzymes in fish. Common carp (7.5-30 g) were fed diets with different contents of protein (fish meal as protein source) and carbohydrate (potato starch as carbohydrate source). Maltase, amylase and protease activities of intestine showed adaptation to the dietary change within a week; higher activities were found in groups fed with 40-60% starch (Kawai and Ikeda, 1972). Onishi et al. (1976) reported that in common carp, amylase and intestinal protease activities reached maximum levels at 5-7.5 h after first feeding. Amylase activity depends on the natural diet of each species, herbivorous and the omnivorous fish having more activity than carnivore (Kuz’mina, 1978, 1986; Hofer et al., 1982). In grass carp, the pattern of distribution and activity of the digestive enzymes were found to depend on the type of diet ingested by the fish. The presence of cellulase activity suggested the necessity for providing cellulose as an ingredient in the diet of grass carp. The presence of high amylase and
protease activity in fish from the culture pond suggested that incorporation of animal protein, e.g. fish meal, is necessary for the preparation of a diet which would suit the enzyme pattern of the grass carp. Highest lipase activity was recorded in the hepatopancreas of fish (Das and Tripathi, 1991). It was suggested that growth of carp larvae on any artificial diet is not faster than on natural food. Dement’ev (1984) found deviation in type of enzyme activities in silver carp fed with artificial diet as compared to younger fish fed with natural diet. Sharma and Chakrabarti (1999) reported that the digestive tract of common carp larvae fed with live food was longer compared to the larvae fed with artificial diet. Specific proteolytic enzyme activity was also higher in the fish fed with live food compared to the latter group. Enzyme activity showed a direct relationship with the length of the digestive tract (r = 0.95). For better survival of larvae, feeding must be initiated on digestible diets before or very soon after depletion of the endogenous energy sources, yolk and oil (Kim et al., 2001). Mrigal larvae (223 ± 2 mg) were fed with artificial diets varying in protein contents 30, 40 and 50%. Significantly (P < 0.05) higher proteolytic enzyme activity was found in fish fed with 40% protein containing diet compared to other two feeding schemes (Chakrabarti and Kumar, 2001). The development of larva to a fingerling relies on a proper development of digestive functions during larval life and the maturation of digestive tract can be altered by diet composition (Ma et al., 2005). In substrate-based culture systems, application of sugarcane bagasse and paddy straw as substrates influenced the digestive enzyme activities in fringe-lipped carp Labeo fimbriatus (Mridula et al., 2003). Higher protease activity in hepatopancreas, amylase activity in intestine and lipase activity in intestine and hepatopancreas in fish from substrate-based treatments were higher compared to the control treatment could be due to the higher contribution of protein, carbohydrate and fat through the bioflim. Debnath et al. (2007) found that in rohu fingerlings, amylase, lipase and alkaline phosphatase activities were not influenced by the dietary protein, but proteolytic and acid phosphatase activities were influenced (P < 0.05) by dietary protein. Proteolytic activity 2 showed a second order polynomial relationship with dietary crude protein (Y = 0.0734X + 2 4.937X - 68.37, r = 0.97). Acid phosphatase activity showed almost an increasing trend with the protein level. Alkaline phosphatase activity was unresponsive to the dietary protein level. In a comparative study of digestive proteases of adult catla, rohu and silver carp, Kumar et al. (2007) found that total protease activity was significantly (P < 0.05) higher in rohu -1 -1 -1 (1.219 ± 0.059 U mg protein min ) followed by silver carp (1.084 ± 0.061 U mg protein -1 -1 -1 min ) and catla (0.193 ± 0.006 U mg protein min ). Trypsin activity of silver carp and rohu was 89-91% higher compared to catla. Chymotrypsin activity was significantly (P < 0.05) higher in silver carp compared to rohu and catla. These three species have different feeding habits that may influence the protease activity. Mitra et al. (2008) studied the effect of supplementation of ascorbic acid through enriched zooplankton (with 10, 20 and 30% ascorbyl palmitate) on different digestive
enzyme activities of rohu larvae. They found a positive correlation between dietary AA content and enzyme activity of fish. Common carp (1.2 ± 0.07 mg) and catla (0.79 ± 0.1 mg) were cultured under two feeding regimes: live food and refrigerated-plankton food (Sharma and Chakrabarti, 2000, 2009). Proteolytic enzyme activities were significantly (P < 0.05) higher in both species fed with live zooplankton. Zinc lactate was added to the diet of juvenile Jian carp (Cyprinus carpio var. Jian). Trypsin, chymotrypsin, lipase, amylase, + + alkaline phosphatase (AKP), Na , K -ATPase and γ-glutamyl transpeptidase (γ-GT) activities were higher by dietary zinc supplementation than zinc un-supplementation (P < 0.05). These results suggested that zinc could increase intestinal enzyme activities (Tan et al., 2010). The study of effect of UV-B radiation on the digestive physiology of catla larvae showed that amylase, protease trypsin and chymotrypsin activities decreased with the increasing dose of UV-B radiation under laboratory condition (Sharma et al., 2010). Presence of antinutritional factor in feed ingredients affects the digestibility in fish. In common carp the amylase activity was strongly inhibited by wheat flour, an important component of compound fish diets. The amylase inhibitors of wheat may reduce the digestibility of starch in carp (Hofer and Sturmbauer, 1985). Wheat and some other grains contain albumin which inhibits secretion of α-amylase in carps. Sturmbauer and Hofer (1986) have suggested that common carp is capable of compensating for the action of amylase inhibitors in a wheat diet by increasing the rate of secretion of pancreatic amylase. α-amylase collected from gut fluid, gut tissue and hepatopancreas of common carp were inhibited by wheat amylase inhibitor. Residual amylase activity measured after inhibition was proportional to the initial enzyme activity of the sample and inversely proportional to the inhibitor concentration. Inhibition was greatest for amylase from gut tissue followed by amylase from hepatopancreas and least for the amylase from gut fluid (Natarajan et al., 1992). Soy protein concentrate (SPC) was added at different levels (20, 60 and 70%) in the diet of common carp larvae to test the suitability of soy protein as major protein source in carp larval diet. The purified soybean trypsin inhibitor (SBTI) was added to casein based diets. A significant decrease of trypsin specific activity was found in the larvae fed the diets with the highest levels of SPC and of purified SBTI, whereas amylase specific activity was not affected. It was concluded that the growth limitation of carp larvae fed high levels of SPC was not due to SBTI alone but to other antinutritional factors (Escaffre et al., 1997).
MICROBIAL DIGESTION Microbial digestion in fish gut was confirmed by the presence of short chain fatty acids (SCFA) in the fish gut contents. However, very little is known about hindgut fermentation in fish, or its physiological effect on the host fish. Each intra-luminally produced organic acids (succinic or lactic acid) have different nutritional and physiologic effects. The physiological effects of indigestible saccharides would vary, depending on the profile of organic acids produced by microbes in the digestive tract (Hoshi, 1995). The presence of SCFA of common carp indicates microbial fermentation in the gut lumen (Smith et al., 1996). Although amylolytic enzymes such as amylase was not digesting β-starch well, common
carp had higher apparent digestibility of β-starch compared to carnivorous teleost, rainbow trout Oncorhynchus mykiss (Takeuchi, 1991). This suggested that gut microbial fermentation might contribute to the digestion of β-starch in the carp. Das and Tripathi (1991) suggested the presence of both endogenous and bacterial cellulose in grass crap. Kihara and Sakata (2002) suggested that soybean-oligosaccharide and raffinose were potentially highly fermentable oligosaccharides for hindgut microbes of common carp. Chemical structures of oligosaccharides seem to play an important role in the fermentability. The commercial probiotics preparations of Streptococcus faecium improved the growth and feed efficiency of Israeli carp (Noh et al., 1994; Bogut et al., 1998). Saha and Roy (1998) reported that cellulose activity in rohu was largely contributed by the intestinal microflora. Amylolytic, cellulolytic, lipolytic and proteolytic microflora were identified from the gastrointestinal tract of catla, rohu, mrigal, silver carp, grass carp and common carp (Bairagi et al., 2002; Ramachandran et al., 2005). The isolates were qualitatively screened on the basis of their extracellular enzyme producing ability. The selected strains were further quantitatively assayed for amylase, cellulase, lipase and protease activities. Protease activity was exhibited by almost all the bacterial isolates, while strains isolated from grass carp and common carp showed considerable amylolytic and cellulolytic activities. Maximum activity of lipase was exhibited by a strain isolated from silver carp. The study indicated that there was a distinct microbial source of the digestive enzymes - amylase, cellulase, lipase and protease, apart from endogenous sources in fish gut. Feeding of common carp with mixture of lyophilized photosynthetic bacteria and Bacillus sp. (added to the basal diet before feeding) enhanced the amylase, protease and lipase activities. The addition of probiotics highly increased the growth performances and digestive enzyme activities (Yanbo and Zirong, 2006). Saha et al. (2006) isolated bacterial strain CI3 from grass carp, which showed cellulolytic activity. The isolated stain was identified as Bacillus megaterium. Li et al. (2009) isolated six strains of bacteria from the intestine of grass carp. Strains showed different ability to produce cellulase. Ray et al. (2010) isolated ten strains of bacteria from the proximal intestine and distal intestine of catla, rohu and mrigal. Population levels of amylolytic strains were highest in the proximal intestine of catla and lowest in the distal intestine of rohu. Cellulase and protease producing bacteria were highest in the distal intestine and proximal intestine of mrigal, respectively. Appropriate probiotic applications resulted into improved intestinal microbial balance. Thus, leading to improved food absorption, digestive enzymes activities and reduced pathogenic problems in the gastrointestinal tract. The modes of action were as follows: production of inhibitory compounds; competition for chemicals or available energy; competition for adhesion sites; enhancement of the immune response; improvement of water quality; interaction with phytoplankton; source of macro- and micronutrients; enzymatic contribution to digestion.
Characterization of Digestive Enzymes Cohen et al. (1981) found that purified trypsin, chymotrypsin, elastase and carboxypeptidase B of common carp were not stable at a low pH but retain their activity at a neutral pH in the
2+
presence of Ca . Jonas et al. (1983) studied the pH, temperature dependence, heat inactivation and the reactive groups of the active centres of proteolytic enzymes extracted from the alimentary canal of silver carp and common carp. In these species, activation energy values of enzymes, active in a pH range near neutral (7.5) were nearly identical silver carp: 14 kcal/mol; common carp: 15.4 kcal/mol. Heat stability study showed that at 55°C time values for 50% activity loss were 3.8 and 1.3 in the case of silver carp and common carp, respectively. Trypsin and amylase activities of silver carp and bighead carp were investigated in relation to pH in 10 different segments of the gut. Trypsin and amylase of both species had a pH optimum at 8.3 and 7.0, respectively. The relatively low pH of 6.4 in the fore-gut of silver carp did not support an efficient operation of these enzymes, which was compensated by a higher enzyme concentration as compared to bighead carp. The latter, however, showed a higher pH of 7.3 in the fore-gut and thereby maintained similar activities as silver carp (Bitterlich, 1985). The study of Hidalgo et al. (1999) showed that common carp had proteolytic activity in the liver at neutral and alkaline pHs, whereas only very low activity was observed at acid pHs. The highest proteolytic activity in the digestive tract was detected at neutral and alkaline pHs. The bile showed detectable proteolytic activity with an increase in this activity when the incubation temperature was increased to 37°C. Characteristics and functional efficacy of digestive proteases of adult catla, rohu and silver carp were studied (Kumar et al., 2007). The protease activity of rohu and silver carp displayed bell-shaped curves with maximum activity at pH 9; whereas in catla, maximum activity was found between pH 8 and 11. The optimal pH and temperature of the enzyme extracted from the intestine of grass carp were 2.5 and 37 °C, respectively. It retained only 20% of its initial activity after incubating at 50 °C for 30 min. The enzyme lost 81% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride (PMSF). Its Vmax and Km values -1
were determined to be 3.57 mg ml-1 and 0.75 min , respectively (Liu et al., 2008). Cohen et al. (1981) reported that purified trypsin, chymotrypsin and elastase of common carp consisted of four, three and two electrophoretically distinct but enzymatically active fractions, respectively. Carboxypeptidase B consisted of one major and one minor band. The approximate molecular weights of trypsin, chymotrypsin and elastase were 25,000 and for carboxypeptidase B was 34,000. Amino acid analysis of trypsin, elastase and chymotrypsin revealed high similarities to the respective bovine or porcine enzymes. No such similarity was detected in carboxypeptidase B. Amino terminal sequence analysis of the purified chymotrypsin resulted in one main chain resembling that of the B-chain of bovine chymotrypsin. SDS-PAGE of crude enzyme extracts of catla, rohu, mrigal, common carp and hybrid carp showed that in 4-day-old fish bands were fewer in number and their number increased during ontogenic development. Substrate SDS-PAGE also showed the same trends in all these species. The numbers of protease activity bands (zymograms) were minimum on day-4
after hatching and their number gradually increased with age regardless of species of fish. The intensity of bands also increased during ontogenic development (Rathore et al., 2005a, b; Chakrabarti et al., 2006a, b; Chakrabarti and Rathore, 2009). They also reported that high molecular mass bands appeared during the early stages followed by low molecular mass bands. SDS-PAGE showed the presence of several protein bands ranging from 15.3 to 121.9 kDa in enzyme extracts of adults catla, rohu and silver carp. The substrate SDS-PAGE evidenced the presence of various protease activity bands ranging from 21.6-93.7, 21.6-63.8 and 26.7-98.5 kDa for catla, rohu and silver carp, respectively. In pH-stat hydrolysis of Chilean fishmeal showed significantly (P < 0.05) higher degree of hydrolysis compared to soybean meal, silver cup (a commercial fish feed of Mexico) and wheat flour, with enzyme preparations of three fishes. The rate of hydrolysis was significantly (P < 0.05) higher in silver carp compared to others (Kumar et al., 2007). SDS-PAGE electrophoresis of acidic protease extracted from the intestine of grass carp showed that the enzyme was homogeneous with a relative molecular mass of 28,500. Substrate-PAGE at pH 7.0 showed that the purified acidic protease had only an active component. Specificity and inhibiting assays showed that it should be a cathepsin D (Liu et al., 2008). Inhibition of enzyme extract with specific inhibitor helps to classify proteases. Jonas et al. (1983) reported a significance inhibition of protease activity by phenylmethylsulfonyl fluoride (PMSF) in silver carp and common carp, as PMSF selectively modifies the serine side chain located in the active centers of –OH type enzymes. Inhibition of protease activity of adult catla, rohu and silver carp with soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF) revealed the presence of serine proteases. The inhibition of activity with N-α-p-tosyl-L-lysine-chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanychloromethane (TPCK) indicated the presence of trypsin-like and chymotrypsin-like enzymes in all these three carps (Kumar et al., 2007). Similarly in larvae of catla, rohu, mrigal and common carp, inhibition of protease activity bands in substrate SDS-PAGE with SBTI and PMSF revealed the abundance of serine proteases and inhibition of activity bands with TLCK and TPCK evidenced the presence of more than one isoform of trypsin and chymotrypsin in the digestive tissue extract of these commercially important carps (Rathore et al., 2005a, b; Chakrabarti et al., 2006a, b; Chakrabarti and Rathore, 2009) during ontogenic development.
EXPRESSION OF FUNCTIONAL GENES Ruana et al. (2010) examined the gene structures and expression of trypsinogens, as well as the trypsin activities of the herbivore grass carp Ctenopharyngodon idellus and carnivore topmouth culter Culter alburnus. Isolated full-length trypsinogen cDNA clones were 869 bp and 857 bp. The deduced amino acid sequences were 242 aa and 247 aa long, both containing the highly conserved residues essential for serine protease catalytic and conformational maintenance. The results from isoelectric and phylogenetic analyses suggest that grass carp trypsinogen is grouped with teleost trypsinogen group I, while topmouth culter trypsinogen is grouped with group II. The expression pattern of trypsinogen mRNA
was similar between these two species, appearing 2 days post-hatching (dph) and reaching peaks at 11 and 23 dph. The trypsin-specific activities in both species were detected 2 dph and reached the major peaks at 8 dph, however the minor peaks were observed at 20 dph in the grass carp and 17 dph in the topmouth culter. The trypsin-specific activity was significantly higher in the grass carp than in the topmouth culter, which might be attributed to the nature of their different nutritional habits.
CONCLUSIONS The study of digestive enzyme profiles indicates that there is clear difference in the abundance of various enzymes at first feeding, which may be genetically regulated. The enhanced amount of particular enzyme during ontogenic development may be influenced by the diet. Incorporation of beneficial bacteria has significant role in digestion of ingested food. Considerable attention should be given during the formulation of commercial diet for these stomachless fishes.
ACKNOWLEDGEMENTS Authors are thankful to Prof. U. C. Goswami, Gauhati University for his encouragement in writing this article.
REFERENCES Bairagi, A., Ghosh, K.S., Sen, S.K., Ray, A.K. 2002. Enzyme producing bactericidal flora isolated from fish digestive tracts. Aquacult. Int. 10, 109-121. Bitterlich, G. 1985. Digestive processes in silver carp (Hypophthalmichthys molitrix) studied in vitro. Aquaculture 50, 123-131. Bleir, P.U., Pelletier, D., Dutil, J.D. 1997. Does aerobic capacity set a limit on fish growth rates? Rev. Fish. Sci. 5, 323-340. Bogut, I., Milakovic, Z., Bukvic, Z., Brkic, Z., Zimmer, R. 1998. Influence of probiotic Streptococcus faecium M74 on growth and content of intestinal microflora in carp Cyprinus carpio. Czech J. Anim. Sci. 43, 321-235. Buzinova, N.S. 1973. Amylase activity in amurs and carps. Ecological Physiology of Fishes (in Russian). Nauka Press, Moscow, pp. 48-50. Cahu, C., Ronnestad, I., Grangier, V.A, Zambonino-Infante, J.L. 2004. Expression and activities of pancreatic enzymes in developing sea bass larvae (Dicentrarchus labrax) in relation to intact and hydrolyzed dietary protein; involvement of cholecystokinin. Aquaculture 238, 295-308. Chakrabarti, R., Sharma, J.G. 1997. Ontogenic changes of amylase and proteolytic enzyme activities of Indian major carp, Catla catla (Ham.) in relation to natural diet. Indian J. Anim. Sci. 67, 932-934. Chakrabarti, R., Kumar, S. 2001. Effect of feed protein manipulation on the survival, growth, and enzyme activities of Cirrhinus mrigala larvae. In: Hendry, C.I., Stappen, G.V., Wille, M.,
Sorgeloos, P. (Eds.), Larvi 2001, Fish and Shellfish Larviculture Symposium, European Aquaculture Society, Special Publication No.30. Belgium, pp. 129-132. Chakrabarti, R., Sharma, J.G. 2005. Digestive physiology of fish larvae during ontogenic development: A brief overview. Indian J. Anim. Sci. 75, 1337-1347. Chakrabarti, R., Rathore, R.M. 2009. Ontogenic changes in the digestive enzyme pattern and characterization of proteases in Indian major carp Cirrhinus mrigala. Aquacult. Nutr. 16, 569-581. Chakrabarti, R., Rathore, R. M., Kumar, S. 2006a. Study of digestive enzymes and characterization of related proteases during early ontogeny of Labeo rohita. Aquacult. Nutr. 12, 35-43. Chakrabarti, R., Rathore, M.S., Kumar, S. and Mittal, P. 2006b. Functional changes in digestive enzymes and characterization of proteases of silver carp (male) and bighead carp (female) hybrid, during ontogeny. Aquaculture 253, 694-702. Cohen, T., Gertler, A., Birk, Y., 1981. Pancreatic proteolytic enzymes from carp (Cyprinus carpio). II. Kinetic properties and inhibition studies of trypsin, chymotrypsin and elastase. Comp. Biochem. Phys. B 69, 647-653. Cousin, J.C.B., Laurencin, F.B., Gabaudan, J. 1987. Ontogeny of enzymatic activities in fed and fasting turbot, Scophthalmus maximus L. J. Fish Biol. 30, 15-33. Das , K.M., Tripathy, S.D. 1991. Studies on the digestive enzymes of grass carp, Ctenopharyngodon idella (Val.). Aquaculture 92, 21-32. Debnath, D., Pal, A.K., Sahu, N.P., Yengkokpam, S., Baruah, K., Choudhury, D., Venkateshwarlu, G. 2007. Digestive enzymes and metabolic profile of Labeo rohita fingerlings fed diets with different crude protein levels. Comp. Biochem. Phys. B 146, 107- 114. Dement’ev, M.C. 1984. Increasing the efficiency of culture of juvenile carps under fish farming. Authors Abstr. of Candidate Dissertation (in Russian). All-Union Pond Fisheries Research Institute, Moscow, 24 pp. Escaffre, M.A., Zambonino-Infante, J.L., Cahu, L.C., Mambrini, M., Bergot, P., Kaushik, J.S. 1997. Nutritional value of soy protein concentrate for larvae of common carp (Cyprinus carpio) based on growth performance and digestive enzyme activities. Aquaculture 153, 63-80. Fraisse, M., Woo, N.Y.S., Noaillac-Depeyre, J., Murat, J.C. 1981. Distribution pattern of digestive enzyme activities in the intestine of the catfish (Ameiurus nebulus L.) and the carp (Cyprinus carpio). Comp. Biochem. Phys. 70 A, 443-446. Gjellesvik, D.R., Lombardo, D., Walther, B.T. 1992. Pancreatic bile salt dependent lipase from cod (Gadus morhua): Purification and properties. Biochim. Biophys. Acta 1124, 123-134. Green, B.S., McCormick, M.I. 2001. Ontogeny of the digestive and feeding systems in the anemonefish Amphiprion melanopus. Environ. Biol. Fish. 61, 73-83. Gruzdkov, A.A., Zil’ber, Y.D, Lezuitova, N.N. 1986. Physiology and biochemistry of enzyme adaptions. Pancrease, Membrannyi gigroliz i transport: novye dannye i gipotezy (New data and hypotheses), Leningrad, Nauka, pp. 45-51. Hidalgo, M.C., Urea, E., Sanz, A. 1999. Comparative study of digestive enzymes in fish with different
nutritional habits. Proteolytic and amylase activities. Aquaculture 170, 267-283. Hofer, R., Dalla Via, G., Troppmair, J., Giussani, G. 1982. Difference in digestive enzymes between cyprinid and noncyprinid fish. Mem. Ist. Ital. Idrobiol. 40, 201-208. Hofer, R., Sturmbauer C. 1985. Inhibition of trout and carps α-amylase by wheat. Aquaculture 48, 277-283. Hoshi, S. 1995. Oligosaccharides and intestinal physiology. Food Indust. 38, 57-66. Jonas, E., Ragyanssszki, M., Olah, J., Boross, L. 1983. Proteolytic digestive enzymes of carnivorous (Silurus glanis L.) herbivorous (Hypophthalmichthys molitrix Val.) and omnivorous (Cyprinus carpio L.) fishes. Aquaculture 30, 145-154. Kawai, S., Ikeda, S. 1972. Studies on digestive enzymes of fishes. II. Effect of dietary change on the activities of enzymes in carp intestine. Bull. Jap. Soc. Sci. Fish. 38, 265-270. Kihara, M., Sakata, T. 2002. Production of short-chain fatty acids and gas from various oligosaccharides by gut microbes of carp (Cyprinus carpio L.) in micro-scale batch culture. Comp. Biochem. Phys.132, 333-340. Kim, B.G., Divakaran, S., Brown, C.L., Ostrowski, A. 2001. Comparative digestive enzyme ontogeny in two marine larval fishes: Pacific threadfin (Polydactylus sexfilis) and bluefin trevally (Caranx melampygus). Fish Physiol. Biochem. 24, 225-241. Konovalov, Yu.D., Mestechkin, A.Ya. 1975. Activity of peptide hydrolases during embryonic development of common carp. Ontogenez 6, 201–205. Kumar, S., Fernando, L.G., Chakrabarti, R., del Toro, M.A.N., Cordova-Murueta, J.H. 2007. Characterization of digestive proteases of three carps Catla catla, Labeo rohita and Hypophthalmicthys molitrix and in vitro digestibility study. Aquacult. Nutr. 13, 1-9. Kumar, S., Sharma, J.G., Chakrabarti, R. 2000. Quantitative estimation of proteolytic enzyme and ultrastructural study of anterior part of intestine of Indian major carp (Catla catla) larvae during ontogenesis. Curr. Sci. 79, 1007-1011. Kuz’mina, V.V. 1978. Membrane digestion in cyclostomata and fishes. Vopr. Ikhtiol. 18, 684-696. Kuz’mina,V.V. 1980. Season and change of α-amylase activity in bream. Vopr. Ikhtiol.. 17, 111-133 (in Russian). Kuz’mina, V.V. 1986. Obshchie zakonomernosti membrannogo pishchevareniya u ryb i yego adaptivnyye perestroyki (General principles of membrane digestion in fish and its adaptational evolution). Author’s abstract of Doctor’s Thesis, Biological Sciences, 03.00.13. Leningrad. Lauf, M., Hofer, R. 1984. Proteolytic enzyme in fish development and the importance of dietary enzymes. Aquaculture 37, 335-346. Li, H., Zheng, Z., Cong-xin, X., Bo, H., Chao-yaun, W., Gang, H. 2009. Isolation of cellulose-producing microbes from the intestine of grass carp (Ctenopharyngodon idellus). Environ. Biol. Fish 86, 131-135. Liu, Z., Wang, Z., Zhang, J. 2008. An acidic protease from the grass carp intestine (Ctenopharyngodon idellus). Comp. Biochem. Phys. B 149, 83-90.
Ma, H., Cahu, C., Zambonino, J.L., Yu, H., Duan, Q.,Le Gall, M., Mai, K. 2005. Activities of selected digestive enzymes during larval development of large yellow croaker (Pseudosciaena crocea). Aquaculture 245, 239-248. Mitra, G., Mukhopadhyay, P.K., Ayyappan, S. 2008. Modulation of digestive enzyme activities during ontogeny of Labeo rohita larvae fed ascorbic acid enriched zooplankton. Comp. Biochem. Phys. 149, 341-350. Mridula, R.M., Manissery, J.K., Keshavanatha, P., Shankara K.M., Nandeesha, M.C., Rajesh, K.M. 2003. Water quality, biofilm production and growth of fringe-lipped carp (Labeo fimbriatus) in tanks provided with two solid substrates. Bioresource Technol. 87, 263-267. Natarajan, M., Ross, B., Ross, L.G. 1992. Susceptibility of carp and tilapia α-amylase to purified wheat amylase inhibitor. Aquaculture 102, 265-274. Noh, S.H., Han, K., Won, T.H., Choi, Y.J. 1994. Effect of antibiotics, enzyme, yeast culture and probiotics on the growth performance of Israeli carp. Korean J. Anim. Sci. 36, 480-486. Ostroumova, I.N., Dement’eva. M.A. 1981. Beginning of the pancreatic function in the digestive process of carp larvae. Zhurnal Evoliutsionnoi Biokhimii I Fiziologii 17, 302-305. Onishi, T., Murayama, S., Takeuchi, M. 1976. Changes in digestive enzyme levels in carp after feeding – III. Response of protease and amylase to twice-a-day feeding. Bull. Jap. Soc. Sci. Fish. 42, 921-929. Ramachandra, S., Bairagi, A., Ray, A.K. 2005. Improvement of nutritive value of grass (Lathyrus sativus) seed meal in the formulated diets for rohu, Labeo rohita (Halmilton) fingerlings after fermentation with a fish gut bacterium. Bioresource Technol. 96, 1456-1472. Rathore, R.M., Kumar, S., Chakrabarti, R. 2005a. Digestive enzyme profile of Cyprinus carpio during ontogenic development. World Aquacult. 36, 37-39. Rathore, R.M., Kumar, S., Chakrabarti, R. 2005b. Digestive enzyme patterns and evaluation of protease classes in Catla catla (Family: Cyprinidae) during early developmental stages. Comp. Biochem. Phys. B 142, 98-106. Ray, A.K., Roy, T., Mondal, S., Ringo, E. 2010. Identification of gut-associated amylase, cellulose and protease-producing bacteria in three species of Indian major carps. Aquac. Res. 41, 14621469. Ruana, G., Lia, Y., Gaoa, Z.,Wang, H., Wanga, W. 2010. Molecular characterization of trypsinogens and development of trypsinogen gene expression and tryptic activities in grass carp (Ctenopharyngodon idellus) and topmouth culter (Culter alburnus). Comp. Biochem. Phys. B 155, 77-85. Saha, A.K., Ray, A.K. 1998. Cellulase activity in rohu fingerlings. Aquacult. Int. 6, 281-291. Saha, S., Roy, R.N., Sen, S.K., Ray, A.K. 2006. Characterization of cellulase-producing bacteria from the digestive tract of tilapia, Oreochromis mossambica (Peters) and grass carp, Ctenopharyngodon idella (Valenciennes). Aquac. Res. 37, 380-388. Seenappa, D., Devaraj, K.V. 1995. Effect of different levels of protein, fat and carbohydrate on growth, feed utilisation and body carcass composition of fingerlings in Catla catla (Ham). Aquaculture 129, 243-249.
Sharma, J.G., Chakrabarti, R. 1999. Larval rearing of common carp Cyprinus carpio: A comparison between artificial and natural diets under three stocking densities. J. World Aquacult. Soc. 30, 214-219. Sharma, J.G., Chakrabarti, R. 2000. Replacement of live-food with refrigerated-plankton food for Cyprinus carpio (L) larvae cultured with three different types of biological filters. Curr. Sci. 79, 214-219. Sharma, J.G., Chakrabarti, R. 2009. Comparative growth performance and proteolytic enzyme activity of Indian major carp Catla catla larvae, fed with live food and refrigerated plankton food. Indian J. Anim. Sci. 79, 1185-1188. Sharma, J.G., Rao, Y.V., Kumar, S., Chakrabarti, R. 2010. Impact of UV-B radiation on the digestive enzymes and immune system of larvae of Indian major carp Catla catla. Int. J. Radiat. Biol. 86, 181-186. Smith, T.B., Wahl, D.H., Mackie, R.I. 1996. Volatile fatty acids and anaerobic fermentation in temperate piscivorous and omnivorous freshwater fish. J. Fish Biol. 48, 829-841. Sturmbauer, C., Hofer, R. 1986. Compensation for amylase inhibitors in the intestine of the carp (Cyprinus carpio). Aquaculture 52, 31-33. Takeuchi, T. 1991. Digestion and Nutrition. In: Itazawa, Y., Hanyu, I. (Eds.), Fish Physiology, Koseisha-Koseikaku,Tokyo, pp. 67-101. Tan, L-N., Feng, L., Liu, Y., Jiang, J., Jiang, W.-D., Hu, K., Li, S.-H., Zhou, X.-Q. 2010. Growth, body composition and intestinal activities of juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of dietary zinc. Aquacult. Nutr.17, 338-345. Tanaka, M. 1973. Studies on the structure and function of the digestive system of teleost larvae. Ph.D. Thesis. Department of Fisheries, Faculty of Agriculture, Kyoto University, Japan, 136 pp. Ueberschar, B. 1993. Measurement of proteolytic enzyme activity: significance and application in larval fish research. In: Walther, B.T., Fuhn, H.J. (Eds.), Physiological and biochemical aspects of fish development, University of Bergen, Norway, pp. 233-239. Verreth, J., Segner, H. 1995. The impact of development on larval nutrition. In: Lavens, P., Jasper, E., Roelants, I. (Eds.), Larvi’95, Fish and Shellfish Larviculture Symposium. European Aquaculture Society Special Publication No. 24, Gent, Belgium, 229 pp. Volkova, I.V. 1999. Activities of digestive enzymes in plant-eating fish at early phases of ontogenesis. Canadian Science (Biology). Dissertation, Astrakhan. Yanbo, W., Zirong, X., 2006. Effect of probiotics for common carp (Cyprinus carpio) based on growth performance and digestive enzyme activities. Anim. Feed Sci. Tech. 127, 283-292.
