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Background. Microbial and fungal cellulases are known to hydrolyse cellulose, which is ingested as plant material by herbivorous/omnivorous fishes. Microbial enzymes have enormous advantage of being produced in large quantities by established fermentation techniques. The present investigation aims to optimize the environmental and nutritional parameters for fermentation to enhance cellulase production by two bacterial strains isolated from fish gastrointestinal tracts. Materials and Methods. Two bacterial strains, Bacillus subtilis CY5 and Bacillus circulans TP3, isolated from the gastrointestinal tracts of common carp, Cyprinus carpio L., and Mozambique tilapia, Oreochromis mossambicus (Peters, 1852), respectively were identified as potent cellulase producers. Both strains were cultured in tryptone soya broth for 24 h at 32 ± 2°C, when average viable count of 9.75 • 107 cells • mL-1 culture broth was obtained. This was used as the inoculum for the production medium. The fermentation medium was seeded with 1.0%, 2.0%, 3.0%, 4.0%, and 5.0% inoculum (tryptone soya broth) and incubated in static culture at 40°C to standardize the inoculum size for fermentation. The effect of different production parameters, such as fermentation condition, moisture, pH, temperature, inoculum size, and nitrogen sources on cellulase production by the isolated bacterial strains were studied. Results. Cellulase yield was highest (26 U in B. subtilis and 20.2 U in B. circulans) in solid-state fermentation (SSF). Enzyme production in both the isolates increased in an optimum pH range of 7.0 to 7.5. Minimum cellulase production was observed at 45°C, while maximum production was obtained at 40°C. To standardize the fermentation period for cellulase production, production rate was measured at 12-h intervals up to 120 h. Enzyme production increased for 96 h of fermentation in both strains, and decreased thereafter. The enzyme production increased with increased inoculum size up to 3.0 percentage points. Asparagine as the nitrogen source was most effective in B. subtilis CY5, while beef extract proved useful in optimizing enzyme production by B. circulans TP3. Conclusion. The results of this study will help to standardize the requirements for optimum production of cellulase by cellulase-producing fish gut bacteria and might contribute towards better fish feed formulation incorporating plant ingredients, especially in the larval stages when the enzyme system is not efficient.
Background. The information on gut microflora in fish is scanty and there is a paucity of knowledge regarding microbial enzyme activity in fish gastrointestinal tracts. Although some information is available on the enzyme-producing bacteria in fish digestive tracts, almost nothing is known about their distribution in different regions of the gut. In the present study, an attempt has been made to investigate the distribution of enzyme-producing microflora in the foregut and hindgut regions of seven culturable freshwater teleosts. Materials and Methods. Isolation and enumeration of aerobic bacterial flora in the foregut and hindgut regions of the gastrointestinal tracts of seven freshwater teleosts of different feeding habits, namely rohu, Labeo rohita; catla, Catla catla; mrigal, Cirrhinus mrigala; bata, Labeo bata; orange-fin labeo, Labeo calbasu; Nile tilapia, Oreochromis niloticus; and climbing perch, Anabas testudineus, have been carried out. Microbial culture of the gut mucosa on selected nutrient media, following the enrichment culture technique, was done for bacterial isolation. Bacterial isolates were qualitatively screened on the basis of their extracellular enzyme-producing ability. The selected strains were further quantitatively assayed for amylase, cellulase and protease activities. Results. In general, bacterial population was lower in the foregut region of all the seven species of fish examined. Amylolytic strains were present in higher densities in the foregut region of orange-fin labeo and bata (12.20 × 103 CFU·g-1 gut tissue and 11.50 × 103 CFU·g-1 gut tissue, respectively) in comparison to the hindgut region. The cellulolytic population exhibited maximum densities in the hindgut region of bata (7.20 × 103 CFU·g-1 gut tissue) followed by the foregut region of the same fish (5.50 × 103 CFU·g-1 gut tissue). Amylolytic and cellulolytic bacterial flora was not detected in both the fore and hindgut regions of climbing perch. Proteolytic bacterial flora was found in all the species of fish studied and the maximum count was observed in the hindgut region of bata (13.40 × 103 CFU·g-1 gut tissue), orange-fin labeo (9.00 × 103 CFU·g-1 gut tissue), Nile tilapia (8.30 × 103 CFU·g-1 gut tissue) and climbing perch (7.20 × 103 CFU·g-1 gut tissue). Minimum count of proteolytic bacterial flora was observed in the foregut region of all the fishes studied. Peak amylase and cellulase activities were exhibited by bacterial strains isolated from the foregut of orange-fin labeo (266.43 ± 0.15 U) and the hindgut of bata (64.01 ± 0.42 U), respectively. Maximum protease activity was exhibited by a strain isolated from the hindgut region of orange-fin labeo (44.33 ± 0.09 U), followed by the strains isolated from the hindgut regions of climbing perch (32.87 ± 0.12 U), bata (29.71 ± 0.11 U), and Nile tilapia (29.46 ± 0.11 U). Conclusion. The results of the present study indicate that there is a distinct microbial source of digestive enzymes apart from the endogenous sources in fish digestive tracts. The enzyme-producing bacteria isolated from the digestive tracts can be beneficially used as a probiotic while formulating aquafeeds, especially in the larval stages. However, further investigations are required 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.
Background. Because of the advancement of multiple spawning of carps, the importance of a quality larval diet is increasing day by day. The larval fish do not have the necessary enzyme or the amount of digestive enzymes to digest feed at optimum level. Therefore, fermentation of feed ingredients and/or formulated diets by bacterial enzymes to produce simpler forms of nutrients may be beneficial for them. Materials and Methods. Five isocaloric (4.38 kcalּg-1) and isonitrogenous (35% crude protein approximately) experimental diets (D1-D5) containing 32% fish meal, 34% mustard oil cake, 30% rice bran, and 2% cod liver oil, were fermented in vitro with Bacillus circulans cells (at the rate of 108 bacterial cells per g) at 37oC for 1-5 day duration (1 day: D1, 2 days: D2, 3 days: D3, 4 days: D4, and 5 days: D5). The bacterial strain [extracellular enzyme producer Bacillus circulans (Lr 1.1)] used for fermentation was isolated from the intestine of rohu, Labeo rohita fingerlings. The reference diet (RD), containing same ingredients, was not fermented with bacterial cells. Rohu spawn (av. wt. 0.35 Âą 0.01 mg) were fed ad libitum for 21 days in the laboratory condition at 30 minutes interval starting from 0900 h to 1600 h. Results. Fermentation of diets resulted in an increase in crude protein and free amino acid contents and decrease in crude fibre content. Diets D4 and D5 resulted in best growth and survival (98% and 98.33%, respectively) of rohu spawn compared to those fed with the reference diet (RD) and other experimental groups. A significant positive correlation was obtained between RNA : DNA ratio and specific growth rate (SGR) of 18 dietary groups (6 groups in triplicate). Conclusion. Fermentation of feed ingredients may be practiced as a tool for starter diet formulation to obtain better growth and survival of rohu spawn. The enzyme-producing strain, Bacillus circulans could be used to ferment diets for 4 to 5 days to increase the bioavailability of nutrients.
A 21-day feeding experiment was conducted to assess the effects of diets supplemented with fish intestinal bacteria producing extracellular enzyme on growth and survival of rohu, Labeo rohita (Hamilton) spawn (average weight 0.3 mg). Intestinal bacteria were isolated from healthy rohu fingerlings (average weight 3.51 ±0.31 g). Six isocaloric (4.24 kcalּg-1) and isonitrogenous (approximately 35% crude protein) diets (D1-D6) were prepared containing 32% fish meal, 34% mustard oil cake, 30% rice bran, 2% cod liver oil, and 2% vitamin premix. Experimental diets (D2-D6) were supplemented with the isolated bacterial strain Bacillus circulans Lr 1.1 at five different levels (4 × 105, 4 × 106, 4 × 107, 4 × 108 and 4 × 1010 cells per 100 g of feed, respectively). Rearing of spawn on diet D5 (supplemented with 4 × 108 cells per 100 g) resulted in better growth and survival (97.33 ±1.15%) of rohu spawn compared to those with the control diet (diet D1, without any microflora supplementation) and other experimental diets. Diet D5 resulted in significantly higher growth and specific growth rate (SGR) followed by diets D6 and D4. RNA and DNA contents in the spawn were measured as an index of growth. The correlation coefficient (r value) of 0.831 and regression line having a slope of 0.79 suggested a significant positive correlation (0.01 level) between RNA-DNA ratio and specific growth rate.
Background. Yeast extract powder (YEP) is a rich source of several B vitamins and is used mainly in bacterial culture. In the present study, the effect of dietary supplementation of YEP on growth, feed conversion, nutrient ADC, body composition, and digestive enzyme profile of rohu, Labeo rohita (Hamilton, 1822) fingerlings was evaluated. Materials and Methods. Rohu fingerlings (mean individual weight 1.76 ± 0.15 g) were fed, twice daily, fish meal-based 35% protein diets supplemented with YEP (0.1%, 0.2%, 0.3%, 0.4%, and 0.5% on dry matter basis) for 75 days at 3% of body weight, in triplicate. Fish performance in terms of growth, apparent nutrient digestibility, and whole body composition was studied. The intestinal protease and amylase activities were also determined in rohu fingerlings fed YEP-supplemented diets. Microbial cultures of the intestinal mucosa of the fish in all dietary treatments were carried out after the feeding trial in soybean-casein digest agar (TSA), gelatin-peptone (GP), starch (ST) and carboxymethylcellulose (CMC) plates, separately, to determine the protease- amylase-, and cellulase-producing capacities (qualitative) of the isolated organisms. Results. The fish fed YEP-supplemented diets exhibited better growth than the control up to 0.2% level. YEP at 0.1% level resulted in the best performance of the fish in terms of per cent weight gain, SGR, FCR, and PER, followed by the 0.2% level. YEP led to higher deposition of protein and lower deposition of lipid in the carcass. The intestinal protease activity was highest in the fish fed 0.1% yeast, followed by 0.2%. Protease-producing capacity of the intestinal bacterial flora in the in vitro cultures followed the same trend. Conclusion. It appears therefore that YEP in the diets might have some effect on the gut microflora by supplying additional digestive enzymes leading to better nutrient utilization.
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