Digestion of food depends on three main factors: (i) the ingested food and the extent to which the food is susceptible to the effects of digestive enzymes, (ii) the activity of the digestive enzymes and (iii) the length of time the food is exposed to the action of the digestive enzymes. Each of these factors is affected by a multitude of secondary factors. The present review highlights the experimental results on the secondary factor, enzymatic activity and possible contribution of the fish gut microbiota in nutrition. It has been suggested that fish gut microbiota might have positive effects to the digestive processes of fish, and these studies have isolated and identified the enzyme-producing microbiota. In addition to Bacillus genera, Enterobacteriaceae and Acinetobacter, Aeromonas, Flavobacterium, Photobacterium, Pseudomonas, Vibrio, Microbacterium, Micrococcus, Staphylococcus, unidentified anaerobes and yeast are also suggested to be possible contributors. However, in contrast to endothermic animals, it is difficult to conclude the exact contribution of the gastrointestinal microbiota because of the complexity and variable ecology of the digestive tract of different fish species, the presence of stomach and pyloric caeca and the relative intestinal length. The present review will critically evaluate the results to establish whether or not intestinal microbiota do contribute to fish nutrition.

Enzyme‐producing bacteria isolated from fish gut: a review

Phytate formed during maturation of plant seeds and grains is a common constituent of plant-derived fish feed. Phytate-bound phosphorus (P) is not available to gastric or agastric fish. A major concern about the presence of phytate in the aquafeed is its negative effect on growth performance, nutrient and energy utilization, and mineral uptake. Bound phytate-P, can be effectively converted to available-P by phytase. During the last decade, phytase has been used by aqua feed industries to enhance the growth performance, nutrient utilization and bioavailability of macro and micro minerals in fish and also to reduce the P pollution into the aquatic environment. Phytase activity is highly dependent on the pH of the fish gut. Unlike mammals, fish are either gastric or agastric, and hence, the action of dietary phytase varies from species to species. In comparison to poultry and swine production, the use of phytase in fish feed is still in an unproven stage. This review discusses effects of phytate on fish, dephytinisation processes, phytase and pathway for phytate degradation, phytase production systems, mode of phytase application, bioefficacy of phytase, effects of phytase on growth performance, nutrient utilization and aquatic environment pollution, and optimum dosage of phytase in fish diets.

https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1439-0396.2011.01169.x

Phytate and phytase in fish nutrition

The microbes in the gastrointestinal (GI) tract are of high importance for the health of the host. In this study, Roche 454 pyrosequencing was applied to a pooled set of different 16S rRNA gene amplicons obtained from GI content of common carp (Cyprinus carpio) to make an inventory of the diversity of the microbiota in the GI tract. Compared to other studies, our culture-independent investigation reveals an impressive diversity of the microbial flora of the carp GI tract. The major group of obtained sequences belonged to the phylum Fusobacteria. Bacteroidetes, Planctomycetes and Gammaproteobacteria were other well represented groups of micro-organisms. Verrucomicrobiae, Clostridia and Bacilli (the latter two belonging to the phylum Firmicutes) had fewer representatives among the analyzed sequences. Many of these bacteria might be of high physiological relevance for carp as these groups have been implicated in vitamin production, nitrogen cycling and (cellulose) fermentation.

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Pyrosequencing of 16S rRNA gene amplicons to study the microbiota in the gastrointestinal tract of carp (Cyprinus carpio L.)

Culturable autochthonous gut bacteria in Atlantic salmon (Salmo salar L.) fed diets with or without chitin. Characterization by 16S rRNA gene sequencing, ability to produce enzymes and in vitro growth inhibition of four fish pathogens

Species of Bacillus are spore-forming bacteria that are resistant to aggressive physical and chemical conditions, with various species showing unusual physiological features enabling them to surviv...

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Genus bacillus, promising probiotics in aquaculture: Aquatic animal origin, bio-active components, bioremediation and efficacy in fish and shellfish

Isolation and enumeration of amylase, cellulase and protease-producing autochthonous bacteria in the proximal intestine (PI) and distal intestine (DI) of three species of Indian major carps, catla (Catla catla), mrigal (Cirrhinus mrigala) and rohu (Labeo rohita), were investigated using the conventional culture-based technique. Population levels of amylolytic strains were the highest in the PI of catla and the lowest in the DI of rohu. The highest viable count of cellulase and protease-producing bacteria was recorded in the DI and PI of mrigal respectively. Among the bacteria isolated, 10 strains (five from PI and five from DI) were selected as potent enzyme producers according to a quantitative enzyme assay. The chosen strains were further identified by 16S rRNA gene sequence analysis. The five strains isolated from catla showed high similarity to Citrobacter sp. clone W2, Enterobacter sp. JA24, Bacillus coagulans strain TR, uncultured bacterial clone Hel3bc04 and Bacillus cereus strain UST2006-BC004. The four strains isolated from mrigal were most closely related to Bacillus sp. KCd2, uncultured bacterial clone Hel3bd09, B. cereus strain BU040901-020 and Citrobacter freundii strain YRL11, while the strain isolated from rohu probably belonged to Bacillus sp. GV.

Identification of gut-associated amylase, cellulase and protease-producing bacteria in three species of Indian major carps.

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 enzymeproducing 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). Conclusions. 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.

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Distribution of enzyme-producing bacteria in the digestive tracts of some freshwater fish

Isolation and enumeration of phytase-producing bacterial flora in the foregut and hindgut regions of the gastrointestinal tracts of 10 culturable freshwater teleosts of different feeding habits, namely rohu (Labeo rohita), catla (Catla catla), mrigal (Cirrhinus mrigala), bata (Labeo bata), kalbasu (Labeo calbasu), Nile tilapia (Oreochromis niloticus), climbing perch (Anabas testudineus), common carp (Cyprinus carpio), silver carp (Hypophthalmichthys molitrix) and grass carp (Ctenopharyngodon idella), have been carried out. Microbial culture of the gut mucosa on selected nutrient media following the enrichment culture technique was performed for bacterial isolation. The bacterial isolates were screened on the basis of their enzyme-producing ability. The bacterial population on the tryptone soya agar (TSA) plate was maximum in the hindgut region of bata, followed by mrigal and minimum in the foregut region of Nile tilapia. In modified phytase screening medium (MPSM), phytase-producing strains were recorded at higher densities in the foregut region of mrigal and grass carp and minimum in the foregut region of bata. In case of the hindgut, maximum phytase-producing strains were present in grass carp and mrigal and minimum in rohu. In general, in MPSM, the bacterial population was lower in the hindgut region of all the 10 species of fish examined. The phytase-producing ability of the selected 31 strains (16 from the foregut and 15 from the hindgut region) was determined by clearing zones on phytate-containing plates. Among these isolates, 22 strains (12 from the foregut and 10 from the hindgut region) were selected as potent phytase producers according to a quantitative enzyme assay. The highest phytase activity was observed in the bacterial strains LF1 and LH1 isolated from the fore and the hindgut regions of rohu respectively. Both the strains were identified as Bacillus licheniformis on the basis of phenotypic characteristics as well as 16S rDNA sequence analysis.

Phytase-producing bacteria in the digestive tracts of some freshwater fish.

Isolation and enumeration of aerobic bacterial flora in the foregut and hindgut regions of the gastrointestinal tract of bata, Labeo bata, were carried out on selected nutrient media following the enrichment culture technique Four 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 Amylolytic strains were present in higher densities in the foregut region, whereas the cellulolytic and proteolytic populations exhibited maximum densities in the hindgut region Peak amylase, cellulase, and protease activities were exhibited by bacterial strains BF2 and BH4, isolated from the foregut and hindgut regions, respectively Both the strains are Gram-positive rods, motile, and capable of forming endospores They can tolerate a wide range of temperatures (25–42 C and 25–55 C in cases of BF2 and BH4, respectively) and pH (5–11) On the basis of phenotypic characteristics and 16S rDNA sequence analysis, the strains were identified as Bacillus licheniformis (BF2, GenBank Accession Number: EF635428) and Bacillus subtilis (BH4, GenBank Accession Number: EF032683) The study provides scope for further research on the use of the enzyme-producing fish gut bacteria in aquafeeds, especially for the larval stages

Characterization and Identification of Enzyme-producing Bacteria Isolated from the Digestive Tract of Bata, Labeo bata

The culture conditions for extracellular protease production by two bacterial strains, Bacillus licheniformis BF2 and Bacillus subtilis BH4 isolated from the foregut and hindgut regions, respectively, of bata (Labeo bata, Cyprinidae) were optimized to obtain maximum level of protease. Both strains were cultured in TSA broth for 24 h at 37 ± 2 °C. 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 (TSA) and incubated in static culture at 37 °C to standardize the inoculum size for fermentation. The effects of fermentation condition, moisture, pH, temperature, inoculum size, and nitrogen sources on protease production by the isolated bacterial strain were studied. The strain, BH4, produced more protease in comparison to the strain BF2 isolated from the same fish in solid-state fermentation. Enzyme production in both the isolates increased in an optimum pH range of 6.0–6.5. Maximum protease production by both the strains was obtained at 40 °C. To standardize the fermentation period for protease production, production rate was measured at 12-h intervals up to 120 h. Enzyme production increased for 72 h of fermentation in both strains, and decreased thereafter. The enzyme production increased with increased inoculum size up to 3.0 percentage points. Beef extract as the nitrogen source was most effective in optimizing enzyme production by both the isolated strains. The information generated from the present investigation will help to standardize the requirements for optimum production of protease by protease-producing fish gut bacteria.

Optimization of Culture Conditions for Production of Protease by Two Bacterial Strains, Bacillus licheniformis BF2 and Bacillus subtilis BH4 Isolated from the Digestive Tract of Bata, Labeo bata (Hamilton)

Sabyasachi Mondal

Papers

Identification of gut-associated amylase, cellulase and protease-producing bacteria in three species of Indian major carps.

Distribution of enzyme-producing bacteria in the digestive tracts of some freshwater fish

Phytase-producing bacteria in the digestive tracts of some freshwater fish.

Characterization and Identification of Enzyme-producing Bacteria Isolated from the Digestive Tract of Bata, Labeo bata

Optimization of Culture Conditions for Production of Protease by Two Bacterial Strains, Bacillus licheniformis BF2 and Bacillus subtilis BH4 Isolated from the Digestive Tract of Bata, Labeo bata (Hamilton)