Characterization and Identification of Gut-Associated Phytase-Producing Bacteria in Some Freshwater Fish Cultured in Ponds

Abstract

Background. Phytase produced by gut bacteria increases the availability of phosphorus and other important nutrients in ruminants by virtue of enzymatic hydrolysis of the phytic acid, an antinutritional factor present in the majority of plant feedstuffs. This topic, however, has been insufficiently investigated in fish. This study was intended: to evaluate the presence of phytase-producing autochthonous bacteria in the gastrointestinal (GI) tracts of 14 freshwater teleost fishes; and to identify the most promising phytase-producing strains by phenotypic characterization and 16S rDNA. Materials and methods. The GI tracts were removed and divided into proximal (PI) and distal (DI) intestine. Homogenates of intestinal segments were spread onto sterilized tryptone soya agar and modified phytase screening media (MPSM) plates to determine autochthonous culturable heterotrophic and phytase-producing microbiota, respectively. Data were presented as log viable counts (LVC) g-1 intestine. Out of 95 phytase-producing isolates, primarily selected 32 isolates were studied for phytase-assay using MPSM broth. Promising phytase-producing isolates were evaluated for other exo-enzymes (amylase, cellulase, protease, lipase) using 4 selective media. Two most promising phytase-producing isolates were identified by phenotypic characterization and 16S rDNA. Results. Population of heterotrophic bacteria was highest (LVC = 8.29 g-1 intestine) in the DI of Gudusia chapra followed by DI of Hypophthalmichthys molitrix (LVC = 6.82 g-1 intestine). However, more than log 4 reduction of the phytase-producing bacteria was observed compared to heterotrophic bacteria. Phytase-producing microbiota was highest in the PI of G. chapra (LVC = 3.95 g-1 intestine) followed by PI of Labeo calbasu (LVC = 3.78 g-1 intestine). The strain LB1.4 isolated from DI of Labeo bata showed highest phytase activity (2.33 ± 0.006 U · mL–1) followed by the strain GC1.2 (2.19 ± 0.018 U · mL–1) isolated from PI of G. chapra. Both isolates were efficient in producing other exo-enzymes. Phenotypic characterization and nucleotide homology analysis revealed that the isolates LB1.4 and GC1.2 were similar to Bacillus subtilis and Bacillus atrophaeus, respectively. Conclusion. Autochthonous phytate degrading bacteria were present in the GI tract of fish that might endow ecological advantages to overcome the anti-nutritional effects of plant phytate.