Assessment of Phytase Producing Ability of Marine Fish Intestinal Bacteria and Yeasts  

Ariole C. N  , Uchegbu S. C  
Department of Microbiology, University of Port Harcourt, P.M.B. 5323, Port Harcourt, Rivers State, Nigeria
Author    Correspondence author
International Journal of Marine Science, 2016, Vol. 6, No. 40   doi: 10.5376/ijms.2016.06.0040
Received: 11 Aug., 2016    Accepted: 14 Oct., 2016    Published: 27 Oct., 2016
© 2016 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Ariole C.N., and Uchegbu S.C., 2016, Assessment of Phytase Producing Ability of Marine Fish Intestinal Bacteria and Yeasts, International Journal of Marine Science, 6(40): 1-6 (doi:10.5376/ijms.2016.06.0040)

Abstract

The phytase producing bacteria and yeasts associated with the intestinal tract of two marine fish species (Liza grandsquamis and Ethmalosa fimbriata) from New Calabar River in the Niger Delta were established. Spread plate technique was employed for isolation of phytase producing bacteria and yeasts using modified phytase-screening medium (MPSM). The pH of the medium was adjusted to 7.0 and 4.5 for isolation of bacteria and yeasts respectively. The isolates were further evaluated for quantitative phytase assay with MPSM broth. The phytase producing bacterial genera in the digestive tract of fish species were identified as Bacillus, Pseudomonas and Enterobacter while the isolated phytase producing yeasts genera were Saccharomyces and Candida. Among bacterial phytate degraders, Bacillus sp. isolated from Liza grandsquamis intestine showed highest phytase activity (50.09 ± 0.15 U/ml) while Enterobacter sp. isolated from Ethmalosa fimbriata intestine showed the lowest phytase activity (2.75 ± 0.32 U/ml).  Among yeasts phytate degraders, Saccharomyces sp. isolated from Liza grandsquamis intestine showed highest phytase activity (12.5 ± 0.27 U/ml) while Candida sp. isolated from Ethmalosa fimbriata intestine exhibited the lowest phytase activity (3.66 ± 0.71 U/ml). These genera of aerobic microorganisms may take part in phytate degradation in the intestine of marine organisms. Their ability to degrade phytate may provide environmental benefits to defeat the plant phytate anti-nutritional effects.

Keywords
Marine fish; Intestinal tract; Bacteria; Yeast; Phytase activity

Introduction

The main phosphorus (P) reservoir in plant feed materials is phytic acid (Myo-inositol 1, 2, 3, 4, 5, 6-hexakis-dihydrogen phosphate) (Oatway et al., 2001). The release of phosphate from phytic acid is initiated by a hydrolytic enzyme known as phytase (E. C. 3. 1. 3. 8. myo-inositol hexaphosphate phosphohydrolase). Phytate, apart from acting as phosphorus reservoirs, bind a significant portion of the micro-elements, proteins, carbohydrates and amino acids transferring them into complex insoluble conglomerates (Onyango and Adeola, 2012). A promising way out to these problems is phytases which have been reported to boost phosphate consumption effectiveness from phytate in feed materials and to reduce phosphorus environmental contamination (Broz et al., 1994; Pen et al., 1993).

 

A vital nutrient for fish and other livestock is phosphorus which plays a key function in cell formation. It is an important element for fish reproduction, growth and skeletal development (Asgand and Shearer, 1997). The release of elevated concentrations of soluble phosphorus from fish farms into aquatic environment leads to phytoplankton growth which causes concentrations of dissolved oxygen to fluctuate (Li et al., 2004). Inclusion of exogenous phytase in fish (Buruah et al., 2007; Debnath et al., 2005a; b; Sardar et al., 2007; Cao et al., 2008), pig (Han et al., 1997) and poultry (Lei and Stahl, 2000) diets has been reported to improve availability of trace-elements, amino acids, minerals and energy. This also contributes significantly to ecological safety by reducing excretion of phosphorus.

 

Microbes are the most excellent sources for industrial production of phytases because of their high enzyme production and easy cultivation (Li et al., 2008). Phytase-producing symbiotic bacteria isolated from fish intestine can be used as probiotics in aquaculture to inhibit or decrease the toxicity of phytate in the microenvironment of the gut (Khan and Ghosh, 2012). Autochthonous phytase producing gut bacteria in freshwater fishes have been reported (Khan and Ghosh, 2012; Khan et al., 2011; Roy et al., 2009). However, information on phytase producing intestinal bacteria and yeasts in marine fish species in the Niger Delta is scarce.

 

Therefore, in the present study, we evaluated the phytase producing ability of intestinal bacteria and yeasts isolated from two marine fish species (Liza grandsquamis and Ethmalosa fimbriata) from New Calabar River in the Niger Delta.

 

1 Materials and Methods

1.1 Collection of Sample

Healthy live marine fish species (Liza grandsquamis and Ethmalosa fimbriata) were collected from New Calabar River in Nigeria with the assistance of a local fisherman and transported to the laboratory in sterile plastic bags.  

 

1.2 Isolation of phytase producing intestinal bacteria and yeasts

Phytase-producing bacteria and yeasts were isolated using a modified phytase-screening medium (MPSM) described by Khan and Ghosh, 2012. The medium contain: 0.01 g FeSO4 7H2O, 10 g glucose, 10 g urea, 2 g sodium citrate, 3 g citric acid, 1 g MgSO4 7H2O, 1 g (NH4)2SO4, 3 g sodium phytate and 20 g agar per litre of deionized water.

 

Two marine fish species (Liza grandsquamis and Ethmalosa fimbriata) were examined. For each replicate, three samples of fish were used. The average weight/length of Liza grandsquamis and Ethmalosa fimbriata were 22 g/10 cm and 11 g/8 cm respectively. The six fish samples were killed by physical damage of the brain. Prior to dissection, 70% ethanol was used to clean the fish superficially. From each collective gut contents, 1.0 g was taken with sterile precaution and suspended in 9.0 ml sterile normal saline. Serial dilution up to 10-6 was then carried out with the homogenate.

 

For isolation of phytase producing bacteria, 0.1 ml of each dilution was inoculated in triplicate on MPSM (pH 7.0) plates. The plates were incubated at 37oC for 72 h. Colonies with different morphological appearances from a particular plate were streaked singly on MPSM plates to obtain uncontaminated cultures. Based on their morphological and biochemical characteristics, the purified isolates were identified to generic level (Holt et al., 1994; Garrity et al., 2005; de La Maza et al., 2013).

 

For isolation of phytase producing yeasts, 0.1 ml of each dilution was inoculated in triplicate on MPSM (pH 4.5) plates. The plates were incubated at 28oC for 5 days. A portion of each yeast colony which developed was aseptically subcultured into fresh MPSM (pH 4.5) plates for purification. Characterization of yeasts isolates were based on colonial and microscopic examination as well as biochemical features. Identification to generic level was performed using the keys provided by Samson and De Boer, 1995.

 

1.3 Quantitative assay for extracellular phytase production

Phytase-producing bacterial and yeast strains isolated from the fish species examined were further evaluated for quantitative phytase assay with MPSM broth. The method described by Yanke et al., 1998 was employed. Broth culture (1.5 ml) was centrifuged at 18,000 g. The cells were washed with 0.1 M sodium acetate (pH 5.0) and then suspended in 750 µl buffer. Thereafter, 600 µl 0.2% (w/v) sodium phytate which was prepared with 0.1 M sodium acetate buffer (pH 5.0) was added to 150 µl of the cell suspension. It was incubated for 30 min at 39℃. Then 750 µl 5% (w/v) trichloroacetic acid and 750 µl phosphomolybdate colour reagent were added to stop the reaction. The colour reagent was newly prepared by adding 4 vols 1.5% (w/v) ammonium molybdate solution in 5.5% (v/v) sulphuric acid to 1 vol. 2.7% (w/v) ferrous sulphate solution. The colour was allowed to develop for 5 min. Then, the liberated inorganic phosphorous was measured at A840 with a Spectronic UV Spectrophotometer. One unit (U) of phytase activity was defined as 1µg of inorganic phosphorous released per 1 ml of culture filtrate per 1 min (Yanke et al., 1999).

 

1.4 Statistical analysis

Standard deviations for each of the experimental results were calculated using Excel Spreadsheets, with Microsoft excel software. Differences between treatments were examined for significance by one-way ANOVA and P = 0.05 was considered to be statistically significant. 

 

2 Results

2.1 Phytase producing intestinal bacteria

Phytase producing intestinal bacteria from Liza grandsquamis were identified as Bacillus (2 strains), Pseudomonas (1 strain) and Enterobacter (1 strain) (Table 1) while phytase producing intestinal bacteria from Ethmalosa fimbriata were identified as Bacillus (2 strains) and Enterobacter (1 strain) (Table 2).

 

  

Table 1 Phytase activity of bacterial isolates from Liza grandsquamis intestine

 

  

Table 2 Phytase activity of bacterial isolates from Ethmalosa fimbriata intestine

 

2.2 Phytase producing intestinal yeasts

 

The phytase producing intestinal yeasts from Liza grandsquamis were identified as Saccharomyces (2 strains) and Candida (1 strain) (Table 3). The phytase producing intestinal yeasts from Ethmalosa fimbriata were identified as Saccharomyces (2 strains) and Candida (1 strain) (Table 4).

 

  

Table 3 Phytase activity of yeast isolates from Liza grandsquamis intestine

 

  

Table 4 Phytase activity of yeast isolates from Ethmalosa fimbriata intestine

 

2.3 Quantitative phytase activity

Among bacterial phytate degraders, Bacillus sp. isolated from Liza grandsquamis intestine showed highest phytase activity (50.09 ± 0.15 U/ml) while Enterobacter sp.isolated from Ethmalosa fimbriata intestine showed the lowest phytase activity (2.75 ± 0.32 U/ml) (Tables 1 and 2). Among yeasts phytate degraders, Saccharomyces sp. isolated from Liza grandsquamis intestine showed highest phytase activity (12.5 ± 0.27 U/ml) while Candida sp. isolated from Ethmalosa fimbriata intestine showed the lowest phytase activity (3.66 ± 0.71 U/ml) (Table 3 and Table 4).

 

3 Discussion

Three bacterial genera Bacillus, Pseudomonas and Enterobacter isolated from Liza grandsquamis intestine (Table 1) and two bacterial genera Bacillus and Enterobacter isolated from Ethmalosa fimbriata intestine (Table 2) were found to utilize phytate for growth. A phytase producing bacteria such as Bacillus licheniformis isolated from Labeo rohita (Roy et al., 2009) and Rhodococcus sp. MTCC 9508 isolated from Catla catla (Khan et al., 2011) have been reported. It has also been reported that Acinetobacter sp., B. subtilis, B. cereus, B. thuringiensis and Bacillus sp. isolated from the gastrointestinal tract of Atlantic salmon (Salmo solar) that was fed with or without diet supplemented with chitin demonstrated phytase activity (Askarian et al., 2012). Phytase producers such as Brochothrix thermosphacta and Brochothrix sp. from gastrointestinal tract of Atlantic cod were also reported (Askarian et al., 2013).Bacillus atropheus and Bacillus subtilis isolated from Gudusia chapra and Labeo bata respectively also exhibited phytase activity (Khan and Ghosh, 2012). Several authors suggested that enzymes produced by such intestinal microorganisms may have a major function in digestion (Ariole et al., 2014; Ghosh et al., 2002a; b; Ray et al., 2010; 2012).

 

Two yeast genera Saccharomyces and Candida isolated from Liza grandsquamis (Table 3) and Ethmalosa fimbriata (Table 4) were found to utilize phytate for growth. A number of marine yeast strains from the intestine of marine fish (Acanthogobius hasta and Hexagrammos otakii) and sea cucumber (Holothuria scabra) have been recognized as phytase producers (Li et al., 2008). Such phytase producing microorganisms might aid in breakdown of phytate in the intestines of the host animal (Li et al., 2008).

 

The measured levels of phytase activity (Tables 1, 2, 3 and 4) suggest that fish gut bacteria and yeasts are a prospective source of phytase that can be commercially developed. Exploitation of such gut microbiota detected in the present study for breakdown of phytate in feed materials of plant source is possible for the improvement of the nutritive value of feeds rich in phytate. Results of the current study suggest that symbiotic phytase-producing bacteria and yeasts from fish intestine can be exploited as probiotics in aquaculture that may reduce the toxicity of phytate in the microenvironment of the gut and reduce faecal phosphorus pollution into the aquatic environment.

 

Authors’ contributions

SCU collected the samples and performed the experiment. CNA conceived, designed and supervised the study and also wrote the manuscript. All the authors read and approved the final manuscript.

 

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