MATERIALS AND METHODS
The Ethics Unit of the Research and Innovation Committee of Samuel Adegboyega University approved the study
The study was carried out at College of Basic and Applied Sciences, Samuel Adegboyega University, Ogwa in Esan
West Local Government Area of Edo State, Nigeria.
Sample collection and preparation
Eight cockerels were purchased from Global Poultry, Uromi, Esan North East Local Government Area, Edo State,
Nigeria. The gastrointestinal tracts of the eight chickens were aseptically collected in ten sterile plastic bags and
transported to the laboratory in ice packs for microbiological analysis. The samples were represented with codes A-H. The
duodenum, ileum and cecum represented with codes d, i and c for each of the eight samples were removed separately
under sterile conditions to give a total of twenty-four samples.
Enumeration and isolation of bacteria
Ten grams of the duodenum, ileum and cecum respectively for each sample was weighed aseptically and transferred
into a sterile beaker containing 100ml of normal saline. Six-fold serial dilution (10-1 to 10-6) was made using normal
saline. An aliquot of 1 ml of the appropriate six-fold serial dilution (10-2) of the intestinal samples were inoculated into the
de Man Rogosa and Sharp (MRS) agar plates using standard pour plate method and incubated anaerobically at 37°C for
36 hours. Visible discrete colonies on inoculated plates were counted using the colony counter and expressed in colony
forming units per millilitre (cfu/ml) of the intestinal sample. Discrete colonies were selected and purified by subculturing
in MRS broth. Further purification was carried out by repeated streaking on freshly prepared MRS agar plates. The pure
isolates were stored at 4°C using MRS agar slants.
Characterization and identification of bacterial isolates
Pure cultures of all isolates were characterized and identified by means of their cultural, morphological, physiological
and biochemical characteristics using Bergey’s manual of systematic Bacteriology (Holt et al., 1994)
Phytase activity screening
The isolated pure strains were screened for the production of extracellular phytase using phytase specific medium
(Chunshan et al., 2001). The phytase screening medium was prepared by dissolving 3g glucose; 1g Tryptone; 1g sodium
phytate; 0.3g Cacl2; 0.5g MgSO4; 0.04g MnCl2; 0.0025g FeSO4; and 15g agar in 1 litre of distilled water. The pure cultures
were streaked at the centre of the plate and the plates were incubated at 370C for 62 hours as described by Kumar et al.
(2011). The plates were then observed for formation of clear zone around the colony. A clear zone around the colony
indicates positive result. Only those with zones greater than 6mm in diameter were recorded as significant.
The mean, standard error of mean, one way ANOVA and Tukey’s Post Hoc analysis were done using IBM SPSS
Statistics 23 software for Windows. P value ˂ 0.05 was statistically significant.
The total bacterial count from the duodenum, ileum and cecum of the eight chicken samples are presented in Table 1. A
total of fifty-seven bacteria isolates were randomly selected based on distinct colony morphology and purified. The
morphological, physiological and biochemical characteristics of the pure isolates revealed that 49.12% of the bacterial
isolates were white, viscous, entire, glistering and raised. 10.53% were creamy, viscous, entire, glistering and flat. 26.32%
were white, viscous, entire, glistering and raised. 12.28% were white, dry, entire, rough and raised. 1.75% were creamy,
viscous, entire, glistering and raised. Nineteen out of the fifty-seven bacterial isolates were presumed as lactic acid
bacteria on the basis of gram stain reaction, catalase production and oxidase activity. The isolates were gram positive
short rods and cocci, catalase negative and oxidase negative. Further presumptive tests including growth at temperature
100C and 450C, growth at pH 4.5 and 6.5, gas production from glucose and ability to ferment various carbohydrates
(lactose, maltose, sucrose and glucose) performed indicated that growth was recorded for all the isolates at pH 4.5 and
pH 6.5 at 450C only. The isolates were identified as Lactobacillus, Lactococcus and Enterococcus species. The percentage
occurrence of the lactic acid bacteria isolates is shown in Figure 1. Thirteen out of the nineteen lactic acid bacteria
isolates showed phytase activity by hydrolyzing sodium phytate to form a clear zone around the colony (Table 2). Five
bacterial isolates, all Lactobacillus species, (Dc2, Dd2, Dd4, Fd1 and Fc3) had a significantly different (p˂0.05) ability to
hydrolyze phytate by forming a clear zone > 6mm.
Citation: Daodu AA, Olumuyide GD, and Edemhanria L (2020). Isolation of extracellular phytase producing lactic acid bacteria from the gastro intestinal tract of poultry