Volume 10, Issue 3: 144-149; May 27, 2020  
ISSN 2228-7701  
Aima Airenobuwa DAODU1, Gbemisola Deborah OLUMUYIDE1, and Lawrence EDEMHANRIA2  
1Department of Biological Sciences, Samuel Adegboyega University, PMB 0001, Ogwa, Nigeria  
2Department of Chemical Sciences, Samuel Adegboyega University, PMB 0001, Ogwa, Nigeria  
Email: ledemhanria@gmail.com; Phone: +2349055159224;  
: 0000-0002-6034-3780  
Supporting Information  
ABSTRACT: Bacterial phytases and phytase-producing bacteria are of great industrial significance in the poultry  
industry and also in phosphorus pollution management. This study was designed to isolate and screen for  
phytase producing lactic acid bacteria from the duodenum, ileum and cecum of eight healthy cockerel samples.  
Standard microbiological procedures were followed to isolate phytase producing lactic acid bacteria using de  
Man Rogosa and Sharp (MRS) agar while extracellular phytase screening was done using phytase specific  
medium. The range of total microbial count obtain was highest at the cecum (2.85±0.11 to 4.34±0.12 log10  
cfu/ml), lower at the duodenum (2.02±0.11 to 4.27±0.20 log10 cfu/ml) and lowest at the ileum (2.00±0.21 to  
4.19±0.25 log10 cfu/ml). Nineteen bacterial isolates were identified as lactic acid bacteria on the basis of  
morphological, biochemical and physiological characterization and later identified as Lactobacillus species  
(78.94%), Enterococcus species (15.78%) and Lactococcus species (5.26%). Thirteen out of the nineteen lactic  
acid bacteria showed phytase activity. Low phytase activity was observed in eight of the lactic acid bacteria  
isolates while five of the isolates produced significant extracellular phytase activity (>6mm). The most  
predominant Lactobacillus species were also found to be the most potent phytase producers. This can be  
exploited for industrial production of phytase in upgrading the nutritional status of feed and combating  
phosphorus pollution from poultry waste.  
Keywords: Phytase, Gastrointestinal tract, Lactic acid bacteria, Phosphorus pollution, Poultry industry.  
Phosphorus is an important nutrient stored in the form of phytic acid (Myo-inositol 1,2,3,4,5,6-hexakis dihydrogen  
phosphate) in cereals, legumes, and oilseed crops (Azeke et al., 2007). Phytic acid acts as antinutrient constituent in  
plant-derived food and feed as it forms complexes with proteins, amino acids, and various metal ions (Astley and Finglas,  
2016; Nissar et al., 2017). The bound phosphorus is poorly available to monogastric animals such as pigs, poultry and  
fishes, due to lack of production of phytases in the gastrointestinal tract (Jacela et al., 2010; Abdel-Megeed and Tahir,  
2015). Excretion of the undigested phytate poses a serious phosphorus pollution problem contributing to eutrophication in  
areas of intensive livestock production (Singh et al., 2011; Abdel-Megeed and Tahir, 2015). The enzyme phytase  
hydrolyzes the ester bond in phytic acid to liberate inositol and inorganic phosphate (Nissar et al., 2017). It can be sourced  
from some plants, animal tissues and microorganisms; microbial sources are however more promising for the  
commercial production of phytases (De Angelis et al., 2003).  
Phytases have been obtained mainly from filamentous fungi (Maller et al., 2013); it has also been detected in  
various bacteria species such as Bacillus, Pseudomonas, Escherichia coli, Enterobacter, Klebsiella, Lactobacillus  
sanfranciscensis as well as anaerobic rumen bacteria, particularly in Selenomonas ruminantium, Megasphaera elsdenii,  
Prevotella sp., Mitsuokella multiacidus and Mitsuokella jalaludinii (Shim and Oh, 2012).  
Species of lactic acid bacteria (LAB) belonging to numerous genus under the family of Lactobacillaceae have been  
widely applied in food fermentation worldwide due to their widely known status as generally recognized as safe (GRAS)  
microorganisms (Hayek and Ibrahim, 2013). They are also recognized for their fermentative ability which contributes to  
enhancing food safety, improving organoleptic attributes, enriching nutrients and increasing health benefits (Sharma et  
al., 2012; Steele et al., 2013). There are only few reports of phytase producing lactic acid bacteria available in literature,  
therefore this present study was designed to isolate phytase producing lactic acid bacteria from the gastrointestinal tract  
of poultry. The addition of phytase to poultry feed will improve the nutritional quality of feed by increasing the amount of  
free phytate phosphorus in poultry diet and diminishing the necessity of addition of inorganic phosphate to animal feed,  
thereby combating phosphorus pollution associated with the feed and poultry industries.  
Citation: Daodu AA, Olumuyide GD, and Edemhanria L (2020). Isolation of extracellular phytase producing lactic acid bacteria from the gastro intestinal tract of poultry  
Ethical Approval  
The Ethics Unit of the Research and Innovation Committee of Samuel Adegboyega University approved the study  
Study Area  
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.  
Data analysis  
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  
Table 1 - Total bacterial counts (Log10 cfu/ml) from the gastrointestinal tracts of samples.  
2.02 ± 0.11  
2.85 ± 0.11  
2.26 ± 0.17  
3.77 ± 0.12  
4.27 ± 0.20  
2.03 ± 0.15  
2.57 ± 0.38  
3.57 ± 0.37  
3.85 ± 0.33  
2.00 ± 0.21  
2.98 ± 0.25  
4.11 ± 0.20  
3.23 ± 0.22  
4.05 ± 0.13  
4.19 ± 0.25  
3.65 ± 0.25  
3.88 ± 0.14  
3.37 ± 0.14  
4.34 ± 0.11  
3.53 ± 0.12  
3.90 ± 0.18  
3.69 ± 0.11  
Values are mean ± standard error of mean of triplicate determinations. - = Absent, A-H = Isolation codes for the 8 chicken samples.  
Table 2 - Phytase screening of lactic acid bacteria from the gastrointestinal tract of poultry samples  
Isolation code  
Hydrolysis of phytate  
Clear zone (mm)  
Probable bacterial species  
5.13 ± 1.02a  
3.24 ± 0.86 b  
9.26 ± 2.11 c  
11.21 ± 1.32 d  
5.42 ± 1.51 a  
8.25 ± 0.93 e  
4.18 ± 1.44 f  
5.22 ± 0.76 a  
8.33 ± 0.43 eg  
2.38 ± 1.63 h  
7.44 ± 0.88 ei  
5.23 ± 1.05 a  
6.00 ± 1.32 a  
KEYS: A-H= isolation codes for the 8 chicken samples, + = positive, - = negative, d= duodenum, i= ileum, and c=cecum. Values with different  
superscript are significantly different (p˂0.05).  
Figure 1 - Percentage occurrence of lactic acid bacteria isolates from gastrointestinal tract of poultry samples  
Citation: Daodu AA, Olumuyide GD, and Edemhanria L (2020). Isolation of extracellular phytase producing lactic acid bacteria from the gastro intestinal tract of poultry  
The bacteria growth recorded in the duodenum, ileum and cecum of all the chicken samples had different growth count  
range (Table 1). The variation in the microbial population suggests that each region developed its own unique bacterial  
community due to the pH of the stomach contents, the toxicity of bile salts, fermentative metabolism and the relatively  
swift flow of the digesta in the gastrointestinal tract (Walter, 2008). This result agrees with the findings of other  
researchers available in literature. Jiangrang et al. (2003) reported differences in the diversity of bacterial floras in the ilea  
and ceca of maturing broiler chickens; Bjerrum et al. (2006) investigated microbial communities in the ileum and cecum  
of broiler chickens, they reported that lactobacillus species dominated the chicken ileum while the cecum harbored more  
diverse microbial community and Abbas et al. (2007) identified various levels of abundance of different lactobacillus  
species from the crop of 1- and 5- week old broiler chickens using 16s rRNA gene sequence.  
The isolates were identified as Lactobacillus species, Lactococcus species and Enterococcus species. Previous studies  
confirm the existence of these organisms in the gastrointestinal tract of chicken (Lan et al., 2003; Sonplang et al., 2007).  
The Lactobacillus species were more dominant because of their ability to adhere to the surface of the non-secretary  
epithelium lining of these sites, which enables the bacteria to form a biofilm-like structure that provides a bacterial  
inoculum of the digesta (Salas-Jara et al., 2016). Different studies on the microbiota of the gastrointestinal tract of poultry  
have pointed out the predominance of lactobacilli in chicken crops and intestine (Beasley et al., 2004; Bakari et al.,  
Thirteen out of the nineteen lactic acid bacteria showed phytase activity, suggesting that they could be a potential  
source of phytase to be used in improving the nutritional quality of poultry diet and decreasing the amount of phosphorus  
released to the environment (Hill et al., 2007; Abdel-Megeed and Tahir, 2015). Five of the Lactobacillus species were  
found to be the most potent phytase producers. Phytase producing ability of lactic acid bacteria has also been reported in  
some previous studies. Raghavendra and Halami (2009) isolated forty lactic acid bacterial strains with phytate degrading  
ability while Anastasio et al. (2010) reported the use of lactic acid bacteria to improve mineral solubilization during dough  
fermentation due to their production of phytate-degrading enzymes.  
In this study, phytase producing lactic acid bacteria were isolated from the gastrointestinal tract of healthy cockerels.  
These findings can be further explored in the industrial production of phytase. It will be of immense benefit to the poultry  
industry. Feed supplementation with phytase will help to improve the nutritional status of the feed. This also has  
implication for environmental management as it would lead to a reduction in phosphorus pollution.  
Competing interest  
The authors declare that they have no competing interest.  
The research was done following ethical procedures.  
Authors’ contribution  
AAD, LE designed the experiment; AAD, GDO carried out data collection; LE performed data analysis; AAD, GDO and  
LE contributed in manuscript preparation and approval for publication.  
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Citation: Daodu AA, Olumuyide GD, and Edemhanria L (2020). Isolation of extracellular phytase producing lactic acid bacteria from the gastro intestinal tract of poultry