Molecular Identification of Lactic acid bacteria Isolated from Lactating Cattle Lower Gut as a Potential Probiotic Species

Cattle are reared for meat and milk production to fulfill the dietary requirements of growing human population all over the world. It is indispensable to increase milk production and cattle health to fulfill the demands of human beings. Performance of dairy animals primarily dependent on balanced gut microbiota. Healthy microbial balance is essential to obtain maximum return from dairy animal (Malmuthuge, 2017). Therefore, substantial and keen interests on gut microbiota have been depicted in the recent years among scientific communities. Microbial diversity inside gut includes many beneficial bacterial species known as probiotics.

Bacterial species belonging to different genera (Bifidobacterium, Lactobacillus, Enterococcus and Pediococcus) are accepted as animal probiotics (Kery et al., 2018). Probiotic species have tendency to adapt to the environment in the cattle gut due to tolerance to bile acid, affinity to glycoprotein and intestinal mucosa. They also have properties of hydrophobicity, aggregation for colonization, mucin binding property and ability to adhere to intestinal epithelium of the host (Manhar et al., 2016). They synthesize bactericidal proteins and bacteriocins which are unfavorable for pathogens. In addition to above stated characteristics probiotic also have ability to exist during food processing and storage. They are viable at freezing temperature (Makete, 2017).

In Pakistan, different products containing probiotics are available in local market with different brand names such as EcotecTM. These products may not be very effective for our local breeds due to high cost. The economic importance of probiotic feed additives depend on different factors such as cost of microbial strain used in feed additive, type, age, diet and geographical site  of animal breed fed with probiotic supplement (Sandes et al., 2017). In this scenario, there is a need to isolate and identify probiotic strains suitable for local use and probiotic feed supplement formulations affordable by a common man. Therefore, present study was planned to get better understanding of bacterial strains indigenous to cattle gut. This is one of the most important step in the development of indigenous probiotics feed supplement, because each strain is important in its functional point of view.

Sample collection
 
Thirty (30) fecal samples were aseptically collected by hand directly from deep in the rectum of Sahiwal cattle, 26 ±1 months of age from National Agriculture Research Center (NARC), Islamabad.
 
Isolation of the bacteria
 
For dilution, 1gm of sample was mixed in 10 ml phosphate buffer saline (PBS) and vortexed for 15-20 minutes. DeMan, Rogosa and Sharpe (MRS) agar media was used for growth and isolation of beneficial bacteria from the diluted samples. 100 µl of each diluted sample was spread on MRS agar and then incubated at 37°C for 24-28 h.
 
Morphological and biochemical characterization of isolates
 
To obtain pure isolates, colonies from incubated plates were sub-cultured on the MRS agar media. The pure isolates were selected morphological identification. Some colonies of microbes were selected for different biochemical tests. Bacterial colonies indicated as catalase and oxidase negative were further tested for probiotic tests.
 
Determination of Probiotic Potential
 
Enzymatic potential
 
To detect the cellulolytic ability of the isolate, media described by Manhar et al., (2016) was used. To determine amylolytic activity, starch (1g) and nutrient agar (1g) were dissolved in distilled water (100 ml) and pure bacterial culture was grown on it. Proteolytic activity of the isolates was determined by using method as described by Marlida et al., (2014).
 
Acid tolerance
 
The acid tolerance was determined by preparing the MRS broth tubes of different pH (4.0, 5.0 and 6.0). 1 ml fresh pure culture was put into the MRS broth and incubated at 40°C aerobically. Pour plate method was used for counting of viable bacteria at 0, 2 and 4 hours and incubated at 40°C under aerobic conditions for 48 h and Colony forming unit per milliliter (CFU/ml) was calculated.
 
Antibiotic resistance
 
Disc diffusion method was used for determination of the antibiotic resistance (Olatunde et al., 2018). Antimicrobial activities of the bacterial strains was done by using the pathogenic strains such as, Listeria monocytogenes, E.coli and Staphylococcus aureus based on Shakira et al., (2017).
 
Tolerance to lysozyme, pepsin and Pancreatin
 
Tolerance to lysozyme, pepsin and Pancreatin was assessed following the method reported by Santiago et al., (2018).
 
Auto-aggregation and hydrophobicity assay
 
Auto-aggregation assays were done by using the method described by Collado, Meriluoto and Salminen (2008) while hydrophobicity assay was done by the method given by Collado et al., (2008).
 
Molecular identification
 
The bacterial strains (PBC-3, PBC-6 and PBC-9) were identified by using 16S rRNA gene sequencing. All bacterial strains sequences were submitted to NCBI for getting the accession numbers. Phylogenetic tree was prepared based on unambiguously aligned 16S rRNA gene sequence of three bacterial species identified in the study using MEGA-X (Molecular Evolutionary Genetics Analysis) software.
 
Statistical Analysis
 
The experimental results were expressed in form of mean value of CFU/ml ± standard deviation of the data. Significant differences between means were found by one way analysis of variance (ANOVA) by using SPSS software. P value <0.05 was considered statistically significant. 

Many probiotic supplements are commercially available in market but their probiotic potential in feed of local breed is uncertain. There is need to investigate indigenous probiotic species from local animal breed. The probiotic strains isolated from native organism of same ecological niche may be more compatible with gut microbiota (Shakira et al., 2017). Therefore, the current experiment was performed to isolate probiotic from Sahiwal breed of lactating cattle. Twenty pure bacterial cultures were isolated and examined on the basis of morphological and biochemical characteristics (Fig 1; Table 1) and Gram staining (Fig 2). The presence of Lactobacilli in the gut of Sahiwal cattle was also substantiated by previous findings which summarizes morphological and biochemical tests results according to Bergey’s manual of systematic bacteriology (Chowdhury et al., 2012). As a result of morphological, biochemical and probiotic tests three strains (PBC-3, PBC-6 and PBC-9) were identified as probiotics. According to data on NCBI, Strain PBC-3 showed 100% homology with Enterococcus faecalis (Table 2). Enterococcus faecalis was also isolated and identified from cattle fecal samples by Jackson et al. (2011). Strain PBC-6 showed 100% homology with Pediococcus acidilactiti (Table 2). Rodriguez et al. (2009) also isolated Pediococcus acidilactiti from cattle gut. Strain PBC-9 showed 100% homology with Pediococcus pentosaceus (Table 2). To the best of my knowledge, the species Pediococcus pentosaceus has not been reported so far from fecal samples of lactating cow making the present information novel regarding this species. In literature, scientists studied this strain in cattle milk (Verma et al., 2017) but in this context Pediococcus pentosaceus was isolated from cattle feces because cattle gut is rich source of bacterial diversity which may be varied due to different factors. These factors include breed differences, diet of animal, management practices and methodology of study, geography, age of cattle (Weese et al., 2017). The phylogenetic tree (Fig 3,4,5) depicted evolutionary history of all three bacterial species.















Tolerance against low pH is noticeable trait of probiotic bacteria. Since, to reach the intestine, probiotics have to survive the acidic environment of stomach. The pH value in stomach can be very low as 1.0 but mostly during in vitro assays pH value 3.0 to 4.0 is preferred because below pH 3.0 the viability of bacterial strains decreases (Naeem et al., 2018). All selected bacterial strains showed tolerance to wide range of pH from 4 to 6.  PBC-6 and PBC-9 strains showed significantly (p<0.05) good results at low pH followed by PBC-3 strain (Table 3). It was observed (Fig 6,7) that maximum viability (log CFU/ml) of PBC-3 strain was observed at pH 5 after 3 hours and can grow up to pH 4 when observed at different time intervals (0, 2 and 4 hours of incubation). It was observed that PBC-6 strain show more viability (log CFU/ml) at pH 6 and maximum CFU value after three hours of incubation (Fig 8). It was observed that PBC-9 strain show maximum viability (log CFU/ml) at pH 6 after 3 hours time. The viable bacterial count at pH 5 to 5.5 on MRS media was in accordance with the findings of Morandi et al., (2005).









The enzymatic activity of probiotic strains is also a vital phenomenon which increases feed utilization efficiency (Shakira et al., 2017). Strain PBC-3 (Enterococcus faecalis) showed positive proteolytic activity (Table 6). This bacterial strain contains very efficient proteolytic system which is helpful to reduce allergenicity of milk protein (Biscola et al., 2016). PBC-6 (Pediococcus acidilactiti) showed positive results in amylolytic, proteolytic and cellulolytic activity. Amylase enzyme is produced by bacterial strain hydrolyzes and ferment starch and convert it into lactic acid (Abassiliasi et al., 2017). The isolated PBC-6 strain also showed cellulolytic activity as reported in previous findings (Jason et al., 2018). In agreement with this context, some researchers also found positive enzymatic potential in Pediococcus acidilactiti (Jason et al., 2018). Strain PBC-9 (Pediococcus pentosaceus) has ability to produce cellulase enzyme which is responsible for cellulolytic activity of bacteria (Table 6). Similar results were reported by Lee et al., (2016). 
       
Antimicrobial activity is very important property for unique probiotics. Antimicrobial impact of PBC-6 and PBC-9 isolates are persistent by producing important organic acids, hydrogen peroxide and some antimicrobial substances and bacteriocins (Table 4). Similarly good results were seen in term of antibiotic tests (Table 5) Atienza et al., (2013). The hydrophobicity and auto-aggregation tests are the indicative of adhesive capacity for the probiotic selection.  The PBC-6 and PBC-9 isolates showed good hydrophobicity, as well as auto-aggregation property in present study (Table 7) as also reported by Osmanagaoglu et al., (2010). The microbial surface characteristics were noted in order to see the interactions between bacteria and interface. From safety point of view, the hemolytic activity and antibiotic resistance of our strains were determined. The results (Table 5) showed that the antibiotic resistance was seen in PCB-3 strain (Izumi et al., 2005).