Detection and molecular characterization of butyrate-producing genes in probiotic lactic acid bacteria for use in livestock

Butyrate-producing gut microflora synthesizes and secretes butyrate which serves as source of energy and stimulates rumen development in young animals. The present study was undertaken to characterize and screen butyrate-producing lactic acid bacteria (LAB) for probiotic use in animals in order to manipulate their gut flora for the benefit of host health and productivity. Twenty strains of LAB isolates from faeces of West African Dwarf (WAD) goats were screened for probiotic potentials. The potential probiotic LAB were characterized by 16S rRNA gene sequencing method. Polymerase chain reaction was then used to detect the butyrate kinase (buk) gene in probiotic LAB strains. The 16S rRNA gene sequencing identified the potential probiotic LAB as strains of Lactobacillus acidophilus, L. reuteri, L. plantarum, L. helveticus and L. fermentum. Their probiotic potentials were demonstrated by their ability to tolerate low pH, bile acid and lysozyme. The PCR analysis revealed that gene encoding butyrate kinase is present in only Lactobacillus plantarum PLB5. The study revealed that these LAB strains could be developed into useful probiotics in improving the health, nutrient digestibility and growth performance of livestock, but only L. plantarum PLB5 possesses the ability to produce butyrate.

Introduction economy of West Africa (Adebayo and Chineke, proskauer test and sugars fermentation tests were then 2011).
carried out on the probiotic bacterial isolates. Prevention or control of enteric diseases in animals has been reported to be achieved by the Molecular characterization of probiotic lactic acid incorporation of antibiotics in the feed of the bacteria by 16S rRNA gene sequencing animals. However, the demand for withdrawal of The genomic DNA of the probiotic lactic acid antibiotics from the feed of farm animals has bacterial strains was extracted using Cetyl Trimethyl increased the interest of using alternative natural Ammonium bromide (CTAB) method as described by products that allow maintenance of high Chen et al. (2006) and Sridhar et al. (2010) with little productivity and reduction of morbidity and modifications. mortality in intensive farms. Probiotics as feed An overnight broth culture (2.0ml) was centrifuged at o additives might benefit the host animal by 14,000rpm at 25 C for 1 minute to pellet the cells. The promoting its growth by competing with harmful supernatant was carefully discarded and the pellets gut flora, and stimulating the immune system were re-suspended in 400µl of pre-warmed CTAB buffer thereby increasing resistance to infectious agents and vortexed gently. 75µl of 10% SDS was added to the (Cross, 2002;O'hara et al., 2006).
suspension. Thereafter, 12µl of lysozyme solution Short-chain fatty acids such as butyrate, (400mg/ml) was added to each cell suspension and propionate and acetate are the end-products of the mixed gently. The suspension was heated in a water o dietary carbohydrate breakdown by anaerobic bath at 65 C for 30 minutes. It was then allowed to cool. bacteria in the gastrointestinal tract (GIT) of 10µl of 20mg/ml of proteinase K solution was added to o animals and humans (Raz et al., 2007). These fatty the suspension and incubated at 37 C for 30 minutes. acids, most especially butyrate, are the preferred After incubation, 500µl of chloroform was added and sources of energy (Hold et al., 2003). Butyrate mixed thoroughly. The cell suspension was then supplies energy and stimulates rumen development centrifuged at 10,000rpm for 10 minutes and the in young animals (Defrain et al., 2004 suspension was centrifuged at 10,000rpm for 10 Less consideration has been given to the minutes and the supernatant was carefully discarded. possible use of butyrate-producing bacteria as The pellet was rinsed with 500µl of 70% ethanol, mixed probiotics for animals. This study was therefore thoroughly and then centrifuged at 10,000rpm for 10 undertaken to characterize probiotic lactic acid minutes, so as to remove residual contaminants. The bacteria by 16S rRNA gene sequencing method as supernatant was discarded and the DNA pellet was airwell as to screen for the butyrate-producing lactic dried for 1hr. Finally, the DNA pellet was re-suspended acid bacteria for probiotic use in animals, using in 200µL of nuclease-free water. polymerase chain reaction (PCR) method to amplify The concentration and purity of DNA extracted bacterial gene encoding butyrate kinase.
from each isolate was determined using NanoDrop Lite Spectrophotometer.

Materials and Methods
The 16S rRNA genes of the probiotic LAB strains Bacterial strains and growth conditions were amplified by polymerase chain reaction using a Twenty lactic acid bacteria (LAB) were pair of 16S rRNA universal primers designated as 27F isolated from faeces of West African dwarf (WAD) . conditions. These isolates were screened for Polymerase chain reaction was performed in a probiotic activity by subjecting them to series of in-20µl reaction volume containing 2.0µl of template DNA (1µg), 10.0µl of 2× PCR master mix (Norgen Biotek vitro probiotic tests such as tolerance to low pH, Corporation, Canada) which contains Taq DNA tolerance to bile, tolerance to lysozyme, antibiotics polymerase, dNTPs, reaction buffer, MgCl , KCl and PCR resistance patterns, antibacterial activity against 2 pathogenic bacteria of WAD goats and haemolytic enhancer/stabilizer; 1.0µl of forward primer (2.5µM), test using the methods described by Oloyede and 1.0µl of reverse primer (2.5µM) and 6.0µl of nuclease-Afolabi (2013)  The amplified polymerase chain reaction (PCR) elongation at 72 C for 2 minutes. Reactions were o products were analysed in a 1.0% (w/v) agarose gel terminated at final extension of 72 C for 5 minutes. The electrophoresis in 1x TAE buffer at 100V for 1 hour. PCR products were visualized by electrophoresis on a A 1kb DNA ladder (O'GeneRuler) was used as 1% (w/v) agarose gels, stained with ethidium bromide molecular size marker. The gel was then stained in the presence of 100bp PCR sizer ladder (Norgen with ethidium bromide solution, visualised in a gel Biotek Corporation, Canada). Electrophoresis was documentation system and photographed. The performed at 100V for 1hour. amplicons were purified and sequenced. Sequence assembly and alignment were carried out using CLC Results bio software. Gene sequences were compared with Characterization of probiotic Lactic acid bacterial strains GenBank database at National Centre for Eight out of the twenty lactic acid bacteria Biotechnology Information (NCBI) using BLASTn (LAB) strains isolated from faeces of WAD goats were search tool to identify the strains. The phylogenetic observed to possess probiotic characteristics. tree was constructed by the neighbour-joining Microscopically, the probiotic isolates were Gram method using CLC bio software. The topologies of positive rods, non-motile, catalase negative and lack trees were evaluated by bootstrap analysis of the endospore. The PCR products obtained following sequence data with the software based on 100 amplification with the 16S rRNA primers were estimated random resamplings.
to be 1,500bp in size (Plate 1). The results of the phenotypic characteristics and sequence similarity (%) Amplifications of gene encoding butyrate kinase by BLASTN in GenBank of the National Center for Biotechnology Information (NCBI) library are shown in (buk gene) by Polymerase chain reaction Tables 1 and 2. Genotypic identification results showed The presence of gene encoding the that the probiotic lactic acid bacteria isolated from synthesis of butyrate i.e. butyrate kinase gene (buk faeces of WAD goats belonged to the species of gene) in probiotic lactic acid bacterial strains was Lactobacillus. The strains showed 95-100% 16S rRNA determined by polymerase chain reaction (PCR). sequence homology to Lactobacillus fermentum (2 The nucleotide sequence of the forward primer used in PCR of butyrate kinase gene was 5'-TGC isolates), Lactobacillus acidophilus (2 isolates), GTC AAA TCT TGG TGG AA-3' and that of the Lactobacillus reuteri (1 isolate), Lactobacillus plantarum reverse primer was 5'-AAG TAC CTC CAC CCA GGT (2 isolates) and Lactobacillus helveticus (1 isolate) GT-3' (Raz et al., 2007).
( Table 2). These isolates were able to grow at pH 1.5, Each of the polymerase chain reactions was 2.0 and 2.5 for about 4 hours incubation (Data not performed in a 10µl reaction volume containing shown). The isolates also fulfilled other in-vitro 1.5µl of template DNA (1µg), 5.0µl of 2× PCR probiotic criteria such as tolerance to bile acids and master mix (Norgen Biotek Corporation, Canada) lysozyme. which contains Taq DNA polymerase, dNTPs, The phylogenetic tree of the isolated probiotic r e a c t i o n b u f f e r, M g C l , K C l a n d P C R 2 lactic acid bacteria, based on 16S rRNA sequences enhancer/stabilizer; 1.0µl of forward primer (Neighbor-Joining analysis of CLC bio software) (2.5µM), 1.0µl of reverse primer (2.5µM) and 1.5µl grouped them in three clusters (Figure 1). of nuclease-free water. PCR reactions were carried  In recent years, worldwide interest in the use of probiotic bacteria for health promotion and disease prevention has increased significantly in scientific community, consumers and food producers (Bujnakova et al., 2013). This interest is based on the knowledge that the microorganisms to be used as probiotics will possess suitable properties that may have beneficial effects on animal and human health. These properties include bile tolerance, gastric juice resistance as well as the production of antimicrobial compounds such as lactic acid that inhibits the growth of other microorganisms (Hoque et al., 2010). These Table 2: Molecular identification of probiotic lactic acid bacteria isolated from faeces of WAD goats based on 16S rRNA gene sequencing method Figure 1: Neighbor-Joining phylogenetic tree of the 16S rDNA sequences of the probiotic lactic acid bacteria isolated from faeces of WAD goats characteristics allow the probiotic organisms to be polymerase chain reaction (PCR) results using butyrate established in the intestinal tract as well as enable kinase (buk) primer sets for detecting gene that codes them to survive, grow and perform their beneficial for synthesis of butyrate confirmed that butyrate kinase action in the gastrointestinal tract (GIT) of the hosts gene was found in only one lactic acid bacterial isolate (Oloyede and Afolabi, 2013). Therefore, probiotic (Lactobacillus plantarum PLB5). Since the LAB used in cultures must survive in the environment with this study were isolated from the faeces of WAD goats, gastric and bile acids. Results from this study the result of the present study is consistent with the revealed that 8 out of the 20 (40%) lactic acid previous reports that most gut microbes possess the bacteria (Lactobacillus acidophilus PLB1, L. genes for butyrate kinase (Barcenilla et al., 2000;Louis acidophilus PLB4, Lactobacillus reuteri PLB2, et al., 2004), but not in agreement with the report of Abbas (2009) who found gene for butyrate production Lactobacillus plantarum PLB5, Lactobacillus (buk gene) in none of the LAB tested. However, other plantarum PLB6, Lactobacillus fermentum PLB3, investigators had found butyrate kinase (buk) genes in Lactobacillus fermentum PLB8 and Lactobacillus isolates of Enterococcus faecalis (Louis et al., 2004) and helveticus PLB7) isolated from faeces of West Enterococcus durans with probiotic characteristics from African Dwarf (WAD) goats could tolerate acidic medium and gastric bile, thus, these LAB strains human faeces (Raz et al., 2007). Although, studies have would be able to survive high acidic environment in reported that lactobacilli and Bifidobacteria do not the stomach and high concentration of bile produce butyrate, the presence of butyrate kinase gene components in the intestine when animals consume in Lactobacillus plantarum PLB5 revealed that very few feeds containing these organisms. These results strains of lactobacillus could be able to produce therefore corroborate the previous results of butyrate. This fatty acid provides energy to the animals Oloyede and Afolabi (2013) that isolated strains of for carrying out other metabolic activities in the body, Lactobacillus acidophilus and Lactobacillus enhancing the ruminal fermentation and the growth performance of the animals. Furthermore, butyrate plantarum with high probiotic characteristics from provides a defence against cancer and ulcerative colitis raw milk of WAD goats. In addition, many strains of in animals and humans, as well as used to promote the these lactic acid bacteria have also been reported beneficial response of the immune system (Abbas, by many authors as probiotic bacteria and to a 2009). This acid also reduces the adhesion of harmful certain extent, are used in the production of bacteria such as E. coli, Salmonella spp, Shigella spp and probiotic preparations that are used in animals and human health (Bhattacharyya, 2009;Sarkono et al., other pathogens on mucosal surfaces of the animals 2010; Oloyede and Afolabi, 2013). thereby affecting their colonization. It also lowers the Bile resistance of some organisms has been pH of the GIT of the animals, thereby affecting the reported to be related to specific enzyme activity, survival of the pathogens in the GIT of the animals and bile salt hydrolase (BSH), which helps to hydrolyse therefore, improving the health of the animals. conjugated bile, thus reducing its toxic effect. The Therefore, the use of Lactobacillus plantarum PLB5 study therefore revealed that the potential probiotic observed in this study to possess butyrate kinase lactic acid bacterial isolates which were able to enzyme, as probiotics in livestock in Nigeria will not only survive high concentration of bile acid had high bile improve their growth performance, but also improve salt hydrolase (BSH) activity. BSH activity has their health. mostly been found in organisms isolated from the In conclusion, the probiotic butyrate-producing intestines or faeces of animals, which is in Lactobacillus plantarum PLB5 isolated in this study accordance with the results of this study. could potentially make a significant contribution to the The taxonomy of lactic acid bacteria based growth of livestock industry in Nigeria. However, in-vivo on comparative 16S ribosomal RNA sequencing selection studies are needed to ascertain the analysis has revealed that some taxa generated on colonization and adherence capacities of Lactobacillus the basis of phenotypical features do not plantarum PLB5 as well as its safety before the design of correspond with their phylogenetic relations.
any animal intervention trials. Molecular techniques are therefore important for the specific characterization of lactic acid bacterial Acknowledgement strains. In this study, the 16S rRNA gene The authors acknowledge the supports of the amplification using 27F and 1492R primers revealed Biotechnology Centre and Laboratory of Microbiology that the probiotic isolates were lactic acid bacteria department, Federal University of Agriculture, and the lengths of amplification were 1,500bp.
Abeokuta, Nigeria for providing the enabling facilities These sizes were similar to the sizes previously for carrying out this research study. obtained by many authors. Similarly, the