Isolation And Identification Of A Lactate-Utilizing, Butyrate-Producing Bacterium From Porcine Feces And Its Metabolic Characteristics In Virtrol

The distal mammalian intestine represents an anaerobic bioreactor programmed withan enormous population of bacteria, which provide us with genetic and metabolic attributeswe have not been required to evolve on our own, including the ability to harvest otherwiseinaccessible nutrients. The anaerobic microbial communities produce a large amount ofSCFA as their main non-gaseous fermentation end products. SCFA are assimilated by themammalian host, and provide a high proportion of the total energy gained from the diet inherbivores, especially ruminants. The overall contribution of SCFA towards the host'senergy requirement is far lower in non-ruminants, but they have an important influence oncolonic health. In particular butyrate is the preferred energy source for the colonic mucosaand has been implicated in protection against colitis and colorectal cancer.In the first study, a lactate-utilizing, butyrate-producing bacterium strain LB01 wasobtained from the feces of an adult pig by using the modified Hungate technique. Twentyfive bacteria were isolated and purified with the YCFA media supplemented with lactate asthe single energy and carbon source. Lactobacillus bacteria were eliminated by the MRSsolid medium, and then obligate anaerobic bacteria were selected with the YCFA andM₂GSC media. The selection method was accompanied with the GC that could determinethe composition of the short chain fatty acids. A high butyrate-producing bacterium wasfinally obtained, which produced more than 10 mmol/L butyrate in the fermentation liquidat 24h.The second study was designed to identify the morphology, biochemical and moleculartraits of strain LB01. Strain LB01 was an obligately anaerobic and Gram positive bacteriumwith no spore and flagella, and can utilized glucose, maltose, fructose and lactate, whichresembled the traits of Megaphaera. Identification access of strain LB01 was EF053126.The result of the 16S rRNA BLAST revealed that strain LB01 had a closest relative withUncultured rumen bacterium 3c3d-18 and Megasphaera hominis (unpublished),respectively99.3% and 99.0%. Moreover, strain LB01 had a closer relative with M. endensii, with 98% homology. The identification demonstrated that strain LB01 belongs toMegasphaera.The third study was designed to investigate the metabolic characteristics of strainLB01 by means of the fermentation system in vitrol. Media supplemented with glucose,maltose, fructose and lactate supported the growth of strain LB01, but media supplementedwith maltose, fructose and lactate better supported it. Media singly supplemented withacetate failed to support the growth of strain LB01, but media supplemented with acetateand glucose can better support it than glucose, indicating that strain LB01 can synthesizebutyrate at the cost of acetate. Strain LB01 tolerated to low pH, and improved the pH oflower media with the growth. Owe to distinct energy and carbon source, the composition ofshort chain fatty acid was evidently discrepant.The last study was designed to investigate the cross-feeding between strain LB01 andlactobacillus strain Kg. Strain K9, cultured in the isolated YCFA medium, produced a largeamount of lactate by glucose fermentation pathway and reduce the pH of liquid. StrainLB01, cocultured with strain K9, was able to efficiently consume the lactate produced bystain K9, blocked the lactate accumulation in media and decelerated the pH decline becauseof lactate consumption by strain LB01, where butyrate was the main composition of shortchain fatty acids. The result of MRS amounting method revealed that strain K9 in coculturewas more than that in isolated media, indicating the cross-feeding between strain LB01 andstrain K9.