| The mammalian intestine inhabits of many species of microorganisms,the number is up t0 1013-1014 CFU/mL,and the microbial metabolic activity plays an important role in maintaining intestinal health.Some of intestinal microorganisms' metabolism,such as Lactobacillus,Bifidobacterium,Enterococcus,will produce large amounts of lactate,which mainly represents for two configurations:L-lactate and D-lactate,and the accumulation of D-lactate would led to serious consequences to animals.In fact,the concentration of lactate in healthy animals' fecal samples is very low,this is because lactate can be digested and metabolized by other bacteria in the gastrointestinal(GI)tract.These bacteria can not only metabolize excess lactate in the digestive tract,prevent lactic acidosis,maintain normal pH of GI tract,but also produce a large number of short chain fatty acids(SCFAs),which have an important impact on the health of large intestine.Until now,study on lactate-utilizing bacteria of monogastric animal is rarely reported,especially the D-lactate-utilizing bacteria isolated from pig.Therefore,our study focus on the utilization of different isomers of lactate by fecal microbiota of piglets firstly,then isolate and identify D-lactate-utilizing bacteria from pig feces and study the metabolic charactristics in vitro.In the first section,we aimed to investigate the characteristics of piglets'fecal microbiota utilizing different isomers of lactate in vitro.L-lactate,D-lactate,DL-lactate was added as substrate in the fermentation in vitro and piglets' feces were prepared as inoculum.The concentrations of lactate were determined during the fermentation.The composition of bacterial communities in the inocula and 24 h fermentation fluids was analyzed by pyrosequencing.The results showed that after 24 h fermentation,no significant difference in relative abundances of all phylums was observed among the three substrate groups.On genus level,however,lactate substrates significantly affected the composition of bacterial community.Significantly lower contribution of Coprococcus,Subdoligranulum to total bacteria in D-lactate group was found as compared to L-lactate and DL-lactate mixture groups.On the contrary,the abundance of Ruminococcus,unclassified Clostridiales and unclassified Lachnospiraceae in D-lactate group was significantly higher than the other two groups(P<0.05).After 24 hours,Coprococcus Desulfovibrio and Oscilibacter were enriched by more than ten times,while the abundance of genus Blautia,Clostridium,Dorea,Faecalibacterium,unclassified Erysipelotrichaceae and unclassified Peptostreptococcaceae decreased to 0.1-0.5 of those in the inocula.The results suggest that lactate accumulation in the hindgut is related to the cooperation between microbiota members,and regulating bacterial community may be a possible way to control lactate accumulation.In the second section,we aimed to isolate and identify the D-lactate-utilizing bacteria from pigs.Fresh feces were inoculated to the YCFA culture media added with D-lactate as the sole carbon source.After enrichment,isolation and purification in anaerobic condition,25 strains were gained in the experiment.By measuring the characteristics of utilizing D-lactate of these 25 strains within 24 h,6 strains(J1,J2,J3,J4,J5 and J6)which could make full use of D-lactate within 24 h were picked.Conventional identification and molecular biology identification were used to identify the six D-lactate-utilizing bacteria.Results showed that the six strains were identified to:Bacteroides fragilis J1(the representative strains of Bacteroides,gram negative bacilli);Bacteroides acidifaciens J2(similar to the characteristics of Bacteroides fragilis);Veillonella denticariosi J3(similar to Veillonella paroula);Veillonella caviae J4(gram-negative anaerobic bacteria);Bacteroides uniformis J5(gram-negative,non-spore,obligate anaerobic bacteria);and Megasphaera elsdenii J6.The six strains could utilize glucose,maltose,lactate and other carbon source,and produce large amounts of gas.In the third section,we aimed to investigate the fermentation characteristics of the 6 different D-lactate-utilizing bacteria in vitro and preferential utilization of different lactate isomers of Megasphaera elsdenii J6.Isolates were inoculated on YCFA medium containing 25 mmol/L of D-lactate under 37?.Samples were harvested at each time point after incubation to measure growth rates,D-lactate and SCFAs concentrations.All strains had a significant ability to utilize D-lactate,which was concluded after in vitro fermentation with 25 mmol/L D-lactate as the sole carbon source.Of all six strains,Megasphaera elsdenii J6 showed the highest efficiency of D-lactate utilization and produced a higher ratio of butyrate in total short chain fatty acids(SCFAs).Thus,the in vitro fermentation characteristics of this strain in D-lactate,L-lactate and DL-lactate mixtures(D-lactate:L-lactate=1:1,D-lactate:L-lactate=1:2)were further studied.The results showed that M.elsdenii J6 preferred utilizing D-lactate than L-lactate and DL-lactate,and produced more SCFAs such as acetate,propionate and butyrate when using D-lactate as the sole carbon source(P<0.05).D-lactate dehydrogenase activity was significantly higher than L-lactate dehydrogenase activity in all substrate groups(P<0.05).When glucose was used as the carbon source,a rapid decrease in the glucose concentration with the increase in lactate was found after incubation.After that,the lactate concentration decreased rapidly with the exhaust of glucose,which suggested that M.elsdenii J6 represented the characteristics of primary and secondary metabolism.The results suggest that the administration of D-lactate-utilizing bacteria such as M.elsdenii J6 may have a potential advantage in the alleviation of D-lactic acidosis in the animal gut. |
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