Study On The Screening,Identifying And Application Of Low-temperature-tolerant Lactic Acid Bacteria From Forage Silage In Tibet

The objective of this study was to evaluate the fermentation characteristics of perennial ryegrass(Lolium perenne Lam.),tall fescue(Festuca arundinacea Schred.),alfalfa(Medicago sativa L.),common vetch(Medicago sativa L.)and bulrush(Phragmites australis)silages in Tibet,and then isolating,identifying and selecting low temperature-tolerant lactic acid bacteria from these silages.Further,effects of the selected lactic acid bacteria on fermentation quality of Italian ryegrass at different temperature(10??15? and 25?)in Tibet Finally,we studied the effect of the selected lactic acid bacteria on fermentation quality of tall fescue and oat in Tibet.The aim of this study was to explore lactic acid bacteria(LAB)that adapt to grow under the special climate conditions and provide theoretical basis for the production of LAB additives in Tibet.1 Study on the fermentation characteristics of main grasses in TibetThe objectives of this experiment was to analysis the chemical and microbial compositions of perennial ryegrass,tall fescue,common vetch,alfalfa and bulrush,then study their effects on the fermentation quality of silages.The results showed that five grasses had high dry matter(DM)and water soluble carbohydrate(WSC)contents,and perennial ryegrass,tall fescue and bulrush had low value of buffering capacity,which met the requirements of well preserved silage.But common vetch and alfalfa had high value of buffering capacity.However,the counts of epiphytic aerobic bacteria,mold and yeast of all fresh grasses were more,but the counts of lactic acid bacteria were less,which can not ensure successful fermentation.After 45 days of fermentation,all the values of pH were higher than 4.2(4.3?4.9),the contents of lactic acid were low(3.19?77.88)in perennial ryegrass,tall fescue,common vetch,alfalfa and bulrush silages,and the contents of butyric acid were high in perennial ryegrass and tall fescue silage.In summary,the results showed that it is difficult to ensile perennial ryegrass,tall fescue,common vetch,alfalfa and bulrush in Tibet as influenced by the shortage of counts and activity of epiphytic LAB..2 Isolation and identification of lactic acid bacteria from grasses silage in TibetA total of 20 homofermentiative LAB strains were isolated from grasses silage.Strains AG11,LCG9,CG35,T22,TG20,LG4,LLG7 and LL8 were identified as P.pentosaceus,strain LTG7 as P.acidilactici,strain LA3 as L.curvatu,strains LI3 and LIG5 as L.casei,strains 15 and LG2 as L.paraplantarum,strains LAG1,LCG3,I2,TG1 and L2 as L.plantarum,strain LT4 as L.pentosaceu according to conventional microbial identification methods and 16S rRNA sequence analysis.All the selected lactic acid bacteria have the characteristics of tolerance of low temperature,low and high pH value and high concentration of NACIl.Strains 15,LI3,LTG7 and LCG3 were selected as the optimal strains which could be used as additives to produce silage in Tibet due to their tolerance of low temperature,higher lactic acid producing ability and faster growth rates.And they were used in the following research.3 Effects of lactic acid bacteria on the fermentation quality of Italian ryegrass in Tibet at different temperatureThe aim of this experiment was to investigate the effect of L.paraplantarum 15,L.casei LI3,P.acidilactici LTG7 and L.plantarum LCG3 and commercial inoculants G on the fermentation quality of Italian ryegrass in Tibet at different temperatures.The results showed that all LAB inoculants significantly(P<0.05)improved the fermentation quality as indicated by higher(P<0.05)DM,WSC and lactic acid contents and lactic acid/acetic acid ratios,lower(P<0.05)pH values,volatile fatty acid(VFA)contents,ammonia nitrogen/total nitrogen(AN/TN)ratios and counts of aerobic bacteria,mold and yeast of treated silages as compared with the control silage at 10? 15? and 25 ?,then all LAB inoculants significantly(P<0.05)improved the fermentation quality of Italian ryegrass in Tibet at 10?,15? and 25?.In addition,strains 15,LI3,LCG3 and commercial inoculants G showed better effects on improving fermentation quality than LTG7,and strain 15 was the best among all the inoculants.4 Effects of lactic acid bacteria on the fermentation quality of tall fescue and oat in TibetThis part included two experiments.The aim of experiment 1 was to investigate the effect of L.paraplantarum 15,L.casei LI3,P.acidilactici LTG7 and L.plantarum LCG3 and commercial inoculants G on the fermentation quality of tall fescue in Tibet.After 35 days of fermentation,the results showed that all LAB inoculants significantly(P<0.05)improved the fermentation quality as indicated by higher(P<0.05)DM,WSC and lactic acid contents and lactic acid/acetic acid ratios,lower(P<0.05)pH values,volatile fatty acid(VFA)contents,ammonia nitrogen/total nitrogen(AN/TN)ratios and counts of aerobic bacteria,mold and yeast of treated silages as compared with the control silage.In addition,the silages inoculated with strains 15,LI3,LTG7 and LCG3 showed better effects on improving fermentation quality than commercial inoculants G,and strain 15 was the best among all the inoculants.The aim of experiment 2 was to investigate the effect of L.paraplantarum 15,L.casei LI3,P.acidilactici LTG7 and L.plantarum LCG3 and commercial inoculants G on the fermentation quality of oat in Tibet.After 35 days of fermentation,the results showed that the fermentation quality of silage with uninoculant was bad as indicated by high pH value and AN/TN ratio.Whereas,applying LAB inoculants significantly(P<0.05)improved the fermentation quality as indicated by higher(P<0.05)DM,WSC and lactic acid contents and lactic acid/acetic acid ratios,lower(P<0.05)pH values,volatile fatty acid(VFA)contents,ammonia nitrogen/total nitrogen(AN/TN)ratios and counts of aerobic bacteria,mold and yeast of treated silages as compared with the control silage.Silages inoculated with strains 15,LI3,LTG7 and LCG3 showed better effects on improving fermentation quality than commercial inoculants G,and strain 15 was the best among all theinoculants.In conclusion,the results of our study showed that the effect of inoculants L.paraplantarum 15,L.casei LI3,P.acidilactici LTG7 and L.plantarum LCG3 on improving fermentation quality of tall fescue and oat silage were better than commercial inoculants G.Inoculants 15,LI3,LTG7 and LCG3 could be used as biological additives to produce silage in Tibet,and 15 was the best among all the inoculants.