| The Tibet Plateau is extremely high areas with annual average temperature of below 0?.The silage fermentation at natural low temperature is insufficient and corruptible,however less relevant research about the mechanism of silage at low temperature were reported.In order to improve the silage quality at the Tibet Plateau and establish a low-temperature-ensiling system,this paper mainly carried on the following five parts research:(1)The multiple wheatgrass samples were collected from most regions in Qinghai Province and Lhasa in Tibet Autonomous Region,from which 112 lactic acid bacteria were isolated and identified to species or subspecies by 16S rRNA gene combined with morphological characteristics and physio-biochemical characteristics.The abundant epiphytic lactic acid bacteria resources of indegious wheatgrass in Tibet Plateau were found,of which 39.13%were Leuconostoc,21.74%were Lactococcus,17.39%were Enterococcus,and the rest were Weissella?Carnobacterium and Pediococcu.Their tolerance to salt,acid-base,high/low temperature and bacteriostatic activity were tested,three representatives have wide spectrum antibacterial activity with effective inhibition on Micrococcus luteus?Escerichia coli?Staphylococcus aureus?Salmonella enterica?Listeria monocytogenes?Pseudomonas aeruginosa and Bacillus subtilis,of which the two isolates QZ311(Pediococcus cellicola)and QZ1137(Leuconostoc mesenteroides)can survive in 3.0%and 6.5%NaCl cultures,low temperatures of 5? and 10?,high temperature of 45? and 50? conditions,and have strong receivable ability of pH changes between pH 3.5-pH 9.(2)The microorganism profile,fermentation quality and rumen digestibility in vitro of maize-stalk silage at different maturity stages were studied.Maize-stalk samples were harvested at the stages milk-ripe,dough,fully ripe,and fully ripe exposed to air for 3 or 10 days.Silage pH,ammonia-N and chemical composition were measured.Represented isolates from the raw materials were categorised into five profile clusters:Leuconostoc citreum(23.1%),Weissella paramesenteroides(15.4%),Lactococcus garvieae(23.1%),Enterococcus faecalis(1.7%),and Lactobacillus paraplantarum(30.8%).The total numbers of lactic acid bacteria in silages with raw materials reaped at the stages of the milk-ripe,dough,fully ripe,and fully ripe exposed to air for 3 or 10 days,respectively,were approximately 8,6,10,3.5 and 10 log CFU/g.The dominant lactic acid bacteria groups of maize-stalk silage at different stages were varying.The pathogens such as Escherichia coli,aerobic bacteria,Filamentous fungi and yeasts were found in the silages from different reaptimes.There were significant differences(P<0.001)in crude protein,ether extract,dry matter,acid detergent fibre and organic matter of silage at different stages;however,no significant difference at P = 0.05 was observed in dry matter digestibility after 24 h of fermentation in vitro.(3)Lactic acid bacteria can be used as silage additives to ensure rapid and vigorous fermentation at early stages of ensiling.We presumed that the optimal inoculation dosage for forage at ambient temperature would be different from that at cold temperature.In this study,the effects of inoculum dosage on the fermentation quality of oat(Avena sativa L.cv.Qinghai)and wheat(Triticurm aestivum L.cv.Yumai No.1)silage at room temperature and 4? were investigated in laboratory experiments.Little or no improvement in silage quality occurred upon increasing the inoculum dosage at room temperature(15-38?).By contrast,a lower pH and ammonia-N content,and higher lactic acid bacteria counts and lactic acid content were observed at 4?.Furthermore,Escherichia coli was effectively inhibited at the lower temperature,while silage quality was positively correlated with increasing inoculum dosage(P<0.05).These results suggest increasing the inoculum dosage could improve the quality of silage at lower temperatures of 4?,while an appropriate dosage was a key factor for silage at room temperature.Oat forage could be used at low temperature but was not suitable for ensiling at room temperature without wilting due to the high moisture content(86.55%).QZ227(Lactobacillus plantarum)isolated from the root of wheat in alpine regions displayed superior antibacterial properties against yeast at room temperature and Escherichia coli at 4? compared with commercial strain FG1.This is the first study to explore the effects of inoculum dosage on silage quality at low temperatures.(4)Six representative lactic acid bacteria isolates from the Tibet Plateau with low-temperature resistant,acid-stable,strong acid-producing ability and broad-spectrum antibacterial function were selected to ferment mature oat at constant low temperature.The nutritional compositions and the microbiological variations in the fermentation process were analysed,in order to provide useful references for natural low-temperature ensiling.The six lactic acid bacteria isolates were as follows:QZ311(Pediococcus cellicola),QZ1137(Leuconostoc mesenteroide s),QZ227(Lactobacillus plantarum),QZ613(Lactococcus lactis).QZ251(Enterococcus,mundtii)and QZ666(Lactococcus lactis subsp.lactis).QZ221,QZ251,QZ613 and QZ666 as single addictives while the QZ311 and QZ1137 as the mixed addictives,were inoculated to the mature oat ensiling with a concentration of 10 logCFU/g FM refering to the maturity choices and inuculum dosage advices.Under the constant low temperature of 5?,pH of silage inoculated with QZ227 and QZ666 were reduced to 3.94 and 3.79,respectively,at 7th day,while the silage treated with QZ251 and QZ613 were reached to 3.81 and 4.12 after 30 days.The pH of silage treated with mixed inocula were 4.26 at 30th day.The Escherichia coli,Bacillus cereus,Clostridium,mold and yeast were inhibited effectively by the lactic acid bacteria addictives at 7 and 30 days.No significant effect on crude protein and crude fat were found in the fermentation process.The six representative isolates presented potential benefits to natural low-temperature ensiling as they reduced the pH and inhibited the pathogens of mature oat silage at constant low-temperature.(5)Five different species of selected broad-spectrum antibiotic lactic acid bacteria isolated from extremely high-cold areas were used as starters to ferment indigenous forage oats and wheatgrass under rigid alpine climatic conditions.The five isolates were Lactobacillus plantarum QZ227,Enterococcus mundtii QZ251,Pediococcus cellicola QZ311,Leuconostoc mesenteroides QZ1137 and Lactococcus lactis QZ613,and commercial Lactobacillus plantarum FG1 was used as the positive control and sterile water as the negative control.The minimum and maximum temperatures were-22 ? and 23 ? during the fermentation process,respectively.The pH of wheatgrass silage fermented by the QZ227 and FG1 inocula reached the expected values(4.15)although the pathogens detected in the silage should be further investigated.All of the inocula additives used in this study were effective in improving the fermentation quality of oat silage as indicated by the higher content of lactic acid,lower pH values(4.17)and significant inhibition of pathogens.QZ227 exhibited a fermentation ability that was comparable with the commercial inoculum FG1 for the whole process,and the deterioration rate was significantly lower than for FG1 after storage for 7 months.The pathogens Escherichia coli,mold and yeast were counted and isolated from the deteriorated silage.Escherichia coli were the main ammonia-N producer while Filarmentous fungi and yeast produced very little.In this study,the representative lactic acid bacteria isolated from the Tibet Plateau with strong acid-producing ability,low temperature resistance,and broad-spectrum inhibition on pathogens were used as starters,refering to the dynamic variations of corn silage from different maturity and oat silage at constant low temperature,on basis of the inoculum dosage choices results,the excellent isolates were used to ferment the mature oat and wheatgrass in the Tibet Plateau,the feasible ensiling system for natural low temperature was initially established. |
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