Screening Of Lactic Acid Bacteria For Low Temperature Fermentation From Silage Of Elymus Nutans Growing On The Tibeten Plateau And Study On Its Fermentation Properties

In this study, the grass of Elymus nutans growing on the Tibetan Plateau was ensiled without any additives under different temperatures (5℃,15℃and25℃), and after60d of fermentation, fermentation quality of the ensiled grasses was evaluated; meanwhile, lactic acid bacteria from silages ensiled at different fermentation stages(2,4,6,8,12,14,30,60d)and temperatures(5℃,15℃,25℃) were isolated and identified by using16SrRNA in order to investigate lactic acid bacteria diversity in Elymus nutans silages; thereafter, the isolated lactic acid bacteria with better growth performance at low temperatures of10℃and15℃were selected and applied to Elymus nutans at ensiling. Subsequently, effects of the selected lactic acid bacteria on fermentation profiles of Elymus nutans silages stored at15℃and25℃were evaluated by using the same species of commercial inoculants as control. The aims of present study were to explore indigenous and suitable lactic acid bacteria strains that can be used for high quality silage making under low temperature climate of the Tibetan Plateau, and to provide lactic acid bacteria germplasm and theoretical basis for making high quality silage under the harsh environment of the Tibetan Plateau. The results showed that:1. After60d of fermentation, pH value of the silage stored at5℃declined to6.08, and lactic acid concentration was0.34%(%DM), which suggested that the silage almost did not ferment; for the silage ensiled at15℃, its pH value declined to5.68, and the lactic acid concentration increased to1.44%(%DM); while silages kept at25℃had good fermentation profiles with pH value of4.27and lactic acid concentration of6.21%(%DM). Statistical results showed that pH value of silage kept at25℃was significantly lower than that of the silage kept at15℃and5℃(P<0.05), and higher concentration of lactic acid was observed in silage kept at25℃than those kept at15℃and5℃(P><0.05).2. Totally108strains of lactic acid bacteria were isolated from different stages of fermentation at different temperature, and based on16S rRNA sequence analysis and phylogenetic tree, these108strains were classified into6genera and18species. The6genera of lactic acid bacteria include Lactobacillus, Leuconostoc, Weissella, Enterococcus, Lactococcus and Pediococcus; Leuconostoc and Lactobacillus were the dominant genus, which accounted for82.4%of the total isolated strains; the18species of lactic acid bacteria comprised by Lactobacillus casei, Lactobacillus plantarum, Lactobacillus brevis Lactobacillus curvatus, Lactobacillus crispatus, Lactobacillus coryniformis, Lactobacillus sakei, Leuconostoc mesentercides, Leuconostoc lactis, Leuconostoc citreum, Leuconostoc garlicum, Weissella cibaria, Weissella viridescens, Enterococcus faecium, Enterococcus durans, Pediococcus pentosaceus, Pediococcus acidilactici and Lactococcus lactis. The lactic acid bacteria found in this experiment covered almost all common lactic acid bacteria genera found in silages, but species were fewer as compared to the reported lactic acid bacteria from silages.3. In combination with the screening results of culture medium and fermented green juice, we found that LS-53showed obvious growth advantage at15℃(P<0.05), and PP-63grew faster in the early stage of fermentation, while LP-21made pH value dropped to3.9at15℃and achieved the maximum number of viable cellseory. Therefore, these three strains were selected as preferred candidates of lactic acid bacteria strains for low temperature fermentation of ensiling forage.4. physiological and biochemical characteristics experiment showed PP-63isolated from Tibetan Plateau can ferment raffinose, lactose, sorbitol, melibiose and sucrose, and LS-53isolated from Tibetan Plateau could ferment cottonseed sugar, laetrile, rhamnose, lactose, sorbitol, xylose, arabinose, melibiose and sucrose, but the same species of commercial strains isolated from low land could not use these sugars. Growing ability at different temperatures and pH value experiments showed that LS-53isolated from Tibetan Plateau could not grow at50℃, and the same species of commercial strain isolated from low land had a weak growth at50℃. Hence, compared with the commercial strains, lactic acid bacteria isolated from Tibetan Plateau showed specificities in physiological and biochemical characteristics.5. Inoculation of these three strains into Elymus nutans at15℃and25℃ensiled for50d, we found that LS-53significantly reduced silage pH value, propionicacid concentration and ratio of ammonia nitrogen/total nitrogen at15℃(P<0.05), salvaged more water-soluble carbohydrate and crude protein; Application of LP-21and PP-63as a combined inoculant to Elymus nutans significantly improved lactic acid concentration (P<0.05), resulting a lower ratio of ammonia nitrogen/total nitrogen, saved more crude protein and significantly reduced neutral detergent fiber content (P<0.05) as compared with the same strains of commercial inoculant. The above results indicated that three of the selected strains can improve silage quality of Elymus nutans growing on the Qinghai-Tibetan Plateau at low temperature, but these strains have no obvious advantages at25℃in comparsion with the commercial inoculants.