| In this study, the grass of Elymus nutans growing in four different regions of the Qinghai-Tibetan Plateau(Tianzhu, Guoluo, Dangxiong, Naqu) was ensiled, and we extract the total bacterial DNA of the silage at different fermentation stages(14, 30, 60, 90 days) to find microbial biodiversity of the silage by high-throughput sequencing analysis. Meanwhile, dominant lactic acid bacteria(LAB) from the silage were separated and identified; subsequently, all the separated LAB stains were tested based on their low temperature resistant ability and acid producing ability to screen strains with good fermentation performance at low temperature.Thereafter, the selected strain was applied to Elymus nutans as silage additives and fermented at10, 15, 25℃ respectively. The study was conducted to determine the effects of the selected LAB strain on fermentation quality of ensiled Elymus nutans at different temperatures, and to thereby to provide a theoretical basis for developing appropriate lactic acid bacteria for making high quality silage on the Qinghai-Tibetan Plateau. The results are as follows:1.The biodiversity of the Elymus nutans silage of different regions and different fermentation stages showed some differences. In this study, lactic acid bacteria mainly include 6 genera:Enterococcus, Lactobacillus, Pediococcus, Leuconostoc, Weissella and Lactococcus. This 6genera of lactic acid bacteria include: Lactobacillus plantarum, Weissella cibaria, Pediococcus pentosaceus, Enterococcus mundtii, Lactobacillus brevis, Leuconostoc mesenteroides,Lactococcus lactis,et.2.Due to local geographical differences and different climatic conditions, there are also differences on lactic acid bacteria on fresh forage. Elymus nutans collected at Tianzhu containing 45% of Pseudomonas, 17% of Pantoea, 4.21% of Weissella cibaria and 1.03% of Leuconostoc mesenteroides. For Guoluo there contained 45.8% of Lactobacillus. For Dangxiong area, 94.46% of Weissella cibaria was the dominant species in this area. For Naqu, Leuconostoc mesenteroides accounted for 60%.3.Elymus nutans collected at the same area but on different fermentation stages made the differences of microbial species. Just for lactic acid bacteria, in Tianzhu silage fermented to 60 days other bacteria were replaced by lactic acid bacteria, there were 52.14% of Pediococcus pentosaceus, 21.19% of Lactobacillus, 15% of Leuconostoc mesenteroides, after 90 days,Leuconostoc mesenteroides, Lactobacillus coryniformis and Pediococcus pentosaceus accounted for 31.97%,24.32, and 19.67% respectively. In Guoluo, Lactobacillus played an important role,especially Lactobacillus brevis accounted for 60.19%, 71.02% and 73.76% on 14, 60, 90 fermentation stages. In Dangxiong, Weissella was replaced by Lactobacillus and Leuconostoc when fermentation started, after 14 days Lactobacillus accounted for 51.68%, fermented to 30 days it rose to 60.87%, to 60 days Lactobacillus brevis accounted for 82.75%, to 90 days Lactobacillus accounted for 59.61%. In Naqu, after 14 days fermentation, Weissella cibaria accounted for 33.83%, later, Lactobacillus became the dominant lactic acid bacteria accounted for47.51%, 73.57%, 68.87% respectively.4.In this study, totally 92 dominant LAB strains were isolated. These 92 strains belonged to Lactobacillus and Pediococcus, and included 4 species, there were 13 stains of Lactobacillus plantarum, 50 strains of Pediococcus acidilactici, 28 strains of Lactobacillus brevis and just 1strain of Lactobacillus paracasei. Through the screening of acid producing ability and low temperature resistance ability, 6 strains of good performance LAB strains were selected out, they are LB-40, LB-59, PA-60, PA-69, PA-87 and LP-66. LP-66 reduced the green guice’s p H value at10 and 15℃ significantly(P<0.05), PA-87 and LP-66 showed a batter growth advantage at 15 and25℃. PA-87 with the strongest sugar utilization ability compared with laboratory isolates and standard lactic acid bacteria. All 10 strains grown well at 10 to 50℃, and 3.5 to 9.0 p H range, but there are obvious differences on the growth ability between the standard lactic acid bacteria and strains isolated from the Qinghai-Tibetan Plateau.5.LP-66 and ALP purchased from America were inoculated to Elymus nutans and ensiled at 10, 15, 25℃ for 30 days,lactic acid content in LP-66 group was significantly higher than ALP group at 10℃(P<0.05). The content of ammonia nitrogen, NDF and ADF was significantly lower than ALP group at 15℃(P<0.05) and saved more crude protein. After 60 days of ensiling, LP-66 group’s p H value was significantly lower than that of ALP treatment group under 10,25℃. Yeast and mold were not detected in the LP-66 treatment group at 25℃. From these result we can preliminarily demonstrate that LP-66 group showed fermentation advantage at low temperature, can be used as alternative strain for silage making at Qinghai-Tibetan Plateau. |
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