柱花草耐高温优质乳酸菌的筛选及对其青贮发酵品质的改善效果

Screening of high quality thermotolerant lactic acid bacteria in stylo and the improving effect on fermentation quality of its silage

为明确柱花草(Stylosanthes guianensis)青贮饲料中乳酸菌的耐高温情况及其对柱花草青贮品质的影响,采用传统微生物培养、分离、筛选结合16s rRNA基因鉴定技术研究了柱花草青贮饲料中的主要耐高温优质乳酸菌种类与生理生化特性,并用筛选出的优质乳酸菌菌株接种柱花草调制青贮饲料。结果表明:从柱花草青贮饲料中筛选出了7株可耐受45℃高温、7.5%NaCl盐度、pH 4.5酸度、至少可利用11种碳源、接种2 h后均能迅速产酸的优质乳酸菌;基因鉴定表明它们分属于粪肠球菌(Enterococcus faecalis)(S1、S2、S5和S6菌株)、人参土芽孢杆菌(Weizmannia ginsengisoli)(S3菌株)和戊糖片球菌(Pediococcus pentosaceus)(S4与S7菌株) 3个属。粪肠球菌S1菌株与戊糖片球菌S4菌株分别接种于柱花草青贮30 d后,不但显著减少青贮饲料的干物质损失(P <0.05),还使其pH、丁酸与氨态氮含量显著降低(P <0.05),乳酸含量显著高于植物乳杆菌(Lactiplantibacillus plantarum)接种组(P <0.05),且肠杆菌科数量减少,无霉菌和酵母菌检出;特别是粪肠球菌S1接种组的粗蛋白含量显著高于植物乳杆菌接种组(P <0.05),可有效提高柱花草青贮饲料发酵品质并且保存更多的营养成分。

The objective of this experiment was to clarify the thermotolerant lactic acid bacteria in stylo silage and their effects on the quality of stylo silage,traditional microbial culture,isolation and screening.This was combined with 16s rRNA gene identification technology that was used to study the main species of high quality thermotolerant lactic acid bacterium in stylo silage and their physiological and biochemical characteristics.High quality lactic acid bacteria strains were selected as the inoculant for stylo silage making.The results showed that seven strains of high quality lactic acid bacteria were screened from stylo silage,which could withstand 45℃high temperature,7.5%NaCl salinity,pH 4.5 acidity,could use at least 11 carbon sources,and could produce acid quickly after two hours of inoculation.Genetic identification revealed that they were Enterococcus faecalis(strains S1,S2,S5 and S6),Weizmannia ginsengisoli(strain S3),and Pediococcus pentosaceus(strains S4 and S7),respectively.Enterococcus faecalis S1 strain and Pediococcus pentosaceus S4 strain were inoculated to stylo silage and ensiled for 30 d.This not only significantly reduced the dry matter loss of silage(P<0.05),but also significantly decreased the pH value,butyric acid,and ammonia nitrogen contents(P<0.05).The lactic acid contents of inoculation S1 or S4 strain were significantly higher than that in Lactiplantibacillus plantarum strain inoculation(P<0.05).The number of Enterobacteriaceae decreased and there were no molds or yeasts detected among all silages of inoculation treatments.The crude protein content of the Enterococcus faecalis S1 inoculation group was significantly higher than that of the L.plantarum inoculation group(P<0.05),which could effectively improve the fermentation quality of stylo silage and preserve more nutrients.