青贮用乳酸菌的分离鉴定及生物学特性评价

Isolation,Identification and Biological Characteristics Evaluation of Lactic Acid Bacteria for Silage

本研究旨在从构树青贮、玉米青贮及饲喂构树青贮的羔羊粪便中分离鉴定可饲用乳酸菌,为开发构树青贮用乳酸菌提供试验材料。以构树青贮、玉米青贮以及饲喂构树青贮的羔羊粪便为菌株分离来源,使用含有1%碳酸钙的MRS琼脂培养基分离纯化乳酸菌,获得菌株经16S rRNA测序鉴定后对其中可饲用菌株进行生长、产酸性能的筛选,评估在pH 3.0、3.5、4.0条件下存活率和对大肠杆菌、金黄色葡萄球菌和沙门氏菌的抑制能力,并对筛选菌株进行菌株之间拮抗性测定。结果表明:获得可饲用乳酸菌共37株,包括植物乳杆菌(Lactobacillus plantarum)、布氏乳杆菌(Lactobacillus buchneri)、副干酪乳杆菌(Lactobacillus paracasei)、粪肠球菌(Enterococcus faecalis)4种,不同的菌株在生长和产酸性能上表现出差异,在不同pH中存活率和抑菌性能也具有菌株特异性。综合比较不同菌株之间的生物学特性,植物乳杆菌GQ-3-2、GQ-3-29及GQ-5-4具有良好的生长、产酸及抑菌性能;粪肠球菌Y-3-15在发酵2 h进入对数生长期,能够为植物乳杆菌的大量繁殖创造良好环境;布氏乳杆菌GQ-3-21产酸性能良好,在发酵0~16 h pH快速下降,在pH 4.0的条件下存活率大于50%,在pH 3.5的条件下也能够微弱存活,是有潜力的青贮接种菌。最终选择菌株GQ-3-2、GQ-3-29、GQ-5-4、Y-3-15和GQ-3-21作为青贮用乳酸菌复合制剂的候选菌株,5株候选菌株之间不会互相抑制生长。综上所述,本试验筛选获得5株可饲用乳酸菌,其生物学特性良好,菌株之间无拮抗作用,可作为后续开发构树青贮饲料的乳酸菌复合制剂菌种来源。

The objective of this study was to isolate and identify the edible lactic acid bacteria from the feces of Broussonetia papyrifera silage,corn silage and feces of kids fed with Broussonetia papyrifera silage,and to provide experimental materials for developing lactic acid bacteria for Broussonetia papyrifera silage.Using Broussonetia papyrifera silage,corn silage and feces of kids fed with Broussonetia papyrifera silage as the sources of strain isolation,the lactic acid bacteria were isolated and purified by MRS agar medium containing1%calcium carbonate.The obtained strains were identified by 16S rRNA sequencing,and then the feed strains were screened for growth and acid producing performance,the survival rate and inhibition ability to Escherichia coli,Staphylococcus aureus and Salmonella under the conditions of pH 3.0,3.5 and 4.0 were evaluated,and the antagonistic ability among screened strains was determined.The results showed that 37 strains of edible lactic acid bacteria were obtained,including Lactobacillus plantarum,Lactobacillus buchneri,Lactobacillus paracasei and Enterococcus faecalis.Different strains showed differences in growth and acid-producing performance,and showed strain specificity in survival rate and antibacterial performance in different pH.A comprehensive comparison of the biological characteristics of different strains showed that Lactobacillus plantarum GQ-3-2,GQ-3-29 and GQ-5-4 had good growth,acid production and antibacterial performance;Enterococcus faecalis Y-3-15 entered the logarithmic growth phase after 2 hours of fermentation,which could create a good environment for the mass reproduction of Lactobacillus plantarum;Lactobacillus brucellosis GQ-3-21 had good acid production,and pH could decrease rapidly in 0 to 16 hours of fermentation,the survival rate was more than 50%under the condition of pH 4.0,and it could survive weakly under the condition of pH 3.5,so it is a potential silage inoculation bacteria.Finally,strains GQ-3-2,GQ-3-29,GQ-5-4,Y-3-15 and GQ-3-21 were selected as candidate strains of lactic acid bacteria compound preparation for silage,and the five candidate strains could not inhibit each other’s growth.In conclusion,five strains of edible lactic acid bacteria are screened in this experiment,which have good biological characteristics,and there is no antagonism among them,it can be used as the source of lactic acid bacteria compound bacteria for the subsequent development of Broussonetia papyrifera silage.