海南天然饲草青贮饲料中乳酸菌分离鉴定及优良菌株筛选

doi:10.11733/j.issn.1007-0435.2022.10.033

草地学报 ›› 2022, Vol. 30 ›› Issue (10): 2819-2826.DOI: 10.11733/j.issn.1007-0435.2022.10.033

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海南天然饲草青贮饲料中乳酸菌分离鉴定及优良菌株筛选

刘悦¹, 字学娟¹, 陈婷¹, 孙蓉¹, 李茂^2,3, 汤凯⁴

1. 海南大学林学院, 海南儋州 571737;
2. 中国热带农业科学院热带作物品种资源研究所, 海南儋州 571737;
3. 中国热带农业科学院湛江试验站, 海南湛江 524000;
4. 海南大学食品学院, 海南海口 570228

收稿日期:2022-04-01 修回日期:2022-06-23 发布日期:2022-11-05
通讯作者: 字学娟,E-mail:zixuejuan@163.com;李茂,E-mail:limaohn@163.com
作者简介:刘悦(1998-),女,黑龙江绥化人,硕士研究生,从事热带牧草青贮微生物研究,E-mail:ly943959080@163.com
基金资助:
国家自然科学基金项目(31860680)；农业农村部拨改买项目(16220078)资助

Isolation and Identification of Lactic Acid Bacteria Strains in Natural Forage Crops Silages in Hainan

LIU Yue¹, ZI Xue-juan¹, CHEN Ting¹, SUN Rong¹, LI Mao^2,3, TANG Kai⁴

1. College of forestry, Hainan University, Danzhou, Hainan Province 571737, China;
2. Institute of tropical crop varieties and resources, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan Province 571737, China;
3. Zhanjiang Experimental Station, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang, Hainan Province 524000, China;
4. College of food science, Hainan University, Haikou, Hainan Province 570228, China

Received:2022-04-01 Revised:2022-06-23 Published:2022-11-05

摘要/Abstract

摘要： 为筛选适合热带牧草的高温发酵优良乳酸菌，改善青贮发酵品质，本试验从海南岛不同气候区采集野生草本植物斑茅(Saccharum arundinaceum)、蔓生莠竹(Microstegium vagans)和木本植物银合欢(Leucaena leucocephala)、构树(Broussonetia papyrifera)，青贮60 d后分离纯化乳酸菌，以培养36 h条件下最终发酵液pH≤4且OD(Optical density,吸光度)≥2作为条件初筛乳酸菌。用温度T=40℃，45℃，50℃和pH=3.0，3.5，4.0，6.0，8.0，9.0进行复筛，选取OD值较高的菌株作为优势菌株，再对其进行16S rDNA序列分析、生长和产酸速率测定以及生理生化实验。结果表明，本试验共分离到98株天然乳酸菌，经筛选得到4株优良乳酸菌(15B，10D，13C，14B)。经鉴定，10D，13C，14B为植物乳杆菌(Lactobacillus plantarum)，15B为乳酸片球菌(Pediococcus acidilactici)。菌株15B，10D，13C，14B均耐酸、耐高温、且生长及产酸性能优良，具有良好的青贮潜力并对致病菌具有良好的抑制作用，可作为热带地区制备青贮添加剂的备选菌种。

关键词: 天然饲草, 乳酸菌, 高温发酵, 分离培养, 16S rDNA序列分析

Abstract: In order to isolate excellent lactic acid bacteria strains suitable for high-temperature fermentation of tropical forages,Saccharum arundinaceum,Microstegium vagans and woody plants Leucaena leucocephala and Broussonetia papyrifera were collected in different climatic regions of Hainan Island. The strains were isolated and cultured on MRS medium from silage of 60 days,and they were selected under the conditions of pH ≤ 4 and OD ≥ 2 of 36 hours. Then they were re-selected under the conditions of T=40℃,45℃,50℃ and pH=3.0,3.5,4.0,6.0,8.0 and 9.0. The strains with high OD value were selected as the dominant strains. Then the 16S rDNA sequences were analyzed,and physiological and biochemical experiments,growth rate and acid production rate analysis were conducted. In summary,98 strains of natural lactic acid bacteria strains were isolated,and four lactic acid bacteria (15B,10D 13C,14B) were selected. The 16S rDNA sequence analysis showed that the similarity between 15B and Pediococcus acidilactici was 100%;The similarity between 10D,13C and 14B strains and Lactobacillus plantarum was 100%. In addition,15B,10D,13C and 14B showed high temperatures and acidic environment resistance,and had excellent acid production performance. They have good potential for ensiling and inhibitory effect on pathogenic bacteria. They can be used as alternative strains for preparing silage inoculants in tropical regions.

Key words: Natural forage crops, Lactic acid bacteria strains, High temperature fermentation, Isolation and culture, 16S rDNA sequence analysis

中图分类号:

Q939.11+7

刘悦, 字学娟, 陈婷, 孙蓉, 李茂, 汤凯. 海南天然饲草青贮饲料中乳酸菌分离鉴定及优良菌株筛选[J]. 草地学报, 2022, 30(10): 2819-2826.

LIU Yue, ZI Xue-juan, CHEN Ting, SUN Rong, LI Mao, TANG Kai. Isolation and Identification of Lactic Acid Bacteria Strains in Natural Forage Crops Silages in Hainan[J]. Acta Agrestia Sinica, 2022, 30(10): 2819-2826.

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海南天然饲草青贮饲料中乳酸菌分离鉴定及优良菌株筛选

Isolation and Identification of Lactic Acid Bacteria Strains in Natural Forage Crops Silages in Hainan

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