基于分离培养和高通量测序分析一种自然发酵饲料的微生物组成  

Analysis of microbial composition in a naturally fermented feed based on culture isolation and high-throughput sequencing

作　　者：梁国健 洪居恳 丁茂昌 薛明[1] 陈伟哲 梁华芳[1] 温崇庆[1] LIANG Guojian;HONG Juken;DING Maochang;XUE Ming;CHEN Weizhe;LIANG Huafang;WEN Chongqing(Fisheries College,Guangdong Ocean University,Zhanjiang,Guangdong Province 524088,China;Hainan Haichang Prawn Breeding Co.LTD,Wenchang,Hainan Province 571300,China)

机构地区：[1]广东海洋大学水产学院,广东湛江524088 [2]海南海昌对虾繁育有限公司,海南文昌571300

出　　处：《中国饲料》2023年第17期7-14,共8页China Feed

基　　金：国家重点研发计划“蓝色粮仓”科技创新重点专项(2020YFD0900205);廉江市养殖集团有限公司委托项目(009)。

摘　　要：为揭示一种对虾养殖用自然发酵饲料的微生物组成,通过MRS、营养肉汤(NB)、豆芽汁(BSJ)平板分别计数其微生物含量,并对分离代表菌株16S rRNA或5.8S-ITS基因进行测序分析和鉴定;分别利用高通量测序16S rDNA V4区和ITS2区研究发酵饲料细菌和真菌菌群的多样性。分离培养结果表明:MRS、NB和BSJ平板对发酵饲料乳酸菌、细菌和酵母菌的计数结果分别为4.80×10^(7)、5.00×10^(6)、3.95×10^(6) cfu/g;从分离代表菌中鉴定出3种芽孢杆菌,4种分别属于慢生乳杆菌属、宿主关联乳杆菌属、干酪乳酪杆菌属和黏液乳杆菌属的乳酸菌,以及1种酵母菌库德毕赤酵母。高通量测序分析表明:乳杆菌科在发酵饲料细菌群落中占绝对优势,并包含20个分类操作单元(OTU)和9个属,其中乳杆菌属和黏液乳杆菌属相对丰度分别为66.95%和3.36%;假单胞菌科、依格纳季氏菌科和微杆菌科分别为第2、第3和第4优势科,其下的假单胞菌属、食肉蝇单胞菌属、假棍状杆菌属相对丰度分别为11.81%、10.16和3.58%;真菌群落中毕赤酵母科占绝对优势,其次为丝孢酵母科,两者分别完全由毕赤酵母属和丝孢酵母属构成,相对丰度分别为85.73%和12.54%。综上,该自然发酵饲料中富含乳酸菌、芽孢杆菌和毕赤酵母等有益活菌,且乳酸菌种类丰富。To reveal the microbial composition of a naturally fermented feed for shrimp aquaculture.The microbial contents of the fermented feed were determined by plate counting with MRS,nutrient broth(NB)and bean sprout juice(BSJ)media,respectively.And the representative isolates were identified by sequencing analysis of 16S rRNA or 5.8S-ITS gene.The bacterial and fungal diversities of the fermented feed were investigated by high-throughput sequencing of 16S rDNA V4 region and ITS2 region respectively.The results of culture isolation showed that the contents of bacteria,lactic acid bacteria and yeast in the fermented feed were 4.80×10^(7),5.00×10^(6),3.95×10^(6) cfu/g counted by MRS,NB and BSJ plates,respectively.Three kinds of Bacillus,4 kinds of lactic acid bacteria belonging to Lentilactobacillus,Ligilactobacillus,Lacticaseibacillus and Limosilactobacillus respectively,and a yeast species,Pichia kudriavzevii,were identified from the representative isolates.The high-throughput sequencing analysis showed that the Lactobacillaceae was absolutely dominant in the bacterial communities in the fermented feed,which included 20 operational taxonomic units and 9 genera,of which the relative abundances of Lactobacillus and Limosilactobacillus were 66.95%and 3.36% respectively;Pseudomonaceae,ignatzschneria_f and Microbacteriaceae were the second,third and fourth dominant families respectively,and the relative abundances of Pseudomonas,Wohlfahrtiimonas and Pseudoflavivibacter under them were 11.81%,10.16 and 3.58% respectively.In the fungal community,Pichiaceae was absolutely dominant,followed by Trichosporonaceae,which were completely composed of Pichia and Trichosporon,with relative abundance of 85.73%and 12.54%respectively.In conclusion,the naturally fermented feed was rich in beneficial living microflora such as lactic acid bacteria,Bacillus and Pichia,and harboured many kinds of lactic acid bacteria.

关 键 词：发酵饲料 分离培养 高通量测序 乳酸菌 酵母菌 芽孢杆菌 

分 类 号：S816.3[农业科学—饲料科学]