Identification Of 15 Novel Species Of Lactic Acid Bacteria From Pickle And Gastrointestinal Tract Of Honeybee

Pickle is a traditional fermented product with a long history.Cabbage as raw material,adding salt,and other seasoning,using its own attached microorganisms,fermented under natural conditions.Lactic acid bacteria play a leading role in the fermentation of sauerkraut.Honeybee is the most important pollinating insect in the world.Honeybee is the most important pollinating insect in the world.A large number of lactic acid bacteria reside in the gastrointestinal tract of honeybee,which has many functions including providing nutrition and resisting the invasion of pathogenic bacteria.The purpose of this study was to isolate lactic acid b acteria from pickle and honeybee intestines,and to identify 15 potential new species of lactic acid bacteria by means of bacterial polyphasic classification.The classification methods used include:16S r RNA gene sequence analysis,housekeeping gene(phe S,rpo A)sequence analysis,phylogenetic tree construction,genome sequence analysis,average nucleotide identity(ANI),digital DNA-DNA hybridization,phenotypic characterization and chemical characterization.Strains 382-1~T,116-1A~T,381-7~T,203-3~T,218-3~T and 398-2~T belong to Lactiplantibacillus.The sequence similarity of 16S r RNA,phe S and rpo A genes between strains 382-1~T,116-1A~T,381-7~T,203-3~T,218-3~T,398-2~T and type strains of phylogenetically related species Lactiplantibacillus argentoratensis,Lactiplantibacillus pentosus,Lactiplantibacillus plantarum,Lactiplantibacillus paraplantarum,Lactiplantibacillus fabifermentans,Lactiplantibacillus herbarum,Lactiplantibacillus xiangfangensis,Lactiplantibacillus garii,Lactiplantibacillus modestisalitolerans,Lactiplantibacillus plajomi and Lactiplantibacillus mudanjiangensis was97.1?99.9%,78.2?89.9%and 90.1?98.0%,respectively.ANI value was 74.2-91.2%and d DDH value was 19.1-43.3%,indicating that 382-1~T,116-1A~T,381-7~T,203-3~T,218-3~T,398-2~T were new species of lactic acid bacteria.Strain 778-3~T belongs to Paucilactobacillus.The type strains of phylogenetically related species of strain 778-3~T were:Paucilactobacillus hokkaidonensis,Paucilactobacillus wasatchensis,Paucilactobacillus suebicus,Paucilactobacillus vaccinostercus,Paucilactobacillus nenjiangensis,Paucilactobacillus oligofermentans.The sequence similarities of 16S r RNA,phe S and rpo A were97.0?99.4%,78.2?82.1%and 80.0?91.5%,respectively.ANI value was 71.6?78.6%and d DDH value was 18.8?22.9%,indicating that 778-3~T was a new species of lactic acid bacteria.Strains 6-5(1)~T,244-2~T,13B17~T and 866-3~T belonged to Levilactobacillus The sequence similarity of 16S r RNA,phe S and rpo A genes between strains 6-5(1)~T,244-2~T,13B17~T,866-3~T and type strains of phylogenetically related species Levilactobacillus cerevisiae,Levilactobacillus mulengensis,Levilactobacillus koreensis,Levilactobacillus enshiensis,Levilactobacillus fujinensis,Levilactobacillus tongjiangensis,Levilactobacillus yonginensis,Levilactobacillus fuyuanensis,Levilactobacillus parabrevis,Levilactobacillus tangyuanensis was 98.4?100%,81.0?97.1%and90.5?99.9%,respectively.ANI value was 75.7?95.1%and d DDH value was 21.8?61.2%,indicating that 6-5(1)~T,244-2~T,13B17~T,866-3~T were new species of lactic acid bacteria.Strain HN36-1~T belonged to Apilactobacillus.The sequence similarity of 16S r RNA,phe S and rpo A genes between strains HN36-1~T and type strains of phylogenetically related species Apilactobacillus kunkeei,Apilactobacillus apinorum,Apilactobacillus bombintestini,Apilactobacillus micheneri,Apilactobacillus quenuiae,Apilactobacillus timberlakei and Apilactobacillus ozensis was 95.6?99.8%,81.4?94.8%and 85.2?99.5%,respectively.ANI value was 71.6?92.0%and d DDH value was 17.0?45.9%,indicating that HN36-1~T was a new species of lactic acid bacteria.Strains 53-4~T,ZW152~T and 117-1~T belong to Lacticaseibacillus.The sequence similarity of16S r RNA,phe S and rpo A genes between strains 53-4~T,ZW152~T,117-1~T and type strains of phylogenetically related species Lacticaseibacillus hegangensis,Lacticaseibacillus camelliae,Lacticaseibacillus suibinensis,Lacticaseibacillus jixianensis,Lacticaseibacillus nasuensis,Lacticaseibacillus baoqingensis,Lacticaseibacillus manihotivorans,Lacticaseibacillus porcinae,Lacticaseibacillus yichunensis,Lacticaseibacillus rhamnosus,Lacticaseibacillus paracasei,Lacticaseibacillus casei,Lacticaseibacillus chiayiensis and Lacticaseibacillus zeae was95.8?100%,74.7?95.3%and 76.3?99.5%,respectively.ANI value was 68.5?95.4%and d DDH value was 17.0?63.8%,indicating that 53-4~T,ZW152~T,117-1~T were new species of lactic acid bacteria.In this study,15 potential new species of lactic acid bacteria were identified by polyphasic classification methods,and their taxonomic status was determined,and 15 new species of lactic acid bacteria were named.Strains 382-1~T,116-1A~T,387-1~T,203-3~T,218-3~T,398-2~T and 778-3~Twere published in October 2019 and named:382-1~T(Lactiplantibacillus pingfangensis sp.nov.),116-1A~T(Lactiplantibacillus daoliensis sp.nov.),381-7~T(Lactiplantibacillus nangangensis sp.nov.),203-3~T(Lactiplantibacillus daowaiensis sp.nov.),218-3~T(Lactiplantibacillus daoliensis sp.nov.),398-2~T(Lactiplantibacillus songbeiensis sp.nov.),778-3~T(Paucilactobacillus kaifaensis sp.nov.).In April 2020,Zheng Jinshui et al.reclassified Lactobacillus,so the strains 382-1~T,116-1A~T,387-1~T,203-3~T,218-3~T,398-2~T and 778-3~T were now named as:382-1~T(Lactiplantibacillus pingfangensis comb.nov.)?116-1A~T(Lactiplantibacillus daoliensis comb.nov.)?381-7~T(Lactiplantibacillus nangangensis comb.nov.)?203-3~T(Lactiplantibacillus daowaiensis comb.nov.)?218-3~T(Lactiplantibacillus daoliensis comb.nov.)?398-2~T(Lactiplantibacillus songbeiensis comb.nov.)?778-3~T(Paucilactobacillus kaifaensis comb.nov.).Strains HN36-1~T,866-3~T,6-5(1)~T,13B17~T,244-2~T,117-1~T,ZW152~T,53-4~T were published in 2021 and named as follows:HN36-1~T(Apilactobacillus nanyangensis sp.nov.),866-3~T(Levilactobacillus andaensis sp.nov.),6-5(1)~T(Levilactobacillus wangkuiensis sp.nov.),13B17~T(Levilactobacillus lanxiensis sp.nov.),244-2~T(Levilactobacillus qinggangensis sp.nov.),117-1~T(Lacticaseibacillus mingshuiensis sp.nov.),ZW152~T(Lacticaseibacillus suilingensis sp.nov.),53-4~T(Lacticaseibacillus qinganensis sp.nov.).