Identification And Growth Performance Evaluation Of 20 Strains Of Lacticaseibacillus

Within the genus Lacticaseibacillus,three species,including Lacticaseibacillus casei,Lacticaseibacillus paracasei and Lacticaseibacillus rhamnosus,In our previous study,we isolated some Lacticaseibacillus paracasei and Lacticaseibacillus casei strains but at that time,we only conducted 16S r RNA gene sequenceing,which was not enough accurate.The purpose of this study is to more accurately determine the taxonomic status of these strains at the level of species or subspecies,and initially evaluate their growth performance.20 test strains were isolated from traditional fermented yogurt from Inner Mongolia and Tibet.The results of the 16S r RNA gene,8 house-keeping genes(phe S,rec A,atp A,rpo A,rpo B,gyr B,hsp60,tuf)and Whole Genome sequences analysis,indicated that 18 strains including(0901,1056,1095,1378,1391,2053-1.L2524,X0119-1,X0383,X0427,X0479,X0691,X0789,X0799-1,X0799-2,X0873 and Y0506)and Lacticalibacillus paracasei were phylogenetically related;two strains(X0308B and X0308S)and Lacticaseibacillus casei were most closely related.Representative strains 0901 and X0308B were selected and to compared with reference strains:(1)Sequences analysis of the 16S r RNA gene and 8 house-keeping genes indicated that strain 0901 and the type strains of Lacticaseibacillus paracasei subsp.paracasei and Lacticaseibacillus paracasei subsp.tolerans had the highest similarities;strain X0308B and Lacticaseibacillus casei JCM 1134~Thad the highest similarities.(2)The ANI values between strain 0901 and the two subspecies of Lacticaseibacillus paracasei were 98.5%and 98.2%,respectively.and the ANI value between X0308B and Lacticaseibacillus casei JCM 1134~T was 99.9%,which were higher than the interspecific threshold(95%～96%),(3)The d DDH values between 0901 and the two subspecices of Lacticaseibacillus paracasei are 87.4%and 85.3%,respectively and the d DDH value between strain X0308B and Lacticaseibacillus casei JCM 1134~T was 99.9%,which were greater than the interspecies threshold of 70%.To sum up,the 18 strains,represented by strain 0901,belonged to Lacticaseibacillus paracasei;the two strains,represented by strain X0308B belonged to Lacticaseibacillus casei.The inter-specices sequence similarity of house-keeping genes indicated that phe S and rpo A genes could distinguished effectively those closely related specices in the 16S r RNA gene phylogeny,and their interspecies difference were at least 6.8%and 5.6%,respectively.API50CH sugar fermentation showed that the 18 strains of Lacticaseibacillus paracasei were most similar to Lacticaseibacillus paracasei subsp.paracasei JCM 8130~T,and all of them could use D-ribose,D-mannitol,methylα-D-glucopyranoside,amygdalin,arbutin,aesculin,salicin,cellobiose,maltose,sucrose,trehalose,gentiobiose,turanose,D-tagatose,and gluconate.In the temperature tolerance test,the 18 strains of Lacticaseibacillus paracasei and Lactoticaseibacillus paracasei subsp.paracasei JCM 8130~T could not grow after heated at 72℃for 40min,whereas Lacticaseibacillus paracasei subsp.tolerans DSM 20258~T can grow.To sum up,the 18 strains of Lacticaseibacillus paracasei belonged to Lacticaseibacillus paracasei subsp.paracasei,not Lacticaseibacillus paracasei subsp.tolerans.The results showed that:strain 1056 had the best growth vigor and strain X0799-1 had the strongest acid-producing ability among the 18 strains of Lacticaseibacillus paracasei subsp.paracasei;Of the two Lacticaseibacillus casei strains,X0308B had the best growth activity and the strongest acid-producing ability.After 48 hours of fermentation in raw milk,the viable count of strains X0308B,X0308S,X0691 and 0901 were 1.6×10~9 CFU/m L,2.4×10~8 CFU/m L,3.1×10~9CFU/m L and 1.0×10~9 CFU/m L,respectively.This study enriched the resource pool of Lacticaseibacillus casei and Lacticaseibacillus paracasei.The high-resolution house-keeping genes suitable for interspecies differentiation of Lacticaseibacillus were screened,which provided the basis for accurate identification at the species leve L.The distinguishing characteristics between the two subspecies of Lacticaseibacillus paracasei were verified,which laid a solid foundation for the identification at the subspecies leve L.