Comparative Study On Genome And Probiotic Effects Of Five Different Lactobacillus Species

Lactobacillus are beneficial microorganisms that are prevalent in nature and are capable of providing numerous health benefits to animals and humans,and are often considered alternatives to antibiotics in animal agriculture.However,the probiotic capacity of different strains is strain-specific at both the genetic and physiological levels,and their specific probiotic capacity needs to be evaluated in a comprehensive evaluation.In this study,Lactobacillus salivarius 05,Lactobacillus chiayiensis AACE 3,Lactobacillus paracasei XLK 401,Lactobacillus casei AACE 4,Lactobacillus pabuli AACE 5 were compared at the physiological and molecular genetic levels.The probiotic potential of five different species of Lactobacillus were further evaluated based on the growth level,antioxidant system,immune level and intestinal flora of Lactobacillus fancy,so as to explore the commonality and individuality of the probiotic properties of five different species of Lactobacillus at the phenotypic characteristics and molecular genetic level.The main findings are as follows:(1)In vitro probiotic performance analysis of five Lactobacillus species revealed that all five strains had good antibacterial ability and good tolerance to artificial simulated gastrointestinal fluid and bile salts.The autoagglomeration and hydrophobic ability of the five Lactobacillus species differed,but all had some self-agglomeration and hydrophobic ability,among which,L.pabuli AACE 5 and L.salivarius 05 had the best self-agglomeration rate of 74.16% and 71.16%,respectively;L.salivarius 05 and L.chiayiensis AACE 3 had the had the best hydrophobicity,with 55.88% and 31.29%,respectively.Meanwhile,in the PCR amplification assay of drug resistance and virulence genes,only L.salivarius 05 and L.chiayiensis AACE 3 detected positive bands for tetracycline resistance genes,and the rest of Lactobacillus genomes were not detected in PCR related to drug resistance and virulence.In the drug sensitivity assay,only L.chiayiensis AACE 3 was sensitive to tetracycline antibiotics.In addition,all five Lactobacillus species had no hemolytic activity.(2)Whole-genome sequencing analysis of five Lactobacillus species was performed,and the genetic evolutionary relationships of the five species were analyzed by constructing phylogenetic trees based on core genes,covariance and average nucleotide identity(ANI),while probiotic gene annotation of the genome sequences of the five species revealed that they all had probiotic genes associated with acid tolerance,heat tolerance,resistance to oxidative stress,adhesion ability,and self-agglutination ability.Besides,L.salivarius 05,L.chiayiensis AACE 3,L.paracasei XLK 401 and L.casei AACE 4 were found to have gene clusters encoding bacteriocins by anti SMASH,BAGEL4 database prediction.In addition,the annotation of the resistance gene database(ARDB)database revealed that the genome sequence of L.salivarius 05 had vancomycin and tetracycline-related resistance genes;only one resistance gene related to tetracyclines was found in the genome sequences of L.chiayiensis AACE 3 and L.paracasei XLK 401.(3)In the in vivo feeding experiment of Nandan Yao chicken,after adding 5strains of Lactobacillus to the basal diet to feed Nandan Yao chicken,the weight of Nandan Yao chicken could be increased compared with the blank group,and the weight increase effect was the most significant when L.paracasei XLK 401 and L.salivarius 05 were added(P <0.05).In terms of antioxidant levels,feeding 5 strains of Lactobacillus had the effect of increasing the levels of superoxide dismutase(SOD)and glutathione peroxidase(GSH-PX)in the liver,among which L.paracasei XLK401 and L.chiayiensis AACE 3 had the best effect on increasing SOD enzyme activity(P<0.05).L.salivarius 05 and L.paracasei XLK 401 had the best effect on their GSH-PX enzyme activity levels(P<0.05).Meanwhile,all five strains of Lactobacillus significantly reduced malondialdehyde(MDA)levels in Nandan-Yao Chicken(P<0.05).In addition,the five Lactobacillus species did not stimulate immune responses such as serum immunoglobulin(IgG)and cellular inflammatory factors(IL-6、IL-10、TNF-α和 IL-1β)in Nandan-Yao Chicken.(4)The addition of five Lactobacillus species to the basal diet fed to Nandan-Yao Chicken had an effect on their intestinal diversity.Among the dominant flora at the phylum level,the ratio of Firmicutes to phylum Bacteroides was closely related to fat deposition,and the relative abundance of Firmicutes in the L.salivarius05,L.chiayiensis AACE 3 and L.casei AACE 4 groups increased,and the abundance of Bacteroides decreased all decreased.The relative abundance of the phylum Firmicutes decreased in the L.paracasei XLK 401 and L.pabuli AACE 5 groups decreased,and the relative abundance of the phylum Bacteroides increased.At the same time,at the genus level,the relative abundance of Lactobacillus in the chlortetracycline,L.chiayiensis AACE 3,L.paracasei XLK 401,L.casei AACE 4 and L.pabuli AACE 5 groups increased.Compared with the control group and chlortetracycline,the relative abundance of Akkemansia 4 in L.salivarius 05,L.chiayiensis AACE 3,L.paracasei XLK 401 and L.casei AACE 4 groups increased,which was consistent with the trend of intestinal flora changes after feeding poultry with most alternative antibiotic products,which was conducive to regulating the intestinal flora structure of Nandan-Yao chicken and playing an active role in promoting the intestinal health of Nandan-Yao.In summary,firstly,the five strains of Lactobacillus were analyzed based on a combination of genetic level and physiological level,and then further confirmed by in vivo feeding tests on Nandan-Yao Chicken,all five different species of Lactobacillus showed a certain degree of probiotic effect in animals,providing an important reference value for the application of Lactobacillus in livestock and poultry breeding industry.