Evaluation Of Probiotic Potential Of Swine-derived Probiotics

Probiotics are live microorganisms that,when administered in adequate amounts,confer a health benefit on the host.For the past few hundreds of years,probiotics have been mainly applied in the food and health care industry.In recent years,several advantages of probiotics have gradually been discovered,such as preventing animal diarrhea,promoting animal growth,improving immunity,and promoting feed conversion.When probiotics are used as feed additives,problems of drug-resistant strains,antibiotic residues,and environmental pollution caused by antibiotics were effectively reduced.Probiotics have become one of the widely recognized antibiotic alternatives with safety,high efficiency,environmental protection,and green properties.China is a large animal husbandry country,and its demand for probiotics in the aquaculture industry is relatively huge.However,China's feed probiotics market of livestock breeding industry is still in its infancy and there are a few commercialized probiotic products.Therefore,this study aimed to isolate swine-derived probiotics,and screen out safe and efficient probiotics by comprehensively evaluating their probiotic properties both in vivo and in vitro,so as to provide probiotic resources for the development of compound probiotic formulations.The main results are presented as follows:1.32 Lactic acid bacteria and 20 Bacillus were isolated and identified from healthy pig feces samples collected from different regions.The in vitro safety of the strains was evaluated by the hemolysis test and antibiotic sensitivity test.The result showed that 52 newly isolates had not hemolytic activity and shared different sensitivity to 13 antibiotics,which can preliminary determined that those strains are safe.2.Probiotic properties of the new isolates were comprehensively evaluated in vitro by investigating antimicrobial activity against pathogens,tolerance of acid and bile salt,survival in the simulated gastrointestinal tract,the cell surface properties,the adhesion to Caco-2 cells,the capacity to antagonize the adhesion of pathogenic bacteria and the ability to produce amylase and protease.Finally,3 strains with excellent probiotic properties were screened,namely Lactobacillus johnsonii GHZ10 a,Lactobacillus salivarius ZLp4 b,and Bacillus velezensis BSC16 a.The result showed that 3 newly isolates exhibited different antibacterial activities against Escherichia coli,Salmonella typhimurium,Yersinia enterocolitica,Aeromonas caviae,Proteus vulgaris,Shigella bogdii,Staphylococcus aureus,Listeria monocytogenes,Staphylococcus hominis,and Clostridium perfringens;The survival rate was higher than 70.0% after 2 h tolerance in acid(pH 3.0)and pig bile salt(0.3%),respectively;3 strains survive well after exposed in simulated gastric juice for 3 h and simulated intestinal juice for 6 h;3 strains showed high hydrophobicity,self-aggregation,and adhesion to Caco-2 cells;Characteristics of inhibitory adhesion of E.coli,Salmonella and S.aureus to Caco-2 cells by competition,exclusion and replacement were widely observed in 3 candidate strains;GHZ10a and ZLp4 b can produce protease and amylase,and BSC16 a can only produce protease.3.In vivo evaluation of the safety and probiotic features was conducted by feeding mice with low,medium,and high doses of the 3 candidates for 30 days.The result showed that no adverse symptoms was found in mice during the experiment;there were no ocular lesions after dissection,no pathological changes in intestinal sections,and no significant difference in organ indexes;The V/C value(Villi height/Crypt depth)of duodenal of mice in groups supplemented with a low concentration of ZLp4 b and BSC16 a was significantly higher than the control group;The weight gain rate of mice fed with strains GHZ10 a and ZLp4 b was significantly higher than the control group;The levels of IgA,IgG,IgM,IL-2,and IFN-? in the serum of mice fed with B.velezensis BSC16 a were significantly higher than those in the control(P<0.05);L.johnsonii GHZ10 a with different doses can significantly increase the levels of IgM and IL-2 in the serum of mice whereas only a medium dose of GHZ10 a can promote the production of IgG.L.salivarius ZLp4 b with high,medium and low doses can significantly increase the levels of IL-2 in the serum of mice while only high concentrations of ZLp4 b could promote the secretion of IgG and IgM.4.The effect of 3 newly isolates on the intestinal flora of mice was analyzed by high-throughput sequencing technology.The result showed that Firmicutes and Bacteroidetes are the dominant flora in the intestine of mice at the phylum level and Lactobacillus is the dominant flora at the genus level;Compared with the control group,a significant increase in Firmicutes and Lactobacillus was observed in groups fed with L.johnsonii GHZ10a(P<0.05);The diversity of intestinal flora of mice fed with L.salivarius ZLp4 b decreased,and there is a significant increase in the abundance of Alloprevotella,Blautia,Ruminiclostridium and Marvinbryantia at the genus level(P<0.05);The abundance of Alloprevotela,Unidentified Ruminococcaceae,Blautia,Ruminiclostridium,Oscillibacter,Muribaculum,Candidatus Arthromitus and Butyricicoccus of the intestinal flora of mice fed with B.velezensis BSC16 a was significantly increased at the genus level(P<0.05).Generally,this study obtained 3 swine-derived probiotics for further study and application based on their high safety,strong tolerance,extensive antibacterial activity,high adhesion,inhibitory adhesion property of pathogens,characteristics of enzyme production,effects of improving the growth performance,stimulating body immunity,improving intestinal structure,and modulating intestinal flora.