| Cats are one of the increasingly popular companion animals,and with the increase in cat ownership in China surpassing that of dogs in 2022,keeping cats healthy has become a major concern for the pet industry.Intestinal flora is considered as a"metabolically active organ"and is closely related to cat health.Studies have shown that disturbances in the gut flora can affect the health of cats.Since the Ministry of Agriculture and Rural Affairs of the People’s Republic of China implemented the"reduction and ban of antibiotics",the development and utilization of pet probiotics has attracted much attention.Probiotics can not only improve the apparent digestibility of cat nutrients,but also reduce the rate of diarrhea.Cats are specialized carnivores,the structure of the intestinal microflora is different from that of dogs,and the probiotics isolated from the intestine of cats may have a more pronounced probiotic effect on the intestinal tract of cats.However,most of the commercially available probiotic preparations are shared by dogs and cats,and there is less study on the isolation and activity of cat-derived probiotics.Therefore,it is necessary to clarify the species of cat-derived probiotics and analyze the probiotic properties of their metabolites,to screen probiotics that are beneficial to the long-term intestinal health of cats,and to provide candidate probiotic strains for the development of new functional feed additives for cats.This study aimed to screen Bacillus spp.with strong adverse-resistance that could be selected as a candidate for probiotics.A total of 95 cat samples（anal swabs,rectal swabs and feces）were collected,and TSB broth was used to increase the bacteria and TSA plates were used to purify the culture.The samples were firstly processed at high temperature and coated with plates,and single colonies of suspected Bacillus were selected for staining and microscopic examination,and Bacillus spp.that matched the biological morphological characteristics were used for subsequent in vitro experimental studies.Through the observation of colony morphology and microscopic examination results,Bacillus spp.were isolated from the intestinal tract of cats,and Bacillus spp.isolated from self-probiotic products was used as control.Then,using Escherichia coli,Salmonella Typhimurium and Staphylococcus aureus as indicator strains,the bacteriostatic test of Bacillus fermentation broth in vitro was carried out by Oxford cup method,and the Bacillus was screened with the total acid value of the fermentation broth.Then,the isolated strains were identified and grouped by traditional bacterial identification methods（physiology and biochemistry）combined with 16S r RNA and gyr A genes.The spores were tested for enzyme production,growth ability and adverse-resistance in vitro,the antibiotic sensitivity of nutrients was tested,and the metabolites of Bacillus spp.were analyzed by microbial non-targeted assembly method.The results showed that 90 and 3 strains of Bacillus were isolated from samples and commodities respectively;15 strains of Bacillus with inhibitory effect on Salmonella Typhimurium,Escherichia coli and Staphylococcus aureus were obtained by primary screening;Fermentation broth are acidic（p H 5.85±0.5）;The OD600value of the stable growth period of the strain was 1.177±0.2;From fifteen strains of samples and three strains of commercial bacteria,one strain was identified as Bacillus cereus,one strain was Bacillus allegius,two strains were Bacillus Belleterson,four strains were Bacillus licheniformus and ten strains were Bacillus subtilis.Strain 3-1 had strong ability to produce protease,amylase and cellulase in vitro,strain 4-2,4-3,5-3,13,16 had strong ability to produce amylase and cellulase,strain 1-5,8-6,X2 had strong ability to produce amylase and protease,strain 2-3,9-5 produced protease,strain 14-2 produced amylase.The survival rates of spores under high temperature,low acid and bile salt were 90.53%,81.25%and85.66%,respectively.15 strains of Bacillus were highly sensitive to aminogly cosides,β-lactams,macrolides,tetracyclines,quinolones and amide alcohols except to ampicillin（AMP）.Strains 4-2,10 and 13 were the most sensitive.According to the comprehensive comparison,the probiotic activity of strain 2-3,3-1,4-2,5-3,8-6,9-5,14-2 and 16 in the experimental group was stronger,while that of strain X2 in the control group was stronger.Generally speaking,the in vitro bacteriostatic activity and adverse-resistance of the experimental strain were stronger than that of X2,and the enzyme production ability of the experimental strain was similar to that of X2.Based on the liquid chromatography quadrupole tandem time of flight mass spectrometer（LC-QTOF）platform,using TSB broth as the blank control N,the culture filtrate of the above 18 strains was qualitatively and quantitatively analyzed by microbial non-targeted metabolites.A total of 12992 peaks were detected,of which 2397 metabolites were annotated.Principal component analysis（PCA）showed that the first and second principal components of strains B1,B5,B6,B7,B10,B12,B13 and B14 were higher than N,strains B2,B3,B4,B8,B9,B11,B15 and C2were higher than N,and the second principal components of strains C1,C3 and B13 were higher than N.The Kyoto Encyclopedia of Gene and Genome（KEGG Library）annotated1433 metabolic pathways,among which the most annotated metabolic pathways are the biosynthesis of antibiotics and organic compounds and the metabolism of amino acids.Human metabonomics database（HMDB database）annotated a total of 1288 substances,including 1283 kinds of organic matter,mainly organic acids,lipids and carbohydrates.A total of 394 substances were annotated in the lipid metabolism pathway（Lipidmaps）library,including fatty acids,olefins and esters.A total of 174 organic compounds were annotated in the three libraries of KEGG,HMDB and Lipidmaps,including 45 kinds of organic acids（25.86%）,17 kinds of bacteriocins（9.77%）and 11 kinds of amino acids（6.32%）.Compared the organic matter numbers with N,strains B1,B2,B4,B5,B7,B8,B9,B13,B15 and C2 were 54,66,50,53,57,53,56,54,39 and 54.Compared the organic matter numbers with strain C2,strains B2,B4,B5,B7,B8,B9,B13 and B15 were34,20,19,21,6,26,22 and 14.The results showed that 15 strains of Bacillus with bacteriostatic effect,strong adverse-resistance and good enzyme production ability were screened,which met the screening criteria of probiotics.Through metabonomics,Bacillus can produce many kinds of probiotics,such as polysaccharides,organic acids,amino acids,peptides,short-chain fatty acids,bacteriocins,lipids and esters.Among them,B2,B4,B5,B7,B8,B9,B13 and B15 have good probiotic activity and can be used as candidate strains for the development of cat intestinal microecological preparations. |
