| Lactobacillus as a probiotic plays an important probiotic effect in the intestinal tract of animals.Its adhesive properties are the basis for probiotics to successfully colonize the intestines and become one of the important criteria for screening highly beneficial bacteria.Lactobacillus surface protein glyceraldehyde triphosphate dehydrogenase(GAPDH)acts to adhere to the surface mucin of the intestinal tract on the cell surface.However,the transmembrane secretion of GAPDH from the cytosol to the cell surface and the distribution characteristics of the surface of the cells are not clear.It is important for screening and cultivating high-efficiency lactic acid bacteria to elucidate the adhesion of GAPDH to mucoid in pig intestines.The aim of this study is toreveal the secretory properties of surface protein 3-glyceraldehyde dehydrogenase(GAPDH)in different growth stages of Lactobacillus and its adhesion to mucin.Low adhesion ZJ615 and high adhesion ZJ617 Lactobacillus reuteri culture at different times(3,6,9,12,15,18 h),surface protein extracted with lithium chloride,Western blotting quantitative surface protein GAPDH content.Result show that secretion of GAPDH showed differences at different culture times.Fluorescein diacetate(FDA)and propidium iodide(PI)combined with flow cytometry to detect cell membrane permeability;The difference in the distribution of GAPDH on the surface of lactobacilli was verified by immunoelectron microscopic analysis;The two strains were cultured for 18 h(platform stage)and incubated for 1 h at 37?,gram stain analysis of lactobacillus adhesion after antibody blocking surface protein GAPDH,Periodate oxidized mucin and antibody blocking plus oxidation double treatment.Kinetic characteristics of interaction between Lactobacillus and mucin were analyzed by ForteBio in different treatment conditions.Research indicates:1)The GAPDH content of Lactobacillus surface protein was highest at 6h(logarithmic growth phase)(P<0.05),and decreased significantly after entering the late growth stage(12h,15 h,18h)(P<0.05);The content of GAPDH protein in high adhesion Lactobacillus ZJ617 was higher than that in ZJ615(P<0.05).2)Lactobacillus cultured to logarithmic growth phase(6h),cell membrane permeability was the highest(P<0.05),and late growth period(12h,15 h,18h)showed decreased permeability(P<0.05);high adhesion Lactobacillus ZJ617 Cell membrane permeability was significantly higher than that of ZJ615 at different times(P<0.05).The correlations between the secretion of GAPDH and the permeability of cell membrane of ZJ617 and ZJ615 in different culture time were 0.8 and 0.6,respectively.3)When the cell ulite was fixed with mucin,the amount of Lactobacillus adhesion was significantly lower than that of the Lactobacillus,the amount of Lactobacillus adhesion was significantly decreased after mucin fixation,antibody blocking GAPDH,and periodate oxidized mucin(P<0.05);The double treatment of antibody blocking and periodate oxidation,the number of Lactobacillus adhesion was further significantly reduced(P<0.05);Under the above treatments,the adhesion of Lactobacillus high adhesion Lactobacillus ZJ617 was significantly higher than that of low adhesion ZJ615(P<0.05).The kinetic interaction results showed that compared with the control group,the reaction values of each treatment group decreased after the oxidation of periodate,protein removal,heating and antibody closure.It can be seen that the GAPDH content of Lactobacillus surface protein is the highest in logarithmic growth period,and its secretion is positively correlated with cell membrane permeability;The highly adherent Lactobacillus has a surface protein GAPDH,which plays a role in intestinal colonization of Lactobacillus by adhering intestinal mucin. |
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