| Lactobacillus casei belongs to the Lactobacillus and It's a probiotic that is useful for health food.Lactobacillus casei can withstand the defense mechanism of the organism,including the oral cavity of the enzyme,low pH of the gastric juice and the bile acids in small intestine,etc.Lactobacillus casei has the ability to promote cell division,produce antibody immunity,prevent cancer and other pHysiological functions,Because Lactobacillus casei has a significant beneficial effects on its host nutrition,immunity and disease prevention and so on.It is now the focus of people's research,development and production.The latest study found that lactocepin,the serine protease in the outer membrane,can mediate the adhesion of lactic acid bacteria,and in some lactic acid bacteria,lactocepin has a good inhibitory effect to the intestinal inflammation.It can degrade some inflammatory cytokines.Therefore,in-depth study of this cell surface protein can not only understand the probiotic function of probiotics,but also for the development of intestinal immunomodulation formulations to provide a theoretical basis.1 Construction and biological characteristics of prtP mutant of Lactobacillus casei 21858According to GenBank information,the extracellular serine protease lactocepin is encoded by the prtP gene.In this paper,the prtP gene of Lactobacillus casei(21858)was replaced by ?-Red recombination technique to construct the mutant strain of Lactobacillus casei(21858 ?).By measuring the basic biological characteristics of two lactic acid bacteria,it was found that 21858 ?and 21858 growth curve were similar,21858 ? self-aggregation rate are lower than 21858 at all time points,and the longer the gap is more obvious.The co-coagulation rate of 21858 with four pathogens was greater than 21858 ?.The hydropHobicity of 21858 ? is 44.1± 1.5%which is more than 21858(23.9±0.62%).And there was significant difference(P<0.05).The inhibitory effect on pathogens of 21858 is better than 21858 ?,but there are no significant difference(P>0.05).It suggested that the prtP gene had no effect on the growth curve of Lactobacillus casei,and the knockout prtP gene could decrease the self-agglomeration rate of Lactobacillus casei and the co-coagulation rate of the pathogen,increase the hydropHobicity and decrease the inhibitory effect on the pathogen.2 Evaluation of the Adhesion Ability of 21858?In this study,the adhesion ability of 21858 and 21858 ? to intestinal mucus,cell and mouse intestine was determined by establishing in vitro and in vivo adhesion model.The results showed that the adhesion rate of 21858 to intestinal mucus was 46.38 ± 2.3%,and the adhesion rate of 21858 ? to intestinal mucus was 36.65 ± 4.5%,which were significantly different(P<0.05).The adhesion of 21858 to three kinds of cells was found to be greater than 21858 ? by cell adhesion model.And there was no significant difference(P>0.05)between the adhesion of 21858 and 21858? to spleen cells.And the difference to small intestinal epithelial cell and macropHages is significant(P<0.05).We used flow cytometry analysis of Lactobacillus casei in vivo adhesion and found that in the mouse duodenum,jejunum ileum-adhesion rate of 21858 was higher than 21858 ?,but in the colon the adhesion rate of 21858 ? was higher than 21858,and after the gavage of Lactobacillus caseiin,there was an increasing trend in the number of adherent bacteria in all parts of mice intestinal tract.And we found that the number of total Lactobacillus caseiin which were adhered to the ileum were higher than other parts of the small intestine.Suggesting that prtP gene encoding lactocepin can enhance the adhesion of Lactobacillus casei on intestinal mucus,cell surface and mouse intestinal tract.3 Evaluation of the immune efficacy of 21858AThis study found that 21858 group can enhance the body weight of mice,and it was significantly different(P<0.05)with the control group,The effect of 21858 on mice body weight was better than that of 2185 ?.And the longer the effect was more obvious.21858 and 21858 ?have a specific effect on the index of immune organs.21858 and 21858 ? had no significant effect on the thymus index in the short term(10 d)(P>0.05),But in the long-term(20 d)21858 and 21858 ? high-dose groups both had significantly difference in the thymus index(P<0.05)compared with the control group.While the other groups had no significant difference compared with the control group.There was a significant difference in the thymus index between the high dose group and the middle dose group,Suggesting that there is a dose-dependent relationship between Lactobacillus casei on thymus index.In the study of immune cells we found that 21858 and 21858 ? in the entire stage of feeding can improve the conversion of mouse lympHocytes.And the conversion rate in the 21858 group was higher than that in the 21858 ? group at the same time and dose.Activation studies of macropHages suggest that 21858 can activate macropHages by enhancing the energy metabolism or pHagocytosis of macropHages and enhance their ability to treat antigens.But 21858 ? did not significantly enhance the activation of macropHages,Suggesting that 21858 activated macropHage capacity is stronger than 21858?.In this study,CDllc+,CD80+ double positive splenic lympHocytes were detected by flow cytometry.The results showed that the two strains of Lactobacillus casei could continuously increase the number of CD11c+ CD80+ double positive cells in the whole stage of feeding mice,especially in the late feeding stage,and the enhancement ability of 21858 group was better than 21858 ?group.Therefore,Lactobacillus casei can improve the immune function of mice.This suggests that prtP-encoding extracellular membrane protease lactocepin plays an important role in improving immune function in mice. |
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