Screening Of High Adhesive Lactic Acid Bacteria And Its Immunomodulatory Activity Against RAW264.7 Macrophages

Lactic acid bacteria is a kind of microbe beneficial to the health of the host,which plays a probiotic role mainly through colonization in the intestine.The adhesion and colonization of lactic acid bacteria to intestinal epithelial cells can restore or enhance intestinal homeostasis and improve immunity.Therefore,the adhesion ability of lactobacillus is an crucial indicator for evaluating its probiotics.But the effect and stability of some lactic acid bacteria with probiotic function are unsatisfactory.Thus,screening a lactic acid bacteria with high adhesion and immunoregulatory potential is distinctly important for the development of efficient immunoregulatory probiotics.The purpose of this study was to screen high-adhesion strain from 13 strains of lactobacillus and 2 strains of Enterococcus durans preserved in the laboratory and to analyze the factors affecting the adhesion of lactic acid bacteria,and to explore the adhesion mechanism preliminarily.Finally,the immunomodulatory effect of lactobacilli with high adhesion on macrophage RAW264.7 was evaluated.L.acidophilus NCFM with good adhesion was selected as control.High-adhesion lactic acid bacteria strain was screened by measuring the surface hydrophobicity,self-aggregation ability and the ability of adhering Caco-2 cells in 14 strains of lactic acid bacteria,and the relationships between surface properties and adhesion were also analyzed.The results showed that the adhesion rate of L.acidophilus KLDS1.0901 to Caco-2 cells was the highest,being 12.73%.The hydrophobicity of KLDS 1.0901 was 13.88%,which was second only to that of Enterococcus durable KLDS6.0933.L.rhamnosus KLDS1.0205 has the lowest adhesion capacity of 0.13%.There was a certain correlation between the surface hydrophobicity of different strains and the adhesion ability to caco-2 cells.In vitro experiments were conducted to investigate the effects of concentration,growth stage,chemical and enzymatic treatment of L.acidophilus KLDS1.0901 on its ability to adhere to Caco-2 cells.The results showed that both the growth phase significantly affected the adhesion of KLDS1.0901.When the concentration of the bacteria was 108~1010 CFU/m L,the effect on adhesion was not significant.And the adhesion of KLDS 1.0901 to caco-2 cells may be the result of join action of many adhesins,mainly surface layer proteins.The SLP of KLDS1.0901 was initially isolated and identified by transmission electron microscopy and SDS-PAGE.The experimental results showed that KLDS1.0901 had abundant villus-like surface layer protein with a content of about 15.6% and a molecular weight of about 43 k Da.The SLP crude extract could meaningfully inhibit the adhesion of KLDS1.0901 to caco-2 cells,and the higher the concentration,the stronger the inhibitory effect.Flow cytometry was used to analyze the colonization and distribution of untreated,SLP removed and heat-killed KLDS1.0901 in the intestinal tract of mice.The results revealed that KLDS 1.0901 mainly adhered to duodenum and jejunum in the early stage,and tended to adhere to colon over time.While KLDS 1.0901 with the SLP removed and thermal inactivation could adhere to the duodenum in large quantities on the first day,it could only stay in various parts of the intestinal tract for a short time,and the amount of adhesion in various parts of the intestinal tract of them all were expressively reduced as time pass.It was confirmed in vivo that KLDS1.0901 could also adhere and colonize in the intestine of mice for a long time,and the surface layer protein of KLDS1.0901 could also significantly affect its adhesion ability in vivo.The immunoregulatory effect of KLDS1.0901 on macrophage RAW264.7 was explored in vitro.The effects of different doses of untreated KLDS1.0901(LLA),Removing SLP from KLDS1.0901(RSLA)and heat-killed KLDS1.0901(HKLA)on proliferation and phagocytosis of macrophages were determined.Levels of IL-10 and TNF- secretion and its m RNA expression were measured too.The results showed that LLA and HKLA groups were able to significantly increase the proliferation ability of the macrophages,but only the LLA group could enhance the phagocytic activity when the bacteria co-incubated with the cells at a ratio of 10:1 and 1:1.However,for all concentrations,the RSLA group inhibited the activity of macrophages.It was confirmed that the SLP crude of KLDS1.0901 was an important substance to enhance the activity of macrophages,and its proliferation and phagocytic activity was the strongest when stimulated with 500 ?g/m L of SLP.LLA significantly inhibited IL-10 and TNF-? secretion compared to the blank control group when KLDS1.0901 was co-incubated with macrophages at 1000:1.At other concentrations,the secretion levels of IL-10 in LLA,RSLA and HKLR groups were significantly higher than those in the control group,but far below the LPS group.LLA,RSLA and HKLA could induce cells to secrete TNF-a at the same level as LPS group.Compared with the blank control group,both LLA and HKLA significantly up-regulated the expression levels of IL-10 and TNF-? m RNA when the cells were co-cultured with macrophages at 100:1.This was corroborated by the results of the measurement of cytokine levels.The conclusion was that a strain of Lactobacillus acidophilus KLDS 1.0901 with abundant surface layer protein,high adhesion and immunomodulatory potential has been obtained.This strain has the potential to be used as a carrier of mucosal vaccine,and can also be developed into a probiotics which regulate immunity effectively.