Effects Of Surface Layer Proteins From Lactobacillus On The Strains’ Properties And Their Biological Functions

Surface layer proteins（Slps）are commonly presented on the outermost structure of many bacteria and archaea.They aroused widespread concern in the fields of biosynthesis,nanotechnology and so on,due to their unique characteristics of structure and properties.In recent years,the contribution of Slps to the probiotic effects of Lactobacillus has become a hot study area.However,the breadth and depth of the study is still limited,mainly focused on adhesion,aggregation and surface properties of Slps from a small number of Lactobacillus.Herein,we investigated the influence of Slps on the physiological and biochemical properties of Lactobacillus,then studied the relationship between Slps and the beneficial function of Lactobacillus,further analyzed the potential antioxidant effect of Slps,which aimed to provide a theoretical basis for studying the probiotic properties of Lactobacillus and Slps’ biological function.Firstly,we extracted and identified the Slps of L.acidophilus NCFM,L.casei zhang,L.rhamnosus FB6 and L.gasseri FB8 using LiCl treatment and SDS-PAGE,then purified with ion exchange chromatography.Then the influence of Slps on the physiological and biochemical properties of Lactobacillus was investigated.After the removal of the Slps,the growth and acidogenic fermentation of the strains were delayed,but no significant differences were found in the indexes of physiological and biochemical identification of the strains’ carbon and nitrogen compounds use.Under the environmental stress conditions of oxygen,acid,alkali and heat,the survival rate of Lactobacillus decreased by 1-2 log after removal of the Slps（P<0.05）.When at 3% and 5% NaCl concentration,the growth of Lactobacillus was significantly inhibited after removal of the Slps.It could be found that the Slps make a positive contribution to the stress tolerance of Lactobacillus.We next found that the strains’ DPPH.and O₂^-.clearance ability and reducing power decreased by 5.20%-10.29%,7.29%-14.43% and 8.26%-17.30%（P<0.05）respectively after removing the Slps,which indicated that the Slps have a significant contribution to the in vitro antioxidant capacity of the strains.The cell model of H₂O₂-induced HT-29 cells oxidative stress was applied in the paper and then we found that the Lactobacillus carrying Slps could significantly ameliorate the activity of antioxidant enzymes,the accumulation of ROS and the cells apoptosis（P<0.05）.We further explored the protective effect of the purified Slps（100 μg/mL）on cell oxidative damage and their underlying mechanism.MTT assay showed that the Slps-group increased the survival rate of 7.3%-17.8% compared to H₂O₂ group,which meant that the Slps significantly inhibited the oxidative damage（P<0.05）.Subsequent cell morphology experiments indicated that the Slps inhibited H₂O₂-induced cell ulceration and shrinkage.Meanwhile,Slps could also significantly ameliorate the level of intracellular antioxidant enzymes（P<0.05）.The intracellular ROS content was measured by flow cytometry and the results showed that the Slps-group significantly declined the ROS content compared with the H₂O₂ group（P<0.05）.Fluorescence microscopy and flow cytometry were used to qualitatively and quantitatively analyze the apoptotic rate respectively,the results showed that Slps could decreased the apoptotic rate by 11.41%-21.25% compared with H₂O₂ group（P <0.01）.We also found that the Slps could maintain the degree of mitochondrial membrane potential（MMP）and Ca²⁺ concentration（P<0.05,except the effect of Slp from L.gasseri FB8 on the level of MMP）,also significantly suppressed the activation of Caspase-3 and Caspase-9（P<0.05）.In conclusion,the Slps make a positive contribution to the physiological and biochemical properties,stress tolerance and antioxidant activity of the Lactobacillus strains.Purified Slps own the ability of protecting the oxidative damage of cells and can inhibit apoptosis through mitochondrial pathway.This study shows that the close relationship between the Slps and the strains and the antioxidant potential of the Slps,which will be beneficial to the exploration of the function and application value of Lactobacillus and their Slps.