Screening Of Anti-allergic Active Lactic Acid Bacteria And Preparation Of Their Preparations

Probiotics play a pivotal role in human health,and several researches revealed that probiotics have certain preventive and therapeutic effects on allergic diseases;the evaluation of its anti-allergy capability is mainly in vivo,however,the methods of rapid screening and evaluation of functional probiotics by in vitro experiments are rarely reported.Due to the large population and differences in dietary habits in China,foreign probiotics cannot fully satisfy the microbial environment and physiological characteristics of Chinese people,therefore it is necessary to screen out probiotic strains suitable for different consumers from different regions.In this study,lactic acid bacteria（LAB）were isolated from dairy products in mountainous pasture of Hunan province.According to its characteristics,probiotics were preliminarily screened out and identified,and the in vitro anti-allergy screening method was established to identify the potential anti-allergy active LAB.In addition,high-cell-density cultivation and freeze drying technique were used to obtain probiotic LAB preparation with high quantity of live bacteria and anti-allergic activity.Specific research contents and results are as follows:（1）Lactic acid bacteria strains were isolated from cow’s milk samples,and the probiotic characteristics of the strains were evaluated by in vitro stress resistance,adhesion,bacteriostasis and in vitro safety tests,and the LAB strains with potential probiotic functions were screened out and identified by 16S rDNA sequencing technology.The results showed that among the 269 LAB strains isolated from dairy products,20 strains exhibited low pH and high bile salt tolerance;further evaluation of probiotic features showed that the 15strains possessed favorable probiotic characteristics,such as the tolerance to simulated gastric and intestinal fluid（survival rate>50%）,the inhibitory capability toward common pathogens,the surface activity of the strains and the adhesion with Caco-2 cells（adhesion ability>10 CFU/cell）;by 16S rDNA sequencing,5 strains of Lactobacillus plantarum（LZ008E,LZ012E,LZ015E,LZ020E,LZ041E）,4 strains of L.casei（LZ034E,LZ062E,LZ083E,LZ183E）,2 strains of L.paracasei（LZ019E,LZ077E）,1 strain of L.brevis（LZ026E）,1 strain of L.rhamnosus（LZ260E）,1 strain of L.pentosus（LZ002E）,and 1 strain of L.paribrinella（LZ037E）were identified.These 15 strains of potential probiotic LAB could be further examined for anti-allergic activity.（2）Using screened probiotic LAB to do the antiallergic test in vitro,hyaluronic acid enzyme inhibition in vitro experiment is adopted to improve the initial screen,and then use RBL-2H3 cells degranulation model to do the rescreening to select the probiotic LAB which are antiallergic,further more focus on the influences produced by these selected probiotics on protein expression which are involved in the RBL-2H3 cell surface Fc epsilon RI（FcεRI）receptors signaling pathways to identify the initial mechanism of allergy.The results show that using hyaluronidase from 15 probiotic LAB inhibition test in vitro screens out 10 strains with stronger inhibitory activity of（inhibition rate>40%）,and using the RBL-2H3 cells activated degranulationβ-HEX release inhibitory activity after screen out 3 strains had significant inhibitory activity strains,respectively:L.paracasei LZ077E,L.casei LZ183E and L.rhamnosus LZ260E.The anti-allergic mechanism experiment showed that all the three screened strains could effectively reduce the histamine secretion of RBL-2H3 cells,regulate the release of allergy-related cytokines IL-4 and TNF-αto near normal levels,and promote the release of cytokines IL-10.In addition,the strain was able to down-regulate the level expression of p-Lyn/Lyn and p-Syk/Syk proteins in Ig E-mediated RBL-2H3 cells,thus inhibiting basophil activation in allergic reactions.Combining with the results of probiotics,the strain LZ183E was selected for further study.（3）The number of viable bacteria and the value of OD_600nmwere used as the indexes to study the culture conditions of the screened strains.The effects of L.casei LZ183E on its growth under the culture conditions of different carbon sources,nitrogen sources,nutrient factors,initial pH value and inoculation amount were investigated by single factor test and orthogonal test optimization.The key parameters of the optimal culture were glucose（25g/L）,yeast extract（20 g/L）,pumpkin juice（24 g/L）,the culture temperature was 37℃,the initial pH was 6.5,and the inoculation amount was 2%.Under this condition,the number of viable bacteria could reach 9.204±0.036 lg CFU/mL and 1.136 for OD_600nm,which reached the advanced level in China and increased by an order of magnitude compared with the original culture medium.Moreover,the inhibitory activity of hyaluronidase of strain LZ183E was increased by 0.9822%compared with the original culture medium.（4）On the basis of high density culture conditions,the effects of different single freeze-drying protectants on the survival rate of strain LZ183E were studied by vacuum freeze-drying technology.Then,the inhibition activity of hyaluronidase was studied under the optimal compound ratio.The results showed that when trehalose,skimmed milk,xylose oligosaccharide and sodium glutamate were used as freeze-drying protectants,the bacterial survival rate was higher.The optimal ratio was further optimized by response surface method:the dosage of trehalose was 6.0%,the dosage of xylose oligosaccharide was 8.5%,the dosage of defatted milk powder was 11.5%and the dosage of sodium glutamate was1.0%.The survival rate of the bacterial strain in this condition was 98.236±0.137%,which was significantly higher than that of the bacterial agent without protective agent（79.241±0.382%）.In addition,under the optimal conditions,the inhibition activity of hyaluronidase remained high（71.625±1.086%）.The results showed that the protective agent did not affect the antiallergic activity of the strain.