The Effects Of Five Prebiotics On The Growth Of Two Strains Of Probiotic In Vitro And Preparation Of Synbiotic

Probiotics are called active microorganisms which are beneficial to the health of the host.As a dietary supplement,prebiotics can effectively promote the proliferation of probiotics.Synbiotics can effectively improve the host micro-ecological balance,play a probiotic role,and regulate intestinal health.Two strains were isolated and identified from chewable probiotics tablet.By comparing the absorbance values to reflect the proliferation of probiotics,the effects of five prebiotics on the two probiotics proliferation were evaluated in vitro.High performance liquid chromatography(HPLC)and liquid chromatography-mass spectrometry(LC-MS)were used to analyze the metabolic utilization of prebiotics,and two new synbiotic microcapsules were prepared.The main contents and results of this paper are as follows:It was observed by colony morphology that two strains had typical characteristics of lactic acid bacteria,and gram staining showed positive.The results of 16S rDNA sequence analysis showed that LGG018 strains shared more than 99%homology with Lactobacillus rhamnosus ATCC7469T.Considering all the factors,LGG018 was classified as Lactobacillus rhamnosus.NJ018 strains shared more than 99%homology with Bacillus coagulans DSM1T.Considering all the factors,NJ018 was classified as Bacillus coagulans.A method was established to observe the proliferation effect of probiotics by combining plate count and absorbance spectrophotometry.The control group was carried out using MRS broth prepared without any carbohydrate.Single prebiotic solution was added to give a final concentration of 0.5%,1.0%,1.5%,2.0%,2.5%(w/v)in the test broth.The bacterial counts of probiotics an be quickly and accurately determined by measuring the OD values.According to the results of single factor tests,three kinds of prebiotics with better effects on the proliferation of Lactobacillus rhamnosus and Bacillus coagulans were selected.For Lactobacillus rhamnosus,the optimal ratio of prebiotics were obtained by response surface methodology:The concentration of galacto-oligosaccharide(GOS)、Isomalto-oligoisomaltose(IMO)and xylo-oligosaccharide(XOS)was 2.0%.The probiotics density of LGG018 reached the highest,with the OD600 value of 1.134.For Bacillus coagulans,the optimal ratio of prebiotics was obtained by response surface methodology:The concentration of GOS and IMO was 2.0%.The concentration of XOS was 1.0%.The probiotics density of NJ018 reached the highest,with the OD600 value of 1.669.The combination of GOS,IMO and XOS can promote the proliferation of Lactobacillus rhamnosus and Bacillus coagulans.The changes of oligosaccharides of two probiotics were determined by HPLC and LC-MS before and after fermentation.The result indicted that different probiotics have different metabolic utilization of oligosaccharides.The reducing sugar content was reduced mostly after fermentation of LGG018 and NJ018.NJ018 can utilize XOS to increase the content of glucuronic acid after fermentation,and utilize GOS to increase the content of galactose after fermentation.Since the oligosaccharide components in inulin(GFn)and fructo-oligsacchrides(FOS)cannot be detected by PMP,LC-MS method was used to further determine the metabolic utilization for oligosaccharides in the fermentation broth.For the probiotic fermentation broth samples at 0 h,24 h and 48 h,34 target oligosaccharides and organic acids were detected.For LGG018,24 target saccharides and 1 organic acid were detected,of which about 52.00%of oligosaccharides showed a downward trend.For NJ018,a total of 25 target oligosaccharides and 2 organic acids were detected,of which about 31.1 1%of the oligosaccharides increased firstly and decreased afterwards.GFn and FOS with the high polymerization degrees,such as fructoheptasaccharide and fructooligosaccharide DP8,are easily hydrolyzed by glycosidase to produce 1-kestose and nistose trihydrate,so the contents of oligosaccharides with low polymerization degrees increases firstly and decreases afterwards.In order to improve the survival rate of probiotics in the intestinal tract,two new synbiotics microcapsules were prepared by embedding the bacterial suspension containing probiotics.In this study,the number of viable probiotics and encapsulation efficiency were used as the indicators to optimize of wall materials.By single factor test,it was determined that the embedding wall material of LGG018 was whey protein-sodium alginate-hyaluronic acid.The content of whey protein was 6%,the ratio of bacteria to gel was 1:2,and the content of hyaluronic acid was 0.3%.At this time,the number of viable probiotics in the LGG018 microcapsules reached the highest as 10.83 Log10(CFU/g),and the embedding rate was 99.57%.Determined by single factor test,the embedding wall material of NJ018 was the soybean protein-hyaluronic acid-sodium alginate,with soybean protein content of 10%,the bacteria to gel ratio of 1:2,hyaluronic acid content of 0.4%,the number of living bacterium NJ018 microcapsules to the highest as 9.07 Log10(CFU/g),the embedding rate of 99.33%.The high encapsulation efficiency demonstrated the compatibility between wall materials and the probiotic strains.The embedded microcapsules were tested with simulated gastro enteric fluid.After digestion in gastric fluid for 2 h,the survival rate of probiotics microcapsules was higher than that of the commercial bacterial powder.Moreover,the survivability of the two probiotic microcapsules in the intestine are excellent,indicating that the microcapsule can protect the probiotic bacteria,smoothly pass through the gastric fluid to reach the intestinal tract and exert a probiotic effect.