Screening Of Lyophilized Protective Agent For Lactobacillus Reuteri And Its Application In Fermented Milk

This research is supported by the Jilin Provincial School Co-construction Project(SF2017-6-4).Lactobacillus reuteri,as a lactic acid bacteria colonized in the intestines of humans and mammals,has a certain probiotic effect.Lactobacillus reuteri F-9-35 used in this experiment was selected after ground mutagenesis of Lactobacillus reuteri.It not only can tolerate the harsh environment of low pH and bile salts,but also plays a role in relieving gastroenteritis.In this experiment,some biological properties of Lactobacillus reuteri F-9-35,screening and optimization of lyophilized protective agents,tolerance and storage stability of lyophilized fungal powder,and its application in the preparation of fermented milk This section explores.The main findings are as follows:1.Lactobacillus reuteri F-9-35 was cultured at 37°C for 10 hours and entered a stable phase.16s rDNA sequencing of the strain after 10 consecutive subcultures showed that F-9-35 had good genetic stability.The results of Lactobacillus reuteri F-9-35’s tolerance to acid and bile salts showed that F-9-35 was better tolerated under the conditions of pH=3,4 and 0.1%bile salt.After 3 h of culture The lg values became0.97,1.06and 1.02 times the initial values,but the tolerance was significantly weaker at pH=2 and at 0.3%and 0.5%bile salts than the former.Compared with the ground strain GS-23,it was found that the lg value of F-9-35 was significantly higher than that of GS-23 after 3 h of culture at pH=2 and 0.3%and 0.5%bile salt.2.In this experiment,trehalose,maltodextrin,straw mushroom polysaccharide extract,flammulina velutipes polysaccharide extract,pleurotus eryngii polysaccharide extract,tremella polysaccharide extract,agaric polysaccharide extract,fish collagen peptide,soybean oligopeptide,pig skin peptide,frog skin peptide and 11 lyophilized protective agents were screened.According to the results of single-factor experiments,three substances,maltodextrin,straw mushroom polysaccharide extract,and fish collagen peptide were selected as the factors optimized for response surface testing.Finally,the protective agent formula of Lactobacillus reuteri F-9-35 was 19%maltodextrin,12%straw mushroom polysaccharide extract,and 10%fish collagen peptide.At this time,the survival rate of F-9-35 was 68.91±2.80%.3.The tolerance of Lactobacillus reuteri F-9-35 lyophilized fungus powder to artificial simulated gastrointestinal fluid was explored.The results showed that the lyophilized lactobacillus reuteri F-9-35 lyophilized fungus powder was cultured in simulated gastric juice.The number of viable bacteria in 4h was 2.68±1.13(×10~9cfu/g),and the number of viable bacteria in simulated intestinal juice for 10h was 2.00±0.17(×10~9cfu/g).However,under the same conditions,the number of unprotected bacteria decreased by two orders of magnitude.Both experiments showed that the tolerance of lyophilized fungal powder was significantly better than that of unprotected fungal bodies.In addition,the change of viable count of Lactobacillus reuteri F-9-35lyophilized powder under different storage conditions was also determined.The lyophilized bacterial powder was still maintained at 10~9cfu/g after 60 days of storage at 4℃and-20℃,and the bacterial count was more than 10~6 after stored at 25℃for60 days,indicating that the lyophilized bacterial powder was stable during storage.4.The fermentation characteristics of Lactobacillus reuteri F-9-35 fermented milk,mixed fermented milk and traditional fermented milk(Streptococcus thermophilus and Lactobacillus bulgaricus)were investigated experimentally and the changes of the indexes during storage.The results showed that the pH of the mixed fermented milk decreased to 4.60 after 4 hours of fermentation at 41°C,and the fermentation time was shorter than that of Lactobacillus reuteri F-9-35 fermented milk(6h)and traditional fermented milk(6h).The fermented milk was stored at 4°C for 4 weeks.The results of changes in various indicators of fermented milk showed that the water holding capacity of the three fermented milks was always above 95%during the storage period.The changes in pH and acidity were the smallest,and the changes in the other two fermented milks were not significantly different.Texture analysis and viscosity measurement results showed that the hardness,gluing and chewing properties of Lactobacillus reuteri F-9-35 fermented milk were higher than those of the other two fermented milks,but the viscosity values were significantly lower than those of the other two fermented milks.Below 2500cp.The viable counts of the three types of fermented milk showed a downward trend during the storage period.Among them,Lactobacillus reuteri F-9-35 had the largest decrease in the number of live bacteria.After 4 weeks of storage,the lg value decreased from9.235±0.015 to 8.21±0.058.During the storage period,the lg value of traditional fermented milk decreased from 9.34±0.033 to 9.19±0.0015.For mixed fermented milk,the lg value of the total lactic acid bacteria decreased from 9.14±0.0050 to8.70±0.0050,and the lg value of Lactobacillus reuteri F-9-35 decreased from8.08±0.010 to 7.00±0.035,but still maintained at 10~6cfu/ml.