High Cell Density Culture Of Lactobacillus Rhamnosus YYJP-2

Lactobacillus rhamnosus,which is one of the third-generation probiotics recognized by the state and the most popular lactic acid bacteria in the world,is isolated from the healthy human gut.In recent years,Lactobacillus rhamnosus has become the favorite species of scientific research personnel,because the Lactobacillus rhamnosushave a lot of functional characteristics,such as regulate intestinal flora,prevent and treat diarrhea,remove toxins from the body,prevent dental caries and improve immunity.C urrently the fermentation products of lactobacillus on the market are mostly Lactobacillus bulgaricus and Lactobacillus acidophilusmore than the fermentation products of theLactobacillus rhamnosus,the main reason is due to Lactobacillus rhamnosuslive the number of bacteria is not high enough.Therefore,the study explored the high density fermentation of Lactobacillus rhamnosus YYJP-2 deposited by our laboratory,and promoted the growth and reproduction of Lactobacillus rhamnosus YYJP-2 to finally reach the high density fermentation.The main contents of this experiment are as follows:(1)In this study,we studied the growth and reproduction of Lactobacillus rhamnosus YYJP-2 by continuous tracing of the growth and reproduction of Lactobacillus rhamnosus YYJP-2 in the first step.And the regular growth curve was drawn and the growth curve was used to determine the best fermentation of Lactobacillus rhamnosus YYJP-2 for 14-16h.The highest live number of bacteria is 18h.(2)The high-density fermentation of Lactobacillus rhamnosus YYJP-2 was to optimize the external conditions of its fermentation.Finally,the optimum fermentation conditions were determined by single factor experiment.The optimized external conditions mainly include fermentation temperature,inoculation amount and initial p H of the medium.The final optimization results for these external conditions:fermentation temperature was 35°C,inoculation volum of 3%,initial p H 6.5.(3)Achieving high density fermentation of Lactobacillus rhamnosus YYJP-2 is to optimize the culture medium for which the strain is to survive.Based on MRS medium,the types and optimum additions of carbon sources were determined by single factor experiment.Many kinds of peptone were used in laboratory.Therefore,the peptone was determined by single factor experiment.And then the peptone,beef extract and yeast powder were determined.The optimum ratio of the mixed nitrogen source was explored by orthogonal test.The carbon source and nitrogen source of the optimized medium were as follows:glucose 20g/L,multivalent peptone 15g/L,beef extract 20g//L,yeast powder 15g/L.(4)The high-density fermentation of Lactobacillus rhamnosus YYJP-2 was toadd a buffer salt systemto remove or reduce the inhibitory effect of lactic acid on the growth of the bacteria.The single factor experiment of four kinds of buffered salts of sodium dihydrogen phosphate/disodium hydrogen phosphate,potassium dihydrogen phosphate/sodium hydroxide,disodium hydrogen phosphate/citric acid,disodium hydrogen phosphate/potassium dihydrogen phosphate was the most favorable cell growth and reproduction of the buffer salt system.The final determination of the buffer system ad ded in the optimized medium wasthat the concentration of potassium dihydrogen phosphate/sodium hydroxide 0.10 mol/L.(5)The high-density fermentation of Lactobacillus rhamnosus YYJP-2 was based on the above results.The carbon source,nitrogen source and buffer salt were used as the influencing factors,the response rate was live the number of bacteria by Box-Benhnken Based on the experimental results,a quadratic response surface regression model was established to find the optimum experimental ratio of carbon source,nitrogen source and buffer salt.The final results were as follows:glucose 40 g/L,nitrogen source of 1.39 times of mixed nitrogen source(polyvalent peptone:beef extract:yeast powder=15:20:15),buffered salt of potassium dihydrogen phosphate/sodium hydroxide 0.05 mol/L.Considering the feasibility of the operation,the glucose was set to 40 g/L,and the nitrogen source was 1.4 times that of the mixed nitrogen source(polyvalent peptone:beef extract:yeast powder=15:20:15),potassium dihydrogen phosphate-sodium hydroxide Buffer salt 0.05 mol/L.Under these conditions,the number of viable bacteria was 9.2×10~9CFU/mL.(6)On the basis of the optimized medium,adding the 50 mL of hydrogen-rich water and it was found that the hydrogen-rich water seriously affected the growth of Lactobacillus rhamnosus YYJP-2,the number of viable cells was reduced by 51.9%.The specific reasons are not yet clear.(7)The content of lactic acid in the culture medium was reduced by 13.3%by determining the content of reducing sugar and lactic acid in the fermentation broth.The contents of potassium,sodium and sodium in the fermentation broth were determined by atomic absorption spectrometry.Magnesium,manganese and other chemical elements,found that part of themagnesium is the use of bacteria,and potassium,sodium,phosphorus content than before the high fermentation,the specific reason is not yet clear,to be follow-up experimental study.