High Cell Density Fermentation Of Lactobacillus Rhamnosus LS-8 And Its Powder Preparation And Application

In recent years,as the potential probiotic function of lactic acid bacteria has been continuously confirmed,the demand for lactic acid bacteria-related products has gradually expanded at home and abroad.Therefore,it is of profound significance to improve the competitiveness of the lactic acid bacteria powder industry in China by using low-cost,safe and edible raw materials to culture and prepare it.In this study,Lactobacillus rhamnosus LS-8 was selected as the research strain.The MRS medium was firstly replaced by raw material,and the fermentation process of Lactobacillus rhamnosus LS-8 was optimized by Plackett-Burman test and response surface methodology;And then the fermentation kinetics of Lactobacillus rhamnosus LS-8 was studied in an automatic fermenter and use the method of fed-batch fermentation to achieve high cell density fermentation;In order to prepare high-activity powder,the freeze and heat protective agents were screened by vacuum freeze drying and spray drying,and the spray drying process was optimized;Finally,the powder was directly applied to yoghurt fermentation application.The specific results are as follows:（1）Simplification and optimization of media ingredients and culture conditions for Lactobacillus rhamnosus LS-8.The optimal culture medium and culture conditions of Lactobacillus rhamnosus LS-8 were realized at the level of the flask.The optimal culture medium was consisting of maltose syrup（50g/L）,whey powder（62.5g/L）,corn steep liquor（55g/L）,Na Cl（1g/L）and lysine（0.05g/L）.And the optimal culture conditions were initial p H（6.3）,inoculum size（3.5%）,fermentation temperature（37°C）.The cell density of Lactobacillus rhamnosus LS-8 was reached approximately 1.64×10⁹（CFU/m L）after fermented for 16 h under the simplified and optimized conditions.Compared with the fermentation using MRS medium,the maximum cell density was enhanced by about 3 times,and the time to enter the stable growth period was shortened by about 12 hours,and the cost of the culture medium was decreased by almost 60%.（2）High cell density fermentation and kinetic analysis of Lactobacillus rhamnosus LS-8.The fermentation process of Lactobacillus rhamnosus LS-8 was determined in the level of fermenter,and the cell density of Lactobacillus rhamnosus LS-8 achieved about 4.5×10⁹（CFU/m L）under conditions of p H（4.7）,neutralizer（Na OH）,oxygenation rate（0.2L/min）and stirring rate（200r/min）.Compared with the fermentation using MRS medium,the maximum cell density was enhanced by about 9 times.At the same time,the kinetic models for cell growth and substrate consumption of Lactobacillus rhamnosus LS-8 were studied by Logistic equation and Luedeking-Piret-Like equation respectively.The R² of each proposed models was greater than 99.00%,suggesting that the model can better describe the cell growth and substrate consumption of Lactobacillus rhamnosus LS-8.And five cycles showed good fermentation stability and repeatability in the repeated fed batch fermentation.（3）Preparation of Lactobacillus rhamnosus LS-8 powder.The freeze and heat protective agents were screened by vacuum freeze drying and spray drying,and the spray drying process was optimized.The activity of vacuum freeze-dried bacterium was 1.6×10¹⁰（CFU/g）when glycerol（3.4%）,trehalose（11%）and gelatin（0.1%）were used as composite freeze protector.And the activity of spray-dried bacterium was 1.5×10⁹（CFU/g）under conditions of concentration ratio（2:1）,inlet temperature（130°C）,outlet temperature（61.5°C）,feeding amount（310m L/h）when trehalose（6%）,maltodextrin（6%）and lactose（3%）were used as composite heat protector.Both dry powders are appropriate for storage in a low temperature environment.And under the condition of-20°C for 90 days,there was no significant change in the activity of freeze-dried powder,and the activity of spray drying powder was still as high as1.0×10⁹（CFU/g）.（4）Application of Lactobacillus rhamnosus LS-8 powder.The fermentation process parameters of self-produced fermented milk were established,and the fermentation performance of the self-produced strain was comprehensively evaluated through sensory evaluation,physical and chemical indicators,and flavor analysis.there was no significant difference（p>0.05）in the sensory scores of the self-made powder fermented milk under the conditions of inoculation（0.15%）,the fermentation time（11h）and the fermentation temperature（41°C）compared with the commercial powder fermented milk.Meanwhile,the number of live bacteria in self-made powder fermented milk was as high as 2.2×10⁸（CFU/m L）.In addition,40 kinds of volatile flavor components were isolated and identified from homemade semolina milk,including acetaldehyde,2,3-butanedione,3-hydroxybutanone,and various volatile acids and other major yoghurt flavor components,suggesting that the self-made powder has the potential to develop functional yogurt.