Screening And Identification Of Plant Fermentation Bacteria And Preparation Of Bacterial Agents

With the progress of society and the development of the times,people pay special attention to their own health.Fruits and vegetables contain a variety of nutrients,and lactic acid bacteria also have many probiotic properties,and the two complement each other.Therefore,lactic acid bacteria fermented fruit and vegetable foods have been favored by more and more people.In this study,a variety of lactic acid bacteria were isolated from naturally fermented pear juice enzymes.The lactic acid bacteria were screened through the growth performance,fermentation performance and tolerance performance of the bacteria,and the combination of bacteria with better fermentation effects on plant raw materials was selected.And the bacterial agent was prepared for the screened strains.The results of this study are as follows:Isolation,purification and identification of lactic acid bacteria from the naturally fermented pear enzymes,and 8 types of lactic acid bacteria were obtained.After RFLP analysis and molecular identification,they were identified as six types of lactic acid bacteria,namely Lactobacillus plantarum(S1),Enterococcus faecalis(S2),Lactobacillus casei(S3),Pediococcus pentosaceus(S4),Leuconostoc fallax(S5),Streptococcus thermophilus(S6).The 12 strains of lactic acid bacteria,including the above six lactic acid bacteria,were screened in terms of growth in the compound fruit and vegetable juice,acid production performance,acid resistance,bile salt tolerance of the strains,and tolerance of artificial gastric juice and artificial intestinal juice.Two sets of bacterial species combinations of three lactic acid bacteria were obtained.The three lactic acid bacteria were Lactobacillus plantarum(S1),Lactobacillus casei(S3)and Pediococcus pentosaceus(S4).The two combinations are Lactobacillus plantarum S1:Lactobacillus casei S3(1:1)and Lactobacillus plantarum S1:Pediococcus pentosaceus S4(1:1).High-density cultivation of three kinds of lactic acid bacteria found that the optimal medium for Lactobacillus plantarum S1 is glucose 20.3 g/L,peptone 10.1 g/L,yeast extract 20.2 g/L,sodium acetate 6.0 g/L,potassium phosphate dibasic 2.0 g/L,ammonium citrate tribasic 2.0 g/L,Mg SO₄ 0.58 g/L,Mn SO₄ 0.25 g/L,1 m L/L Tween 80 and 101m L/L tomato juice were 13.8 times higher than before optimization.The optimal medium for Lactobacillus casei S3 is glucose 21.1 g/L,soya peptone 20.2g/L,yeast extract 10.1 g/L,sodium acetate 6.0 g/L,potassium phosphate dibasic 2.0 g/L,ammonium citrate tribasic 2.0 g/L,Mg SO₄ 0.58 g/L,Mn SO₄ 0.25 g/L,1 m L/L Tween 80and 102 m L/L tomato juice,which are 11.21 times higher than before optimization.The optimal medium for Pediococcus pentosaceus S4 is glucose 25.6 g/L,beef extract15.3 g/L,yeast extract 15.3 g/L,sodium acetate 2.0 g/L,potassium phosphate dibasic 2.0g/L,and ammonium citrate tribasic 6.0 g/L,Mg SO₄ 0.58 g/L,Mn SO₄ 0.25 g/L,1 m L/L Tween 80 and 101 m L/L tomato juice,which are 11.71 times higher than before optimization.Through the exploration of the optimal culture conditions and collection conditions of the three bacteria,the results showed that the culture conditions of Lactobacillus plantarum S1 were 35?,initial p H 6.5,inoculation amount 3%,rotation speed 90 r/min;Lactobacillus casei S3 culture The conditions are 37?,initial p H 7.0,inoculation volume3%,rotation speed 0 r/min;culture conditions of Pediococcus pentosaceus S4 are 35?,initial p H 6.5,inoculation volume 2%,shake flask rotation speed 90 r/min;After high-density cultivation,the highest concentration of Lactobacillus plantarum S1was 1.02×10¹¹ CFU/m L;the maximum concentration of Lactobacillus casei S3 was7.93×10¹⁰ CFU/m L;the maximum concentration of Pediococcus pentosaceus S4 was4.76×10¹⁰ CFU/m L.The centrifugal collection conditions of lactic acid bacteria were 4?,5000r/min,and centrifugation time 20 min.The response surface method optimization of the three kinds of lactic acid bacteria composite freeze-dried protective agent,the best Lactobacillus plantarum S1 protective agent formula is:skimmed milk powder 10%,trehalose 8.14%,glycerin 2.04%,Sodium glutamate 3.04%.The best formula of Lactobacillus casei S3 protectant is:skimmed milk powder 10%,trehalose 10.15%,glycerol 3.05%,and sodium glutamate 1.03%.The best Pediococcus pentosaceus S4 protective agent formula:skimmed milk powder 10%,trehalose 8.17%,Mannitol 3.05%,sodium glutamate 3.03%.Preli minary research on the freeze-drying conditions of lactic acid bacteria,the final optimal preparation process of lactic acid bacteria powder is pre-freezing conditions of-20?3 h,-80?3 h,bacteria mud:protective agent(1:3),lyophilization thickness is 5mm.The number of viable cells of Lactobacillus plantarum powder prepared under the above conditions reached 6.85×10¹² CFU/g;the number of viable cells of Lactobacillus casei powder was 5.45×1012 CFU/g;and the number of Pediococcus pentosaceus powder reached 3.36×10¹² CFU/g.The storage stability and fermentation activity of the prepared lactic acid bacteria powder were verified.The results showed that the viable bacteria rate of the lactic acid bacteria powder stored at room temperature for 28 days was lower than that of the bacteria powder stored at 4?,indicating that the bacteria powder should be in a low temperature environment.Next store.The fermentation activity of lactic acid bacteria powder was identified with the number of viable bacteria and acid production as indicators.It was found that the number of viable bacteria and the acid production of the composite fruit and vegetable juice fermented by the bacteria powder were very high.The number of viable bacteria reached 10⁸ CFU/m L,and the acid production reached It is more than 33 g/L.This study enriched the processing methods of plant food and laid the foundation for its industrialization.