Isolation And Screening Of Low Temperature-resistant Lactic Acid Bacteria And Bacteriostasis Of Its Metabolites

Refrigeration is a way to extend the storage time of food.Although this method can reduce food spoilage,there are some low-temperature resistant spoilage bacteria on the surface of food.In order to effectively inhibit the growth of such spoilage bacteria,the low temperature resistant strains were isolated and screened from the low temperature storage sauerkraut fermentation broth.Study on the low temperature adaptability of strains and enzymes and the bacteriostasis of metabolites produced by strains.The double-layer plate method and the Oxford cup method were used to test the bacteriostasis of common spoilage bacteria in food.The low temperature resistant bacteria are prepared into powder by vacuum freeze drying method,and provide excellent strains for low temperature fermentation and biological preservatives.The results are as follows:Sampling from sauerkraut fermentation broth at storage temperatures of 0?,4?,10?,and 15?,using dilution plate coating and scribing separation methods,Screening of low-temperature lactic acid bacteria in bromomethylphenol green-CaCO3 medium to obtain 7 strains of lactic acid bacteria;Then,the cell growth concentration and acid production were used as indexes to rescreen.One strain of low-temperature resistant lactic acid bacteria SF17 was obtained.At 10?,the strain concentration reached OD600 0.654,and the acid yield in MRS medium 50 g/L.Identification of SF17 lactic acid bacteria as Lactobacillus plantarum based on morphological,physiological and biochemical characteristics and gene sequencing analysis of 16SrDNA conserved regions.Lactobacillus plantarum subsp.plantarum(purchased from China Industrial Microbial Culture Collection Management Center)was used as a control strain to study the cold tolerance of SF17 lactic acid bacteria.After incubation at 10? for 48 h,scanning electron microscopy(SEM)showed that the surface of SF17 was smooth and intact,but substances were secreted.The control strain showed swelling and rupture.Extracellular polysaccharides are generally synthesized under conditions of nutritional imbalance and lower culture temperatures.The exopolysaccharide content of the SF17 strain was determined to be 5.98 mg/L at 10?.Under the induction of moderate stress,the increase of SOD activity can improve the adaptability of organisms.At 10?,the SOD activity of SF17 strain was 28.77 U/mg,and the SOD activity of the control strain remained unchanged at 5.01 U/mg.The significant increase in microbial secreted proteases is a major mechanism for adapting to low temperature living environments.The protease activity of the SF17 strain was 8.4 U/mL at 10?.The antibacterial effect of SF17 lactic acid bacteria on the spoilage bacteria Aspergillus niger,Escherichia coli,Bacillus subtilis and yeast which are common in food was studied by the method of flat plate pairing.The results showed that they all had antibacterial effects.In order to clarify the antibacterial activity of the metabolites of SF17 strain,the SF17 lactic acid bacteria were cultured at 25? for 48 h,and the metabolites obtained by centrifugation were heated,protease,lmol/L NaOH,and the bacteriostatic effect on the four indicator bacteria.When the heating temperature is 85?,Escherichia coli and yeast have a inhibition zone diameter of 11 mm,indicating that the metabolites are still bacteriostatic,and the bacteriostatic substances are not sensitive to heat.In order to further determine whether the bacteriostatic substance is a proteinaceous substance,after further enzymatic hydrolysis of the metabolite by tiypsin,pepsin and proteinase K,the minimum inhibitory circle diameter of the yeast is 12 mm,indicating that the bacteriostatic substance is not a protein substance.After metabolite treatment of SF17 strain metabolites,it was found that when pH?5,the metabolites were bacteriostatic to four indicator bacteria,and the minimum inhibitory zone diameter of yeast was 8.3 mm;when pH>5,bacteriostatic Disappeared,indicating that the main inhibitory substance may be an organic acid or a bacteriostatic substance that only works in an acidic environment.The main organic acids of SF17 lactic acid bacteria fermentation broth were further tested by LC-10A high performance liquid chromatography(HPLC),The results were 23.14 mg/mL lactic acid,9.64 ?L/mL acetic acid,9.12 mg/mL oxalic acid,11.23 ?L/mL propionic acid,6.95 mg/mL succinic acid,5.24 mg/mL malic acid,and 8.42 mg/mL citric acid.When the precursor substance phenylpyruvate 10 mg/mL was added to the MRS medium,the phenyl lactic acid content was 6.24 mg/mL.The lyophilized powder of SF17 lactic acid bacteria was prepared by vacuum freeze-drying method.The lyophilized protective agent was optimized by single factor and L9(34)orthogonally.The best protective agent combination of SF17 lactic acid bacteria was 9%trehalose added,degreased The amount of milk powder added is 3%,the amount of sucrose added is 12%,and the number of live bacteria of freeze-dried powder is 1.82×109 cfu/mL,which provides an excellent,convenient and rapid strain for biological antiseptic bacteria.