Screeging And Application Of High-yield Bacteriocin Producing Lactobacillus Plantarum Strains By Mutations And Breeding

The food safety problems are paid more attention with the developing of people's living standards in recent years.More scholars,at home or abroad,are dedicated to the development of natural antibacterial substances with low toxicity and high efficiency.Lactobacillus can produce many kinds of natural antibacterial substances which are including organic acid,hydrogen peroxide,and bacteriocins et al.Bacteriocin is the hotspot of food preservation field.The bacteria named Lactobacillus plantarum JLA-9 was isolated from pickled cabbage of Northeast China by Laboratory of Enzymic Engineering,Nanjing Agricultural University,and it can produce a novel bacteriocin which strongly inhibited the growth of gram-positive bacteria and gram-negative bacteria.The purpose of this study was to obtaining a parental library with diverse positive mutations through ultraviolet(UV)mutagenesis,nitrosoguanidine(NTG)mutagenesis,60Co gamma gray(60Co?)mutagenesis,and atmospheric and room temperature plasma(ARTP)mutagenesis,and then using the technology of genome shuffling to get a higher-antibacterial-titer strain which is stable heredity.In the end,the strain we screened was encapsulated and applied in food preservation.The main results were as follows:1.Establish a parental library with physical and chemical mutagenesis.The best mutating time was determined to be 14 hours by measuring the growth of Lactobacillus plantarum JLA-9.The strain was mutagenized with UV mutagenesis,NTG mutagenesis,60Co? mutagenesis and ARTP mutagenesis,respectively.Through the measuring of lethal rate curve and positive mutation rate curve,the best mutation condition of UV mutagenesis was determined as follows:the UV power was 18 W,the irradiation distance was 20 cm,and the irradiation time was 90 s;the best mutation condition of NTG mutagenesis was determined as follows:the water-bath temperature was 37 ?,the water-bath time was 20 min,and the concentration of NTG was 4 mg/mL;the best mutation condition of 60Coy mutagenesis was determined as follows:the irradiation dose was 600 Gy,and the dosage rate was 15 Gy/min;the best mutation condition of ARTP mutagenesis was determined as follows:the radio-frequency power was 200 W,the gas flow was 10 SLM,the effective distance was 2 mm,and the irradiation time was 10 s.Eight mutant strains with higher antibacterial titer,named UV90-115,UV90-159,N4-26,N4-27,60Co?600-9,60Co?600-67,ARTP10-37,and ARTP10-61,were obtained through primary and secondary screen using above methods.The antibacterial titer of these strains were increased by 35.64%,23.61%,14.94%,38.79%,31.25%,37.30%,35.02%,and 48.06%,respectively,compared to the wild-type strain.2.Obtain high-yield bacteriocin producing strain by genome shuffling.The conditions of protoplast preparation,inactivation and fusion were researched.Through the measuring of protoplast forming rate and regeneration rate,the optimal conditions for protoplast preparation was determined as follows:the concentration of lysozyme was 20 mg/mL,the enzyme treating time was 20 min,and the concentration of high-sugar buffer was 0.5 mol/L;Through the measuring of protoplast fatality rate,the optimal conditions for protoplast inactivation was determined as follows:the UV irradiated for 210 s,or water bathed for 50 min;Through the measuring of protoplast fusion rate,the optimal conditions for protoplast fusion was determined as follows:the concentration of PEG was 40%,and the fusing time was 6 min.Using the 8 mutant strains above as parent strains,a strain named F4-2 was screened after four rounds of genome shuffling.The antibacterial titer of strain F4-2 was increased to 7374.76 IU/mL,2.35 times compared to the wild-type strain.And the strain F4-2 was stable heredity.3.Optimize of the preparation condition of encapsulated lactobacillus.Through single factor experiment and response surface analysis,the optimal preparation condition of encapsulated lactobacillus was determined as follows:the concentration of NaAlg was 1.94%,the concentration of CaCl2 was 2%,and the concentration of bacteria was 8.53%.In such a condition,the bacteria encapsulated rate can reach to 88.68%.The highest antibacterial activity of encapsulated bacteria was reached when it fermented 48 hours,behind the free bacteria,but the antibacterial activity of encapsulated bacteria was dropped slowly than the free bacteria,which reflected the sustained-release effect.The survival rate of the encapsulated and free bacteria in artificial gastric juice was 95%alike,but in the artificial intestinal juice,the survival rate of the encapsulated bacteria can reach to 21.1%,when the free bacteria was less than 1%,which reflected the protective effect of encapsulation.4.Application of encapsulated lactobacillus in fermented milk.The encapsulated bacteria was added into the homemade yogurt with Escherichia Coli,and then the quantitative variance of microorganism during fermentation and storage was measured;the capsule group had more lactic acid bacteria and less E.Coli quantitaties compared with control group.After 20 days of refrigeration,the lactic acid bacteria quantitaties of the capsule group was 3.16 logCFU/g more than the control group;after 12 days of refrigeration,the Escherichia Coli quantitaties of control group was 3.89 logCFU/g,whereas the capsule group was under detection limit,which reflected the protective effect of encapsulation.The encapsulated bacteria was added into the homemade yogurt without Escherichia Coli,and then the physical and chemical indexes(including fat content,protein content,non-fat milk solids content,pH and titration acidity)during storage was measured;the capsule group had no significant influence on the physical and chemical indexes compared with the control group.