Study On Antibacterial Properties Of Lactococcin036019 And Its Application In Soy Milk

Lactic acid bacteriocin is a polypeptide synthesized by lactic acid bacteria through the ribosome mechanism during metabolism.Compared with chemical synthetic preservatives,it has obvious advantages:it is safe and non-toxic to the human body,not easy to cause drug resistance,easy to digest,will not remain in the body,has no pollution to the environment,and will not affect the original flavor and quality of food.However,due to the limitations of production technology and biological characteristics,most bacteriocins have a series of problems such as low yield,sensitivity to p H and temperature,narrow bacteriostatic spectrum,and weak preservation effect in practical application,so the real application of bacteriocins in the market has certain limitations.Therefore,improving the bacteriostatic performance of bacteriocin has in-depth research value.In the early stage,a strain of lactococcus lactis was screened from naturally fermented kimchi,and it has been determined that this bacteriostatic substance is a protein substance,and has the characteristics of wide antibacterial p H,strong heat resistance and strong storage stability.This project aims to improve the ability of Lactococcus lactis to produce bacteriocins by optimizing the fermentation process,analyze the influence of food matrix on bacteriocin performance,then use fence technology to screen the best synergistic,and finally use immobilization technology to protect bacteriocins and synergies,improve the stability of bacteriocins and extend their action time in food,and apply it in food.so as to provide a theoretical basis and reference scheme for the application of bacteriocins in the food industry.The main findings of this paper are as follows:（1）Optimization of Lactococcus lactis NCU036019 medium formula and culture conditions:The effects of different types of carbon sources,nitrogen sources,inorganic salts,growth factors,surfactants and buffer salts on bacteriocin-producing by Lactococcus lactis were studied using titer and inhibition zone diameters as evaluation indexes and Staphylococcus aureus as indicator bacteria.Optimization by single-factor testing,Plackett-Burman testing,steepest climb experiment,and response surface testing to increase bacteriocin antimicrobial activity.The optimal media components for bacteriocin production:yeast powder added 1.97%,zinc sulfate added 138 mg/L,sodium acetate added 1.00%,mannitol 0.5%,Tween-800.31%,dipotassium hydrogen phosphate 0.5%;Then,according to the single-factor experiment,the inoculation amount,incubation time and culture temperature and other culture conditions were optimized,and the optimal culture conditions of Lactococcus lactis were determined as follows:initial p H was 7.5,inoculation amount was 2%,incubation temperature was 30°C,and incubation time was 20 h.（2）Effect of food matrix on antibacterial activity of bacteriocin and screening of synergistic substances:determine the effect of carbohydrates,lipids,proteins and food additives on bacteriocin antibacterial activity in food matrix.With the increase of concentration,glucose,sucrose and lactose can enhance the antibacterial activity of bacteriocins,but the diameter of the inhibition circle of mannose and starch is gradually decreasing.Corn oil,soybean oil and camellia oil had no obvious effect on the antibacterial activity of bacteriocins.However,the antibacterial activity of rapeseed oil,sesame oil,peanut oil,olive oil and lecithin on bacteriocins gradually decreased,among which olive oil had the greatest negative effect on bacteriocins,and when the amount of olive oil was added 50%,the inhibition zone diameters decreased from 12.20 mm to 7.68 mm.The bacteriostatic activity of whey protein,ovalbumin and casein against bacteriocins gradually decreased.However,soy protein has a significant effect on the bacteriostatic activity of bacteriocin,and when the amount of addition is greater than 3%,bacteriocin has no bacteriostatic activity on the indicator bacteria.Ethanol,sodium dodecyl sulfate（SDS）,and glutathione have less effect on bacteriostatic activity of bacteriocins.Tween-80 and vitamin C enhance the bacteriostatic activity of bacteriocins.With the increase of inorganic salt concentration,the bacteriostatic activity of bacteriocin first increased and then decreased.Screening of lactococcin036019 synergistic substances:The best synergistic substance is determined by determining the minimum inhibitory concentration（MIC）and fractional inhibitory concentration indices（FICI）of bacteriocin and plant extract.The fractional inhibitory concentration indices of carvacrol and vitamin C combined with bacteriocin were 0.375 and 0.5,both less than or equal to 0.5,so that carvacrol and vitamin C had synergistic bacteriostatic effects on bacteriocins.On this basis,the bacteriostatic ability of the above two was evaluated,and it was determined that vitamin C was used as a fence factor to enhance the antibacterial activity of bacteriocins.（3）Research on immobilization technology of lactococcin036019 and its application in soy milk:W₁/O emulsion and W₁/O/W₂emulsion were prepared by adding different types of emulsifiers,different concentrations of emulsifier,and different internal aqueous and oil phase ratios,and the apparent properties,encapsulation rate,stability and bacteriostatic properties of the emulsion were analyzed to obtain the optimal double emulsion preparation parameters were obtained:PGPR addition amount was 8%,and the ratio of internal water phase to oil was 1:4.Then,microcapsules were prepared by spray drying,and the apparent properties,antibacterial activities and storage stability of microcapsules were determined to obtain a biological preservative with excellent antibacterial performance.Finally,when the microcapsule was added to artificially contaminated soy milk,it was found that it could not only significantly reduce the amount of S.aureus in soy milk,but also continuously protect bacteriocin from playing a antibacterial role in soy milk.It indicates that the microcapsule can be used as a biological preservative in food.