Preparation And Application Of Antimicrobial Agents Produced By Probiotics

Biological antibacterial agents as biopreservatives have the advantages of high efficiency,brode spectrum,favorable stability and high safety.Biological antibacterial agents are developing rapidly with the enhancement of people's health awareness.Physical preservation has a safe and reliable effect,but its investment is high and it has certain limitations.Twenty-three kinds of probiotic strains preserved in our laboratory were researched in this study.Probiotics of producing antibacterial agents were screened out by bacteriostasis test and were identified by 16 S rDNA.By single factor,PB(Plackett-Burman)test and response surface methodology to determine the fermentation conditions and optimum medium for the strain producing antibacterial agents.The antibacterial agents were dried by vacuum freeze-drying to obtain powder.Finally,it was applied to preservate bananas and chilled pork.The main results are as follows:1.Conducting an inhibition zone test with the indicator microorganism: Staphylococcus aureus,Escherichia coli,Penicillium,Vibrio alginolyticus,the results showed that probiotics did not inhibit indicator microorganism but centrifuged supernatant(antibacterial agents)by probiotic fermentation have inhibitory effect on indicator microorganism,and its inhibitory effect to the Penicillium is weak.A bacillus with good bacteriostatic activity was screened out by primary and again screening.The strain identified by 16 S rDNA was Lactobacillus rhamnosus.2.Bacteriostasis test showed that antibacterial agents of L.rhamnosus L57 has a broad antibacterial spectrum,which has inhibitory effect on E.coli,S.aureus,Vibrio alginolyticus,Bacillus natto and it has weak antibacterial effect to Penicillium,but it has no antibacterial effect on Aspergillus niger,Saccharomyces boulardii and S.cerevisiae.By eliminating the interference of organic acids,hydrogen peroxide and exopolysaccharides,the study confirmed that protein or peptides in the antibacterial agent has bacteriostasis through ammonium sulfate salting.The antibacterial activity of the antibacterial agent treated with trypsin,pepsin and papain was good.However,antibacterial agents good resistance to pH,temperature and UV,and the antibacterial activity showed the optimum pH ranges from 2 to 4.3.The optimum fermentation conditions and medium composition of L.rhamnosus L57 produced the antibacterial agent were obtained by the single factor,PB test and response surface method.The results showed that the optimum fermentation conditions were: initial pH 6.5,inoculum 5.0%,38? for 21 h,the diameters of inhibition zone of E.coli and S.aureus were 19.24±0.14 mm and 21.59±0.12 mm under these conditions,respectively;compared with the control the diameter of inhibition zone of E.coli(17.46mm)and the diameter of inhibition zone of S.aureus(19.21mm)increased by approximately 10.3% and 12.4%,respectively.The optimal medium composition of glucose 3.2%,soy peptone 2.8%,sodium acetate 0.7%,galacto-oligosaccharide 0.2%,Tween-80 2mL/L,dipotassium hydrogen phosphate 0.3%,manganese sulfate 0.005%,the diameters of the inhibition zone of E.coli and S.aureus were 19.36±0.11 mm and 21.97±0.08 mm,respectively;compared with the control group,the diameters of inhibition zone of E.coli(12.35mm)and S.aureus(15.64mm)were increased by 57% and 41%,respectively.4.The growth and bacteriostatic curve of L.rhamnosus L57 showed that the antibacterial activity was enhanced with the growth of the bacteria,and the antibacterial activity became flattened after 20 h of fermentation.Antibacterial agents powder prepared by freeze-drying with adding the 15% soluble starch to antibacterial liquid.there was no significant difference in bacteriostatic performance between rehydrated and undried L.rhamnosus antibacterial agents.The minimum inhibitory concentration(MIC)of antibacterial agents powder in Luria-Bertani(LB)medium was 0.78 g/L for the E.coli and S.aureus,and the minimum bactericidal concentration(MBC)for the E.coli and S.aureus were 6.25g/L and 3.13g/L,respectively.5.The antibacterial agent freeze-dried powder of L.rhamnosus L57,chitosan,natamycin alone and compound applicated to preservate banana and chilled pork.The results showed that the single antibacterial agent can be to a certain kept fresh on bananas and chilled pork,while chitosan 1.5%,antibacterial agent freeze-dried powder 1.25% and natamycin 0.03% had the preferable effect and extended the shelf life of bananas and chilled pork to 4 d and 6 d compared with the control.In this study,the antibacterial agents and composite biopreservatives were prepared and have advantages of broad spectrum and high safety and could extend the shelf life of bananas and chilled meats,which can provide a theoretical basis for the development of biopreservatives,at the same time,it provides technical references for the preservation of fruits,vegetables and meat by biopreservatives.