Antibacterial Performance And Mechanisms Of Complex Acidifier Consisted Of Essential Oil And Medium-Chain Fatty Acids

At present,in the field of feed processing industry and animal husbandry,many studies are carried out for finding the replacement of antibiotics growth promoter(AGP).The feed acidifier is one of the ideal alternatives to antibiotics.However,the antibacterial performance of organic acids is unsatisfactory.If we want to achieve the same antibacterial effect to AGP,large doses of organic acids or essential oil have to be used.Therefore,the development and utilization of single organic acid or plant essential oil with antibacterial performance must be costly,and has a negative effect on the attractiveness of feed.So,this research focused on the development of complex acidifier consisted of essential oil and middle-chain fatty acids with high antimicrobial capability.More than 12 antibacterial agents,such as plant essential oils and medium-chain fatty acids(esters)were selected for antibacterial activity test,synergistic antibacterial experiments,and then use the orthogonal design experiment to obtain the best combination of antimicrobial formulations,and explore its antimicrobial properties.Finally,the bacteriostatic mechanism of cinnamaldehyde to Salmonella was studied,initially.Methods and results were as follows:(1)The detection of antimicrobial activity of essential oil and medium chain fatty acid(esters)E.coli,Salmonella and Staphylococcus aureus were selected as the indicator bacteria.The inhibition zone diameter and minimum inhibitory concentration(MIC)of each bacteriostat were determined by drilling method and agar plate dilution method respectively.The plate method and agar plate dilution method combined to explore the combined effect of both bacteriostatic.Results indicated that in the tested plant essential oil,the antimicrobial ability against E.coli,Salmonella(G-)were in the following order : cinnamaldehyde> carvacrol> eugenol> thymeol.At the concentration of 2.0%(w/w),guaiacol had little inhibitory effect on the above strains,while cinnamaldehyde had strong inhibitory effect on them,the MIC were all 250μg/m L.In the detected medium chain fatty acids,when the concentration of caproic acid,caprylic acid and capric acid was 0.5%(w / w)respectively,they all had strong inhibitory effect on the three kinds of bacteria.Lauric acid has little inhibitory effect on Escherichia coli and Salmonella,but has a certain inhibitory effect on Staphylococcus aureus;Among the detected medium chain fatty acid esters,Glycerylmonocaprylate had a certain inhibitory effect on Salmonella and Staphylococcus aureus,but little inhibitory effect on Escherichia coli;Lauric acid monoglyceride had a certain inhibitory effect on Escherichia coli and Staphylococcus aureus,but little inhibitory effect on Salmonella;caprylic did not show any inhibitory effect on the three strains.Antibacterial capacity of the above samples was enhanced with the increase of concentration.In addition,the combined effects of hexanoic acid,caprylic acid,capric acid,cinnamaldehyde,thymeol,carvacrol and eugenol were studied.The results showed that the combined use of these samples showed synergistic or additive effects,not antagonism.(2)Determination of compound antacid agents for high antibacterial activity and exploration of its antibacterial activityIn the orthogonal experiment of synergistic antibacterial compounds,the optimum ration of octanoic acid,carvacrol,capric acid,cinnamaldehyde,eugenol and hexanoic acid was 2: 1: 1: 5: 2: 2.The inhibitory circle size of Escherichia coli,Salmonella and Staphylococcus aureus was 17.45 mm,16.12 mm and 27.36 mm,respectively at the concentration of 0.5%(w/w),and the MIC were 250 μg/m L,500μg/m L,500μg/m L,respectively.The effects of temperature on the optimal combination formula were studied.The results showed that the antibacterial activity increased first and then decreased with the increase of temperature.And further explore antibacterial time of the optimal combination formula,The results showed that it could play a bacteriostatic effect in a long time(216h),and the pathogen was not easy to grow back.Three pathogens were treated with cinnamicaldehyde for 4 hours,respectively,in artificial simulated gastric fluid environment,The result showed that the bactericidal rate against Staphylococcus aureus and Salmonella was 100%,and the bactericidal ability against Escherichia coli decreased,but the bactericidal rate still could reach 95.6%;Three pathogens were treated with cinnamicaldehyde for 4 hours,respectively,in artificial simulated intestinal fluid environment.The result showed that the bactericidal rate against Staphylococcus aureus was 100%,and the bactericidal rate against Salmonella and Escherichia coli decreased,but still 93.8% and 98.24%,respectively.(3)Preliminary results of the antimicrobial mechanism of cinnamaldehyde to SalmonellaSalmonella was selected as model bacteria to determine the antimicrobial mechanism of cinnamaldehyde.The ultraviolet(UV)absorption and electrical conductivity of the culture supernatant were used to determine membrane integrity.Scanning electron microscopy(SEM)and transmission electron microscopy(TEM)were performed to observe bacterial morphology.The results indicated that firstly,UV absorptions,conductivity displayed a rapid rise trend,and then until stabilized at a relatively steady state,when the strain exposed to cinnamaldehyde.SEM showed that the treated Salmonella samples exhibited shrink,dry,depression,distortion,the structure had begun to collapse,unlike the control group.TEM showed that the bacteria treated with cinnamaldehyde exhibited cytoplasmic membrane separation from the cell wall,electron density reducation,cytoplasmic conten condensation,almost empty.Flagellum has been off,treated four hours with cinnamaldehyde,evaluated by Phosphotungstic acid negative staining and electron microscopy.The optimal ratio of compound acidifier was octanoic acid,carvacrol,capric acid,cinnamaldehyde,eugenol,caproic acid = 2: 1: 1: 5: 2: 2.The formula had a certain sensitivity to the temperature,high antibacterial properties,and the antibacterial time was long,the indicator bacteria was not easy to grow back.In addition,the combined effects of hexanoic acid,caprylic acid,capric acid,cinnamaldehyde,thymeol,carvacrol and eugenol showed that both of these samples showed synergistic or additive effects,not antagonism.Cinnamaldehyde inhibited Salmonella by destroying the cell membrane and flagella.