| The amount of pigs and poultry in China is huge,and the farming methods are rapidly developing in the direction of scale and intensification.At the same time as the production efficiency is greatly improved,the susceptibility of pigs and poultry to disease is greatly increased due to the increase of stocking density and stress level.The use of antimicrobial agents has become a necessary choice to ensure the production performance of livestock and poultry.As a result,drug residues and pathogen tolerance in pig and poultry products and breeding environments have become increasingly prominent.Studies have shown that cinnamaldehyde,carvacrol,eugenol,thymol and other plant essential oil active substances have a broad-spectrum antibacterial effect.However,there are various kinds of vegetable essential oil additives on the market at present,and the lack of objective and scientific evaluation of its antibacterial effect remains to be studie;in addition,there are few reports on the effect of plant essential oils on intestinal microflora of pigs.In this study,we focus on the above problems and use in vitro fermentation technology to compare the antibacterial activity of different types of encapsulated essential oil additives on the market and their effects on in vitro fermentation parameters,microbiota structure and function.Hopefully,the results from this research may provide theoretical guidance for the application of plant essential oil in animal production.Chapter 1.Study on thermal stability and antibacterial effect of plant essential oil additivesThe aim of this experiment was to compare the thermal stability of 15 kinds of plant essential oil additives for commercially use in pigs and poultry,and to explore their antibacterial activity against common pathogens in livestock and poultry production in order to provide a theoretical reference for the rational use of plant essential oil additives.First,the micro-broth dilution assay was adopted to determine the minimal inhibitory concentration(MIC)of 15 essential oil addives and their chemical components(cinnamaldehyde,carvacrol,eugenol,and thymol)against multi-drug resistant isolates(pig-derived Salmonella,Escherichia coli,Streptococcosis suis,poultry-derived Salmonella,Escherichia coli,and sensitive Escherichia coli ATCC25922).The antibacterial activity of combination testing of cinnamaldehyde,carvacrol,eugenol,and thymol was evaluated by the chequerboard method.Secondly,each of 15 essential oil products was divided into two groups(control group and heat treatment group),after diethyl ether extracted,the concentration of cinnamaldehyde,carvacrol,eugenol,and thymol was determined by means of gas chromatography(GC).Results showed that the active chemical components exhibited high sensitivity against all six strains of bacteria(MIC values ranging from 0.25 mg/mL to 2 mg/mL).The ratio of carvacrol/thymol(2:1)and eugenol/thymol(2:1)showed synergistic effects according to the indices of fraction inhibitory concentration(FICI=0.5).Among 15 essential oil additives,only EG2 had no obvious antibacterial activity for 6 selected bacterial strains,but the other 14 products h ad a good antibacterial capacity with the MIC values ranging from 1 to 32 mg/mL.The total amount of cinnamaldehyde,carvacrol,eugenol,and thymol in 15 additives was 2.64-691.40 mg/g,ranged from 0.2%to 14.11%after heat treatment.All tested plant essential oil additives generally had good thermostability and in vitro antibacterial effects Six additives with most strong antibacterial activity were EG7,EG3,ES2,ES1,EAP2 and EP1,which obvious potential for preventing and treatment of diseases caused by multi-drug-resistant bacteria in pig and poultry.Chapter 2.Effects of plant essential oil additives on in vitro fermentation characteristics and growth of Escherichia coli with pig ileum chyme as inoculaThe aim of this experiment was to explore the effects of 15 essential oil products on the microbial fermentation characteristics and growth of Escherichia coli with pig ileum chime as inocula.Each of selected essential oil products was divided into three group:the control group(0 mg/mL),the 0.5 mg/mL group,and the 1.0 mg/mL group.The ileal chyme and Escherichia coli were used as inoculum.The gas production in the fermentation system was monitored during the fermentation,and the E.coli was also counted in each fermentation system at 0,2,4,8,12 and 24 hours.After the fermentation,the pH of each bottle was measured and essential oil additives particles was separated from liquid to be observed under microscope.The results showed that 15 essential oil additives showed significant changes in color,particles size and breakage after 24 h of fermentation.The effects of 15 plant essential oil additives on the fermentation characteristics showed that widely differences was found among different plant essential oil.The pure plant essential oil type products EG2,EG3,EG7,ES1 and EP1 showed significant effects)on gas production during in vitro fermentation.For the four organic acid compound essential oil products EAG2,EAP1,EAP2 and EAS1,the 0.5 mg/mL and 1 mg/mL dose groups of EAG2 inhibited the microbial gas production during the whole fermentation process,and the total gas production was significantly lower than the control group(P<0.01).EAP1 and EAS1 had no significant inhibitory effect on the kinetic parameters of in vitro fermentation.In addition,EAG2 could inhibit the growth of pathogenic E coli during the whole fermentation process,and the effect was most obvious in 15 tested essential oil additives products.EG3(0.5 mg/mL)was found had no significant inhibition on the growth of E.coli during the fermentation.1 mg/mL of EG3 significantly inhibited the growth of E.coli during 0 to 12 hours of the fermentation(P<0.01).Pure plant essential oil products EG7,EG8,EG2,ES1 and EP1 also inhibited the growth of E.coli during fermentation.In summary,the effects of the 15 plant essential oil additives on the microbial fermentation characteristics in the fermentation system are quite different.The organic acid compound plant essential oil product EAG2 and the pure plant essential oil product EG3 have the best effects.In addition,EAG2(0.5 mg/mL)and EG3(1 mg/mL)can effectively inhibit the growth of E.coli during fermentation.Chapter3.Effect of plant essential oil additives on in vitro fermentation microbiota composition and metabolitesThe aim of this chapter was to compare the effect of 6 selected plant essential oil additives on microbial composition and concentration of SCFAs vitro fermentation system with ileal chyme and E.coli as inocula.The experimental design and grouping were the same as chapter 2.Samples were collected for DGGE analysis,qRT-PCR and SCFA at 0 h and 24 h of fermentation,respectively.The similarity results of DGGE showed that six selected essential oil additives reduced the similarity of microbiota after 24 h of fermentation.The diversity of DGGE results showed that EAG2,EG3,EP1 and EG6 at dose of 0.5 mg/mL and 1 mg/mL could significantly reduce the diversity of microflora in the in vitro fermentation system(P<0.01).Real-time PCR results showed that the addition of 0.5 mg/mL EAG2(P<0.01)or 1 mg/mL EP1 could significantly reduce the number of E.coli in the fermentation system(P<0.05).The addition of EG8(1 mg/mL)significantly increased the population of Lactobacillus(P<0.05),while the addition of 0.5 mg/mL and 1 mg/mL of the other five additives had no significant effect on the growth of Lactobacillus in the fermentation system(P>0.05).After 24 h of microbial fermentation,total short fatty acids,acetic acid and propionic acid in the EAG2 group(0.5 mg/mL and 1 mg/mL)were significantly lower than the control group(P<0.01).In addition,the addition of the six essential oil additives products showed increased the conversion of the product in the fermentation system from propionic acid to acetic acid,in which EG6 and EG8 significantly promote the conversion(P<0.05).In summary,appropriate doses of essential oil additives can inhibit the population and activity of some microorganisms,inhibit the abundance of Escherichia coli and optimize the structure of microflora... |
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