Antibacterial Mechanism Of Medium Chain Fatty Acid And Organic Acid Complex And Its Effect On Preventing Bacterial Diarrhea In Piglets

In modern pig production,early weaning of piglets is usually carried out in order to increase production efficiency and save costs.Early weaning generally leads to weaning stress in piglets,changes in intestinal flora structure,damages intestinal barrier function,increases susceptibility to pathogens and inflammation of the body,causing piglet diarrhea.Adding antibiotics and high doses of zinc oxide to feed are the main means to reduce diarrhea in weaned piglets and promote the growth of piglets.However,the large and irregular use of antibiotics can lead to bacterial resistance,drug residues and environmental pollution.High-dose zinc oxide can prevent piglet diarrhea,but it absorbs less in the body.A large amount of zinc will be excreted in the body with feces and pollute the environment.Therefore,in the context of the ban on the use of antibiotics and the limited use of zinc oxide,new antibiotic alternatives need to be developed to prevent piglet diarrhea.Medium chain fatty acids have good antibacterial activity against Gram-negative bacteria,Gram-positive bacteria,and fungi.The study found that the antibacterial activity of medium-chain fatty acids is negatively correlated with the environmental p H.Organic acids are usually added to piglet feed to reduce the p H of the gastrointestinal tract.Therefore,the complexation of medium chain fatty acids with organic acids may have synergistic antibacterial effects.In addition,studies have reported that medium-chain fatty acids can enhance the expression of antimicrobial peptides in host cells.Antimicrobial peptides have functions such as antibacterial,immunomodulatory and barrier protection.However,the mechanism by which medium chain fatty acids induce the expression of antimicrobial peptides remains unclear.Therefore,this study intends to first carry out in vitro antibacterial tests,screen combinations of medium chain fatty acids and organic acids with synergistic antibacterial activity,and study their direct antibacterial mechanism.Using the small intestinal cell line IPEC-J2 as a material to study the induction effect and potential mechanism of medium-chain fatty acids on host antimicrobial peptides.In this study,we first studied the induction effect and potential mechanism of medium-chain fatty acids on host antimicrobial peptides by using the small intestinal cell line IPEC-J2 as a material.Then we carried out an in vitro antibacterial test to study the antibacterial properties of medium-chain fatty acids and organic acids.Clarify its direct antibacterial mechanism.On this basis,a mouse model of E.coli O157:H7 infection was established to investigate the effect and mechanism of the combination of medium-chain fatty acids and organic acids against E.coli infection.Finally,to confirm the combination of medium-chain fatty acid and organic acid has the effect of relieving early weaning diarrhea through the piglet feeding test.The main findings are as follows:1.Caprylic acid and nonanoic acid upregulate endogenous host defense peptides to enhance the intestinal epithelial immunological barrier function via histone deacetylase inhibition.In this study,the jejunal epithelial cells IPEC-J2 infected with E.coli O157:H7 were used as a model to investigate the effects of caprylic acid and nonanoic acid on intestinal epithelial barrier function and its mechanisms.First,we found that caprylic acid and nonanoic acid can significantly reduce bacterial translocation.The expression of tight junction protein was detected by Q-PCR and Western blot.It was found that caprylic acid and nonanoic acid had no significant effect on the expression of tight junction proteins ZO-1 and Occluding.Subsequent studies have found that caprylic acid and nonanoic acid can increase the antibacterial activity of IPEC-J2 cell culture medium by inducing the expression of host defensins(p BD-1 and p BD-2).Dosing of these treatments also remarkably increased the secretion of the porcine β-defensins 1(p BD-1)and p BD-2.Mechanically,like the histone deacetylase inhibitors,C8 and C9 attenuated the activity of the classical histone deacetylase to facilitate the acetylation level of histone 3 lysine 9(H3K9)at the promoters of p BD-1 and p BD-2,consequently,augmenting the gene expression of p BD-1 and p BD-2.2.Study on antibacterial characteristics and antibacterial mechanism of optimal antibacterial combination of medium chain fatty acids and organic acids.In this test,Escherichia coli were used as test strains.Immunofluorescence,flow cytometry,scanning electron microscopy and transmission electron microscopy were used to study the antibacterial mechanism of selected combinations.The results showed that the synergistic antibacterial effect and best proportiont of octanoic acid + citric acid and nonanoic acid + citric acid on Escherichia coli were determined by the Chou-Talalay index method.Octanoic acid + citric acid and nonanoic acid + citric acid are effective in destroying E.coli cell membranes and cytoplasm,resulting in release of their cellular contents,thereby synergistically enhancing the antibacterial effect,as compared to the medium chain fatty acids and organic acids alone.3.Blend of organic acids and medium chain fatty acids prevents the inflammatory response and intestinal barrier dysfunction in mice challenged with Enterohemorrhagic Escherichia coli O157:H7.Impaired epithelial barrier function disrupts immune homeostasis and increases inflammation in intestines,leading to many intestinal diseases.The blend of organic acids(OAs)and medium chain fatty acids(MCFAs)has been shown to have synergistic bactericidal effect.In this study,we demonstrated that two blends of OAs and MCFAs(OM1 and OM2)could prevent the inflammatory response and intestinal barrier dysfunction in enterohemorrhagic Escherichia coli(EHEC)-infected mice.Treatments of OM1 and OM2 significantly reduced the body weight loss and production of IL-6 and TNF-α induced by EHEC.Mice treated with OM1 and OM2 showed decrease in serum D-lactic concentration,DAO activity and bacterial transfer to liver and spleen.Furthermore,OM1 and OM2 increased the expression of tight junction proteins occluding and ZO-1,mucus protein MUC-2,and host defense peptides m BD1,m BD2 and m BD3.Finally,OM1 and OM2 increased the population of Lactobacillus spp.and Bifidobacterium spp.,but decreased that of E.coli in the cecum.4.The protective effects of blend of organic acids and medium chain fatty acids on the growth performance and diarrhea of post-weaning pigs.Four hundred piglets weaned at 21 d with body weight of 6.82 ±1.00 kg were randomly divided into 4 groups.Two groups with a basic diet plus Zn O(2250 mg/kg)(high Zinc group)or basic zinc chloride(1500 mg/kg)(low Zinc group);The other 2 groups were fed with diets basically included basic zinc chloride(1500 mg/kg)plus lower dosage caprylic acid plus citric acid(combination group 1)or basic zinc chloride(1500 mg/kg)plus greater dosage caprylic acid plus citric acid(combination group 2).Compared with high Zn group and low Zn group,combination group 1and 2 significantly increase growth performance of weaning piglets(P <0.01).Compared with low Zn group,combination group 1and 2 significantly reduce the diarrhea rate of piglets(P <0.01).Adding combination 1 to earn 11.5 yuan more than adding high zinc and 11.63 yuan more than adding low zinc.Adding combination 2 to earn 12.49 yuan more than adding high zinc and 12.62 yuan more than adding low zinc.In summary:(1)Octanoic acid + citric acid and nonanoic acid + citric acid have synergistic antibacterial effect and can exert direct antibacterial action by destroying bacterial cell membrane and cytoplasm.Caprylic acid and nonanoic acid can exert an indirect antibacterial effect by inhibiting HDAC activity and increasing cell defensin expression.(2)Octanoic acid + citric acid and nonanoic acid + citric acid can regulate intestinal flora and alleviate weightlessness,inflammation and barrier function damage in mice caused by EHEC infection.(3)When the dietary zinc level is 1500 mg/kg,adding coated octanoic acid + citric acid can effectively control weaning diarrhea and achieve good economic benefits.