Screening The Traditional Chinese Herbs Against The Main Bacteria In Livestock Feeding And The Anti-bacterial Mechanism

With the development of intensive farming and animal husbandry,the chances of animals being infected by pathogens have greatly increased.In animal husbandry production,antibiotics play an important role in the prevention and treatment of livestock diseases and growth promotion,but long-term use of antibiotics will lead to decreased immunity of animals,increased resistance of pathogenic bacteria and imbalance of intestinal flora,and its negative effects are increasingly apparent.Lots of regulations have been made to ban or reduce the use of antibiotics in animal husbandry both nationally and globally.Therefore,it is urgent to find alternatives instead of antibiotics.As a natural treasure,Chinese herbal medicine has been found many functional and beneficial properties,such as antibacterial,antiviral,growth promotion,immunity and antioxidant,and has the advantages of low toxicity and no resistance to bacteria,which is the best substitute for antibiotics.Seven main and widely used herbs growing in northwestern China were used in this study to test anti-bacterial effect by in vitro method.The active herbal ingredients,and the effect on rumen bacteria composition,anti-bacterial mechanism were studied.This research can shed light on the use of herbs to restrict the growth of bacteria in livestock feeding.The results are as follows:1.Extraction and analysis of the effective components of the herbsSeven major traditional Chinese herbs were used in this study,including Angelica sinensis,Codonopsis pilosula,Astragalus membranaceus,Radix isatidis,Rheum Palmatum,Scutellaria baicalensis and Glycyrrhiza uralensis.Different functional parts from the dried root of herbs were extracted,such as alcohol extract,water extract,polysaccharides,saponins and flavonoids.The results showed that seven herbal active components were classified into 10 categories.Flavonoids and phenols are the main categories.The results by cluster analysis showed that the active ingredients in the seven herbs were divided into four groups according to the similarity of components,A.sinensis and G.uralensis,R.Palmatum and S.baicalensis,C.pilosula,R.isatidis and A.membranaceus.There were five main components in the ethyl acetate extract of R.Palmatum.Rhein and baicalin were the main ingredients.Baicalin and baicalein are the main components in the ethyl acetate of S.baicalensis.There are four main components in G.uralensis ethyl acetate.Mono-ammonium glycyrrhizinate and baicalin are the dominant.2.In vitro antibacterial analysis of the main herbal active ingredientsFour common pathogenic bacteria in animal husbandry,Escherichia coli(CMCC44102),Staphylococcus aureus(CMCC26003),Salmonella(CMCC50041)and Pasteurella pasteurella(CVCC393),were selected as target bacteria.The organic solvent extract and ethyl acetate extract were used to analyze the anti-bacterial effect on petri dish.The results showed that R.Palmatum had inhibitory effect on the four kinds of bacteria.C.pilosula showed inhibitory effect on Escherichia coli.Staphylococcus aureus and Salmonella.S.baicalensis had inhibitory effect on Escherichia coli and Staphylococcus aureus.Among the extracts of R.Palmatum,S.baicalensis,C.codonopsis and G.uralensis,the crude polysaccharides did not show antibacterial effect.The antibacterial effect of ethyl acetate extract was better than that of n-butanol,water extract and alcohol extract.After combing the functional parts in the extracts,the results showed that the combination of R.Palmatum and G.uralensis had the best antibacterial effect.3.Herbal antibacterial effect in in vitro rumen fermentationRumen is an important stomach for ruminants.Due to its complex environment,the herbal antibacterial function should be influenced in the rumen after digestion.According to the previous results,some herbal extract parts were selected for the in vitro rumen antibacterial test,such as the ethyl acetate parts of R.Palmatum and S.baicalensis,and the combination of R.Palmatum and G.uralensis ethyl acetate parts.The colony count results showed that the antibacterial effect in the rumen fluid was dosage dependent.The antibacterial effect was not promoted with the increase of concentration of herbal extract.The growth status of the three bacteria(Escherichia coli,Staphylococcus aureus,Salmonella)were different in the rumen environment.The number of Escherichia coli and Salmonella was significantly higher than that of Staphylococcus aureus,which means that Staphylococcus aureus is much easier to be inhibited in the rumen fluid.The microbiota results showed that the rumen fluid communities were clustered with the ethyl acetate fractions of R.Palmatum,G.uralensis and S.baicalensis in the rumen fluid inoculated with Escherichia coli,Staphylococcus aureus and Salmonella.The treatment groups were significantly differentwith the control group,but there was no significant differences between the herbal treatment groups.The ethyl acetate extract of S.baicalensis increased the abundance of Prevotella,while adding R.Palmatum and G.uralensis ethyl acetate parts reduced the abundance of Rikenellaceae＿RC9＿gut＿group and other fiber degrading bacteria in the fibre decomposition bacaterial family.4.Herbal antibacterial effect in different remen fermenting time in vitroWith the increase of herbal extract concentration,the antibacterial effect did not improve in the remen fluid.The Escherichia coli,Staphylococcus aureus and Salmonella were inoculated in the rumen fluid,and sampled at different fermentation time to analyze the antimicrobial effect and the influence on rumen microbial composition.In addition,different herbal adding programs in the rumen fluid were tested.The results showed that R.Palmatum water extract(100-200 mg/m L)had inhibitory effects on Escherichia coli,Staphylococcus aureus and Salmonella.The ethyl acetate of S.baicalensis at 0.2 mg/m L inhibited Escherichia coli growth,but the inhibiting effect can only consist for around 6-10 hours.The R.Palmatum water extract addition increased the abundance of ruminal bacterias,such as Pseudorbutanvibrio,Streptococcus,Arinovibrio succinate,Rieschoniae RC9 intestinal group and Kristenseniae R-7 group.5.Herbal antibacterial mechanismThe ethyl acetate parts of S.baicalensis and the combination of R.Palmatum and G.uralensis were used to test the mechanism of antibacterial effect on Escherichia coli,Staphylococcus aureus and Salmonella.The fatty acids composition in pathogen cell membrane were analyzed.And the cell structure before and after herbal treatment was observed by electron microscope.The results showed that the herbal ethyl acetate fraction caused the change of fatty acid structure of cell membrane,which was reflected in the change of saturated fatty acid-unsaturated fatty acid ratio,and then caused the loss of membrane substance,leading to cell death.The ethyl acetate of S.baicalensis caused severe morphological deformation and cell membrane rupture of Escherichia coli.The ethyl acetate of R.Palmatum depressed the cell membrane of Staphylococcus aureus and incurred the cytoplasm outflow.