Three Strains Of Lactobacillus Were Isolated,identified And Used To Ferment Traditional Chinese Medicine To Inhibit PEDV Proliferation In Vitro

At present,piglet diarrhea has always been an important problem plaguing pig breeding industry.With the continuous expansion of pig large-scale breeding industry,the prevalence of piglet viral diarrhea disease shows an increasing trend year by year,seriously damaged to the economic benefits of breeding industry.Many studies have shown that probiotics alone with Chinese veterinary drugs or combined with antibiotics and vaccines have significant therapeutic effects on diarrhea of piglets.Probiotics and traditional Chinese medicine not only have the effects of antibacterial,antiviral and enhancing the body’s resistance,but also have the advantages of natural environmental protection,wide sources and various effects,which avoid the disadvantages of antibiotics in clinical application.In this study,Lactobacillus strains with excellent probiotic properties were isolated and identified for traditional Chinese medicine fermentation,and the inhibition ability of PEDV proliferation of Lactobacillus and its metabolites,Chinese medicine decoction and Chinese medicine decoction fermented by Lactobacillus were studied.In addition,network pharmacology and molecular docking technology were used to explore the mechanism of action of dominant Chinese medicine Phellodendron on viral diarrhea of piglets.Trial 1 Isolation and identification of three strains of Lactobacillus and study on their probiotic properties Two Lactobacillus plantarum HN11 and HN22 and a Lactobacillus casei HN12 were identified by culture characteristic,Gram staining and16 S r RNA molecular biological analysis.In this study,the growth curve,acid resistance,bile salt resistance,antibacterial ability,antibiotic sensitivity and animal safety of the isolated strains were studied.The results showed that all the tested strains entered logarithmic growth stage at 4 h and stable stage at 16 h,showing good acid production,acid resistance and bile resistance.Salmonella and Staphylococcus aureus were highly sensitive to the three isolates,and Escherichia coli showed low sensitivity to HN11 and HN22,while Escherichia coli was not sensitive to HN12.The results of antibiotic sensitivity test showed that the isolates were sensitive to a variety of antibiotics,but resistant to lincomycin and norfloxacin.Lactobacillus HN11,HN12 and HN22 suspensions with concentrations of 1.0×108 CFU/m L were administered to mice,and animal safety tests were conducted.It was found that the growth,mental state,visceral and intestinal tissues of mice were normal,indicating that the isolated strain had no toxic effect on mice.The results of this study provide a theoretical basis for the further development and utilization of the Lactobacillus.Trial 2 Anti-PEDV effect of Lactobacillus and its metabolites in vitro After the suspension concentration of Lactobacillus HN11,HN12 and HN22 was adjusted to1.0×108CFU/m L,the supernatant was centrifuged,and the metabolic supernatant products were obtained after filtration by 0.22 μm.The centrifuged bacteria were suspended by PBS to obtain the bacterial suspension.Thermal inactivation product was obtained from bacterial suspension after 100℃ water bath for 10 min.The Lactobacillus suspensions,metabolize supernatant products and thermal inactivated products were inoculated into PEDV-infected Vero by pretreatment,co-treatment and post-treatment.The cell viability in each treatment group was detected by CCK8 kit.The results showed that the isolates suspensions,thermal inactivation products and metabolic supernatant could inhibit PEDV proliferation.The inhibition rates of thermal inactivation products and metabolic supernatant in pretreatment and co-treatment were higher than those in bacterial suspension,and there was no significant difference between the thermal inactivation products and metabolic supernatant products of each isolates under pretreatment and co-treatment(P>0.05).The PEDV copies after PEDV was simultaneously inoculated into Vero cells with Lactobacillus suspensions and its metabolites was detected by fluorescence quantitative PCR method.The results showed that,compared with virus treatment group,PEDV copies was significantly decreased in HN11 supernatant treatment group(P<0.05).Trial 3 Anti-PEDV effect of Chinese herbal decoction fermented by Lactobacillus isolates Meta-analysis was conducted on the efficacy evaluation of Chinese herbal preparation in the treatment of swine viral diarrhea by Rev Man 5.4 software,.The results showed that,compared with chemical drug,the cure rate of Chinese herbal compound preparation on swine viral diarrhea disease was significantly increased(P<0.01).Three kinds of clearing heat and detoxifying and dampening drugs were selected for the antiviral study of the following fermentation single Chinese herbal medicine.On the basis of trial 2,the concentrations of Lactobacillus plantarum HN11 and Lactobacillus casei HN12 were adjusted to 1.0×108 CFU/m L,and the concentrations of Coptis chinensis,Phellodendron and Sophora flavescens decoction were adjusted to 1g/m L,at 4% for 36 h,then the viable bacteria count method was used to detect the viable bacteria number in each fermented Chinese herbal decoction.The results showed that the number of viable Lactobacillus HN11 and HN12 did not change significantly after 36 h fermentation in Coptis chinensis,Phellodendron and Sophora flavescens decoction.The content changes of berberine hydrochloride in Coptis chinensis and Phellodendron decoction before and after fermentation,and the content changes of matrine in Sophora flavescens decoction before and after fermentation were detected by HPLC.The results showed that the content of berberine hydrochloride in the post-fermentation decoction of Coptis chinensis and Phellodendron,the content of matrine in the post-fermentation Sophora flavescens decoction was several times higher than that before fermentation.CCK8 kit was used to detect the maximum non-toxic dose of Coptis chinensis,Phellodendron and Sophora flavescens decoction and fermentation decoction on Vero cell model,and the maximum non-toxic dose of Coptis chinensis,Phellodendron and Sophora flavescens decoction and fermentation decoction was applied to Vero cells simultaneously with PEDV.The results of fluorescence quantitative PCR showed that PEDV copies in all treatment groups were decreased compared with the virus control group,and the effect of Phellodendron decoction was very significant(P<0.001).After PEDV was treated with Phellodendron decoction and fermentation broth by pretreatment,co-treatment and post-treatment,the cell viability in different treatment groups was detected by CCK8 kit.It was found that there was no significant difference in the inhibition rate of PEDV among Phellodendron decoction treatment,and fermentation Phellodendron decoction treatment(P>0.05),and the pretreatment and post-treatment had the best effect,with the inhibition rate up to more than 50%.Trial 4 Explore the mechanism of action of Chinese herb Phellodendron on piglet diarrhea based on network pharmacology and molecular docking technology TCMSP database was used to screen the active constituents of Phellodendron and their corresponding target genes,and Gene Cards,OMIM and NCBI database were used to obtain the related targets of piglet diarrhea.The PPI network was constructed by intersecting the active ingredient target of Phellodendron chinensis with the diarrhea target of piglets.GO function and KEGG pathway enrichment were analyzed by DAVID analysis of core targets.The molecular docking was verified by Auto Tools,and the results were visualized by Py Mol software.The results showed that quercetin,β-sitosterol,isoparpamine,and corneline were the main active components of Phellodendron on piglet diarrhea,which acted through cancer pathway,MAPK signaling pathway,PI3K-AKt pathway,IL17 signaling pathway and other pathways.The targets of Phellodendron were TNF,TP53 and IL-6.The multi-component,multi-pathway and multi-target action mechanism of Phellodendron were explored.In this study,we found that Phellodendron chinensis has anti-inflammatory activity and can participate in the regulation of inflammation,which may be the key factor for Phellodendron to play an antiviral role.In conclusion,Lactobacillus isolated in this study has excellent probiotic properties and anti-PEDV ability,and Phellodendron decoction and Phellodendron decoction fermented by Lactobacillus HN11 and HN12 have significant anti-PEDV effects.The traditional Chinese medicine Phellodendron can act on diarrhea disease of piglets through multi-components,multi-routes and multi-targets.