Study On Traditional Chinese Medicine Preparation Fermented By Lactobacillus From Chicken Intestines And Its Resistance To NDV

Traditional Chinese medicine(TCM),as China's "natural medicine treasure",has the advantages of multiple components,multiple pathways,multiple targets and not easy to produce drug resistance.The Antiviral mechanism of TCM is inhibiting the reproduction of the virus.Lots of TCM have a strong ability to resist Newcastle disease virus(NDV)in vivo or in vitro,just like honeysuckle,radix scutellariae,coptis chinensis,folium isatidis,astragalus,and so on.However,many defects limit the application of TCA,including macromolecular substances which were difficult to be absorbed,low content of active ingredients,and poor bioavailability.TCM has the functions of anti-virus,improving immunity,relieving heat and analgesia,and probiotics can degrade macromolecular substances,promote digestion and absorption,and improve the taste of preparations.After they are made into fermentation preparations,the synergistic or superposition of the pharmacodynamic effects will be realized.This research therefore provided a new path for the development and utilization of TCM.In this study,we analyzed the intestinal microflora of chicken,isolated the probiotics with better biological activity.Chinese traditional medicine with antiviral effect were fermentated by lactic acid bacteria,in order to produce new active substances,improve the release of effective components,enhance their antiviral effect and therapeutic effect on Newcastle disease.The results are as follows.1.The microbiological composition and diversity in duodenum,jejunum,ileum and cecum of SPF white leghorn hensA total of 354,295 gene sequences,515 OTU,which belonging to 12 phyla,9 classes,10 orders,16 families,14 genera and 8 species,were obtained by high-throughput sequencing of 16S rDNA technology.At Phylum classification level,the main intestinal bacteria of SPF white leghorn hens were Firmicutes(58.57%),Bacteroidetes(19.60%)and Actinobacteria(17.71%).At the level of genus,Lactobacillus(42.39%),Clostridium(SMB53,9.87%),Bacteroides(8.66%),Prevotella(3.95%)were dominant.At the three levels of phylum,genus and species,the abundance of duodenum species was the most similar to the colony composition of jejunum,with the larger difference from ileum and the largest difference from cecum.Microbiological diversity analysis of individual intestinal segments showed that the species of cecum were the most abundant,with the Simpson index 0.067599,followed by duodenum,jejunum and ileum.Through the comparison of diversity among 4 intestinal segments,we found that UniFrac diversity index were 0.741,0.656,and 0.646 between the cecum and duodenum,ileum or jejunum when the abundance was considered.UniFrac diversity index between jejunum and duodenum or ileum were 0.273and 0.225.The smallest difference is 0.163,which between ileum and duodenum.2.Isolation and identification of superior lactobacillus from chicken gut17 strains of bacteria were isolated from intestines and feces of chicken,and 6 strains of bacteria were identified by biochemical determination and 16S rDNA method,including 3 strains of Lactobacillus acidophilus.1 strain of Lactobacillus paracasei,1 strain of Streptococcus alactolyticus,1 strain of Streptococcus parasanguis,1 subspecies of Leuconostoc mesenteroides subsp.mesenteroides,and 2 strains of lactobacillus salivary.The growth of Lactobacillus acidophilus 14,Lactobacillus paracasei 11,lactobacillus salivary 2-1-1 and Cl were measured,with an exponential growth period of 10?18h and a stable period of 24?36h.After 36h,the bacteria entered the death period.Lactobacillus acidophilus 14 and Lactobacillus paracasei 11 had the best growth activity,with the highest number of live bacteria 1.0x109CFU/mL.After fermenting 48h,the pH value of LAB fermented liquid were 3.7-4.0.The first 24h was the period when the pH value of the fermented liquid decreased the fastest.Lactobacillus salivary produced more acid.All the 4 strains of lactobacillus had certain antacid effect,and all survived under the conditions of pH 3.5 and 2.5,but all died when pH was 1.5.At pH 2.5,Lactobacillus acidophilus 14 had the strongest antacid activity and the survival rate was 10.1%?13.9%.Lactobacillus salivary showed the best tolerance against bile.At a concentration of 0.3%bile salts,there was still a survival rate of nearly 0.1%.Lactobacillus acidophilus 14 and Lactobacillus paracasei 11 were only resistant to 0.1%bile salts.The fermentation supernatant of LAB can inhibit the growth of E.coli(CMCC44103),unchanged by heating 10 min at 80?,but when raising the pH value to 5.0,the antibacterial effect disappeared.It showed that the antibacterial substance may be acid or working at lower pH values.Lactobacillus acidophilus and Lactobacillus paracasei have been allowed to be used as feed additives,which preferred to be Chinese medicine fermentation strains.3.Transformation of Chinese traditional medicine by intestinal lactobacillusThe chicken Lactobacillus acidophilus and Lactobacillus paracasei fermented the extracts of honeysuckle,geniposide and Epigoitrin,respectively.We did not find the addition or disappearance of the main components peaks of chlorogenic acid,geniposide and Epigoitrin with HPLC method,but there were changes in the peak area of other peaks.A small test extraction process for the compound Chinese medicine was established which prepared JZBQ.The JZBQ by inoculation with fermentation of 5%lactobacillus showed that TCM had slight inhibition on the chicken Lactobacillus acidophilus and no obvious inhibition on the Lactobacillus paracasei,and the growth of the bacterial species was normal.The maximum number of live bacteria exceeded 1.9×109 CFU/mL,and the optimal fermentation time was 36h.The content of chlorogenic acid was increased from 0.18%by 1.8 to 2.4 times after the fermentation of JZBQ.The mixed fermentation was the best,and the optimum fermentation time was 36 hours.4.The safety of FJZBQ to chickenAfter oral administration of FJZBQ at the dosage of 185?15000mg/kg,chicks behaved normally,without death or toxicity symptoms,which showed that FJZBQ was a actually non-toxic veterinary drug(LDs50>15000mg/kg).Similarly,there was no significant difference in hemoglobin,RBC count,WBC count,platelet count and lymphocyte percentage(P>0.05)at the dosage of 3.75?15g/kg in 30 days.But the growth rate was inhibited at the dosage of 15g/kg.According to the dosage of 0.1 mL for each chicken,it was applied for 21 days without toxicity and toxic side effects.The group of FJZBQ was optimal in weight at each week,the survival rate was increased by 1.35%,the weight of each chicken was increased by 90g,and the feed meat ratio was reduced by 0.13 at 40d.5.The Antiviral action of FJZBQNo death was caused by 0.2mL inoculation of chicken embryo with FJZBQ,but death was caused by unfiltered liquid.The inoculation concentration of NDV Lasota virus strains to chicken embryo was 200 EID50(10-7.4/0.2mL).Three approaches were used to verify the efficacy of the two drugs:firstly,NDV and drugs mixed for 1h,then inoculation;secondly,drugs were used before;thirdly,drugs were used after NDV inoculation.FJZBQ reduced the HA titer of NDV from 21.6 to 26.5.JZBQ completely inhibited the propagation of NDV in vitro,by the first method.250 SPF chicken were injected genotype VII NDV(GM2 strain)which was diluted 1000 times at a dosage of 0.2 mL/only.In the prevention group,FJZBQ was taken orally 2 days before the challenge and 5 days in a row.FJZBQ reduced the mortality rate by 76%and 60%as preventive medication at a dosage of 0.5mL and 0.25mL,high dose group delayed the onset of 12 h.The death rate was reduced by 60%and 52%by dosage of 1.0mL and 0.5mL for treatment.The experiment proved that FJZBQ had a good effect of prevention and treatment to genotype ? ND.