| Due to the wide application of antibiotics in livestock and poultry,drug resistance and food safety have become a public concern.Therefore,many researchers are looking for green,safe,efficient,pollution-free,and residue-free antibiotic substitutes,probiotics and Traditional Chinese Medicine(TCM)have become the new focus in the field of animal husbandry research.Probiotics and TCM have their disadvantages in clinical application,such as slow and unstable effect,irritating odor,etc.To solve these problems,TCM could be fermented using probiotics.Astragalus which was a traditional Chinese medicine that has anti-inflammatory,anti-viral,anti-oxidant and immune-enhancing effects.The main active substance was Astragalus polysaccharide.Because of the hindrance of the plant cell walls,the extraction and utilization efficiency of the pharmaceutical active substance were low.Biotransformation using probiotic fermentation was one of the effective ways to improve the utilization efficiency of Astragalus.Although the liquid fermentation of Astragalus has been reported,the more efficient solid fermentation process was rarely involved,Moreover,there was no systematic understanding of the small molecule metabolites after fermentation.At the same time,there was also a lack of a comprehensive and in-depth evaluation of laying hens fed with fermented Astragalus.In this study,Lactobacillus plantarum was used to ferment Astragalus and metabolomics of fermented Astragalus and the evolution of microbial flora were analyzed.Further,it was applied to laying hens to observe its effect on intestinal miacrobiota,production performance,antioxidant function and immune response.The purpose of this study was to understand the metabolites of Astragalus membranaceus solid-fermented by L.plantarum and to elucidate the molecular mechanism of its feeding effect on laying hens.It provided a theoretical basis for the application of fermented Astragalus instead of antibiotics.The main research findings are described as follows.1.Routine analysis of metabolites and changes of flora in fermented Astragalus using L.plantarum by solid fermentation technologyThe Astragalus was fermented by using L.plantarum,E.faecium,and E.faecium + L.plantarum,and control,respectively.The solid fermentation was carried out at 37 ℃ for 30 days,and the fermentation samples were taken on 0,3 and 30 th day,respectively.The active constituents of fermented Astragalus were analyzed byphenol-sulfuric acid method.The changes of organic acid metabolites after fermentation were analyzed by liquid chromatography.the microbial spectrum of fermented Astragalus was evaluated by Aplied Pac Bio single molecule,real-time sequencing technology(SMRT).The results showed that the yield of Astragalus polysaccharides by L.plantarum was 9.43%;the content of acetic acid,methylacetic,aethyl acetic and lactic acid were 1723.01 mg/kg,173.29 mg/kg,616.07 mg/kg and1946.17 mg/kg,respectively;The microbial spectrum analysis in each fermentation group at 3 day showed that the L.plantarum accounted for 23.55% in L.plantarum group,the E.faecium accounted for 31.2% in E.faecium group,and L.plantarum and E.faecium accounted for 29.4% and 8.32% in mixed fermentation group,respectively.The results indicated that the spectrum of bacteria in fermented Astragalus was positively correlated with the access bacteria.On the 30 th day of fermentation,theα-diversity index of L.plantarum was the highest,indicating that its microbial abundance and diversity are more abundant.SMRT analysis can be used as one of the methods to determine the quality standard of the microbial spectrum of fermented Astragalus.2.Metabolomics profiling of Astragalus membranaceus using L.plantarum solid fermentationMetabolomics analysis of fermented and unfermented Astragalus was performed by chromatography-mass spectrometry.The differential metabolites were analyzed by KEGG database.There were 1416 metabolites up-regulated in positive ion mode,935 metabolites down-regulated,1040 metabolites up-regulated in negative ion mode,and809 metabolites down-regulated.Metabolomics analysis showed that the differential metabolites had significant difference before and after fermentation: the amino acids such as acids,lipids and ketones were increased significantly,while olefins were significantly down-regulated after fermentation.15 up-regulated metabolites and 2down-regulated metabolites were identified.The main metabolites are α-diethyl sulfate,2-methyl citric acid,3-isopropenyl-6-oxoheptanoic acid,etc.,which are involved in pyruvate metabolism,propionic acid metabolism,galactose metabolism etc.Therefore,solid fermentation promoted the production of small molecular substances of Astragalus by these metabolic pathways.It was conducive to the discovery of new active pharmaceutical ingredients,and provided reference for the application of fermented Astragalus in animals.3.Feeding effect of L.plantarum fermented Astragalus on laying hensA total of 240 351-day-old Hailan brown hens were randomly divided into 4groups: fermented Astragalus group,Astragalus group,L.plantarum group and control group,with 5 replicates in each group and 12 hens in each replicate,respectively.Egg production rate,feed-to-egg ratio and egg quality were determined at 14 th and 28 th day.The content of catalase(CAT),glutathione peroxidase(GSH-Px),malondialdehyde(MDA),total antioxidant capacity(T-AOC)and superoxide dismutase(SOD)in the serum were measured by ELISA.The m RNA expression levels of Interferon-g(IFN-g),Interleukin-2(IL-2),tumor necrosis factor-a(TNF-a)and inducible nitric oxide synthase(INOS)were detected by real-time fluorescent quantitative PCR to evaluate the antioxidant capacity and immune status of laying hens.The results showed that on the 14 th day of the experiment,although the production performance,egg quality and antioxidant index of the experimental group were not significantly different from the control group(p>0.05),the expression of IFN-g m RNA in the ileum and cecal of the fermented Astragalus group was increased by 1.7-folds(p<0.01)and 2.1-folds(p<0.01),respectively.The m RNA expression levels of TNF-a and INOS in the ileum were significantly increased by 3.1-folds(p<0.001)and 1.2-folds(p<0.05),respectively;The m RNA expression level of IL-2 in the liver and ileum were increased by 1.1-folds(p<0.05)and 2.2-folds(p<0.0001),respectively.The egg production rate of the fermented Astragalus group was 7.14%higher than that of the control group(p<0.05),and the feed-to-egg ratio was decreased by 6.6% at 28 th day(p<0.05).Compared with the control group and unfermented Astragalus group,the CAT content of the fermented Astragalus group were increased by 61.5%(p<0.05)and 63.4%(p<0.05),respectively.The GSH-Px content were increased by 62.4%(p<0.05)and 54.9%(p<0.05),and the SOD content were increased by 68.0%(p<0.05)and 55.4%(p<0.05),respectively.The content of T-AOC in the fermented Astragalus group was significantly increased by 52.6%compared with the control group,and the difference was significant(p<0.05).Compared with the control group,the m RNA expression levels of IFN-g in the liver,spleen,ileum and cecal of the fermented Astragalus group were increased by1.3-folds(p<0.01),1.4-folds(p<0.01),2.3-folds(p<0.0001)and 6.1-folds(p<0.0001),respectively.The m RNA expression of TNF-a were increased by 1.4-folds(p<0.05),1.4-folds(p<0.01),1.9-folds(p<0.0001),and 1.3-folds(p<0.01),and INOS m RNA expression in ileum and cecal,respectively.The m RNA expression levels of IL-2 inliver,spleen,ileum and cecal were increased by 1.2-folds(p<0.05)and 1.5-folds(p<0.001),3.6-folds(p<0.0001)and 1.6-folds(p<0.001),respectively.The results indicated that fermented Astragalus increased the antioxidant capacity and expression of cytokines in laying hens and improved their production performance.4.Effect of L.plantarum fermented Astragalus on intestinal microbiota of laying hensThe ileum and cecal samples were randomly collected from the 14 th and 28 th day of the laying hens.A total of 48 samples were collected from three samples of each intestinal at each stage.High-throughput sequencing of 16 S r RNA was used to analyze intestinal flora changes in intestinal microbiota ileum and cecal.The results showed that the Simpson(0.91±0.04)and Shannon(5.79±0.75)indexes of the cecal in the fermented Astragalus group were the highest after 28 days,indicating that the microbiota was more diverse.The fermented Astragalus reduced the abundance of Firmicutes and Proteobacteria,The additive had little effect on the abundance of Bacteroides.From the "phylum" level analysis,it was found that the abundance of the Firmicutes and Bacteroides in the ileum of the fermented Astragalus group were increased,and the proportion of the deformed bacteria was decreased,indicating that the use of fermented Astragalus could reduce the abundance of harmful bacteria in the ileum,but the cecal had no obvious changes.From the "genus" level analysis,it was found that the genus Lactobacillus was mainly in the ileum,and the genus Bacteroides was mainly in the cecal.The abundance of Lactobacillus in the ileum of the fermented Astragalus group were decreased,and the other three groups were increased significantly after 28 th day.The abundance of Bacteroides in the 4 groups of cecal was improved.The use of Astragalus,L.plantarum and fermented Astragalus had a greater effect on the abundance of ileum,but had less effect on the abundance of cecal.The above results indicated that fermented Astragalus affects the abundance of ileum microbiota in laying hens,which is beneficial to digestion and absorption of small intestine,thus improving the performance of laying hens.In summary,this study combined with the preventive veterinary medicine,fermentation engineering and traditional Chinese medicine to carry out multidisciplinary research,established a solid fermented Astragalus model,determined the content of polysaccharides and organic acids in fermented Astragalus,and firstly mapped the microbial spectrum of fermented Astragalus.It was confirmedthat the dominant flora in the fermentation process was positively correlated with the initial access bacteria.The differential metabolites and metabolic pathways before and after the fermentation of Astragalus were systematically and comprehensively analyzed.15 metabolites were up-regulated and 2 metabolites were down-regulated,In addition,the fermentation strain and fermentation mode affecting the yield of small molecular substances of Astragalus was determined.The application of fermented Astragalus on laying hens demonstrated that fermented Astragalus increased production performance and immunological response by regulating ileum microbiota abundance and the m RNA expression of cytokines in internal organs and intestinal tract.In conclusion,this study systematically carried out the theoretical and applied research on fermented Astragalus,and provided a promising potential to use solid-fermented Chinese medicine such as fermented Astragalus to replace antibiotics in animal production. |
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