| The long-term unreasonable use of antibiotics in the feed industry has brought the problems such as the reduction of the health level of animals,the increase of drug resistance of pathogenic bacteria,and the drug residue exceeded the standard in meat and egg products.The use of antibiotics as feed additive has been banned in 2020.The prohibition of antibiotics is bound to have a certain impact on the growth performance and the health level of animals.Therefore,the development of antibiotic replacement products and the improvement of nutrient digestibility are of great significant to promote the sustainable,healthy,and green development of breeding industry.The fermented feed with antibacterial and antioxidant functions is one of the best alternatives to replace the antibiotics.The fermented feed is rich in beneficial microorganisms and metabolites,with high digestible absorption rate of nutrients,which can effectively improve the nutritional and functional quality of feed.Microorganisms and metabolites in the fermented products obtained by different strains and fermentation processes are different,so as the functions.The metabolic pathways of functional metabolites are unclear.These factors make it difficult to achieve accurate regulation and optimization of the fermentation process.Therefore,in order to increase the quality and function of the fermented feed,the approach was applied in this paper which was comprehensively utilized a variety of physical and chemical detection methods,joined with the multiple omics analysis strategies containing the metabolomics based on ultra-high liquid chromatography-mass spectrometry(UHPLC-MS/MS)and microbial diversity based on 16S r DNA sequencing technology.The correlation between various substances and functions,between microorganisms,metabolites and functions in the fermentation process was analyzed to explore the mechanism of fermentation effects on feed functions.The main results are as follows:Taking the ratio of acid soluble protein-to-crude protein as the index,the combination of different functional strains in the compound agent was optimized as:strain FM-18 2.86×10~8CFU/g、strain JA-14 2.86×10~8 CFU/g、strain DB-7-6 4.28×10~8 CFU/g.The single factor test and response surface method were used to determine the fermentation process conditions of the fermented feed as:inoculation quantity 1×10~9CFU/Kg(dry matter),water content 43%,fermentation temperature 34℃and fermentation time 14 d.The final acid-soluble protein-to-crude protein of the fermentation product reached to(31.82±1.75)%.It increased by 5.31 percentage points compared with that before optimization.The physical state,protein composition,contents of different substances,antibacterial and antioxidant functions of different samples during the fermentation process were detected and analyzed by various physical and chemical detection methods.With the progress of fermentation,the color of the fermentation products became darker.The electron microscope observation showed that the appearance structure of fermented feed was more finely broken and there were fine holes on the surface.The p H value continued to decline,organic acid contents continued to rise,and the acid fragrance was obvious.The activities of protease,cellulase and amylase were significantly higher than that of unfermented samples(P<0.05).The total amino acid content and essential amino acid content increased by 7.11-and 8.56-times,respectively.The contents of glycinin andβ-conglycinin decreased by 82.36%and80.65%,respectively.The antigen activity decreased significantly(P<0.05).The antibacterial and antioxidant functions of fermentation products were significantly improved(P<0.001).The ratio of acid-soluble protein-to-crude protein,digestive enzyme activity,organic acid content,p H value,antigenic protein content were significantly correlated with antibacterial and antioxidant properties of fermented samples(P<0.001).Principal component analysis and comprehensive evaluation index analysis showed that the samples changed greatly within 4 d of fermentation,but there was no significant difference after 10 d of fermentation.Crude protein was extracted by alkaline solution and acid precipitation method.The electron microscopy showed that the structure of crude protein was looser and finely broken,with fine holes on the surface and more irregular structures.The results of SDS-PAGE showed thatαandα’subunit components ofβ-conglycinin were degraded most fully,especially within 4 d of fermentation.FT-IR analysis showed that the ratio of protein withα-helix structure decreased,while the ratio ofβ-fold structure increased after fermentation.Through microbial degradation,the structure and properties of crude protein changed greatly,and the water solubility was improved.Based on UHPLC-triple-TOF-MS/MS system,a total of 1425 metabolites were isolated and identified in the fermented feed,of which 583 were significantly different.The contents of various functional metabolites increased,such as Val-Arg and linoleic acid with antibacterial activity,Leu-Pro-Phe,phenolic acid,5-hydroxyl flavone with antioxidant activity,and peptides with glutamic acid residues.The differential metabolites mainly were enriched in the metabolic pathways:D-glutamine and D-glutamate metabolism,Alanine,aspartic,and glutamate metabolism,Galactose metabolism,and Tricarboxylic acid cycle.Microbiome analysis revealed significant differences in microbial composition before and after fermentation.Firmicutes dominated the final fermentation products.The species of Weissella、Lactobacillus and Pediococcus were significantly enriched.Correlation analysis showed that the content changes of functional substances were significantly positively correlated with dominant bacteria,antibacterial and antioxidant functions(P<0.001).After fermentation feed used for breeding of laying hens,the intestinal contents of microbial compositions and substances changes were analyzed.The results showed that the relative abundance of Firmicutes rise,and differential metabolites significantly enriched in the metabolic pathways of Amino acid metabolic pathways and Protein digestion and absorption pathway.These had promoted effects on the improvement of the growth performance of the chickens.This study preliminarily clarified the generation mechanism of antibacterial and antioxidant functions of the feed fermented by the compound microbial agent,provided reference for quality evaluation and precise quality control of fermented feed,and provided a theoretical basis for the promotion and application of functional,low antigenicity and high digestibility feed. |
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