| Vegetable tannin is a kind of polyphenol compound existing in plants.Tannin is a kind of self protective substance produced by the interaction between plant and environment in the process of evolution,.Tannin is widely distributed in all kinds of plant tissues in nature.Except for the young meristem,almost all plant tissues contain tannin,such as leaves,bark and fruit.Tannin is the fourth major secondary metabolite after cellulose,hemicellulose and lignin.Tannins can be divided into three types according to their structural characteristics and chemical properties:Hydrolyzed tannins(gallnut tannin and ellagin tannin),condensed tannins and compound tannins.Tannin not only tastes bitter and astringent,but also forms insoluble complex with protein,alkaloid,polysaccharide and other macromolecular substances,which significantly reduces the feeding rate of animals.Tannin is considered to be an antinutritional factor because it combines with proteins or some digestive enzymes in intestinal mucosa and affects the permeability of cell membrane,thus reducing the digestion and absorption of nutrients and reducing the digestibility of nutrients.Due to the antinutritional property of tannin,many difficulties have been brought to the development of feed industry,and many protein rich feed raw materials(such as rapeseed meal,soybean meal,etc.)can not be effectively utilized,resulting in the waste of a large number of nutrients.Therefore,tannin removal is of great significance to improve the utilization rate of protein in plant feed raw materials.There are two main biodegradation pathways of tannin by microorganisms:(1)microorganisms secrete compounds to extracellular,which have high affinity with tannin,thus replacing the important enzymes or proteins of microorganisms to bind tannin.(2)Microorganisms can secrete enzymes that can resist tannin toxicity and catalyze tannin degradation.Although many scholars have studied the degradation pathways of tannins,due to the complexity of tannin molecules,these degradation mechanisms can not fully explain the degradation mechanisms of different tannins.The purpose of this study is to isolate and screen tannin degrading strains,optimize the culture and fermentation conditions,and analyze tannin degradation metabolites,so as to provide theoretical basis for tannin degradation mechanism and feed detoxification.The main results are as follows:1.Isolation and identification of tannin degrading strainsThrough enrichment culture,a strain C12 was isolated from soil samples collected from Changde oil press factory in Hunan Province,which could grow with tannin as the sole carbon source.The gene sequence of the strain was obtained by PCR technology,and the similarity of the16S r RNA gene sequence of the strain was compared and analyzed,combined with the verification results of physiological and biochemical indicators,it was found that the 16S r RNA gene similarity of the degrading strain C12 and Agrobacterium pusense LMG25623~T was the highest(98.85%).But the phylogenetic tree results showed that the strain C12 had the highest homology with Rhizobium pongamiae VKLR-01~T,and the confidence level was 100%,so it was named Rhizobium sp.C12.2.Degradation characteristics of tannin by strain C12 and optimization of fermentation conditionsThe single factor optimization method was used to further optimize the growth conditions of Rhizobium sp.C12.The results showed that the optimal culture conditions of strain C12 were as follows:temperature 28℃,p H 5.0,inoculum 3%,and sucrose was the best carbon source.The optimal fermentation conditions were as follows:temperature 30℃,inoculum volume 3%,liquid volume 50 m L/100 m L,fermentation time 72 h.under these conditions,the degradation rate of Rhizobium sp.C12 was 71%.3)Analysis of tannin metabolites and metabolic pathwayFive metabolites of tannin were identified by LC-MS,which were:(1)1,2,6-tri-o-gallic-β-d-pyranoglucose;(2)β-glucuronic acid;(3)glucose;(4)gallic acid;(5)pyrogallic acid.It was found that strain C12 would take advantage of glucose in substrate rather than catalyzing gallic acid to form pyrogallic acid.Therefore,gallic acid(52.65%)and pyrogallic acid(41.19%)were accumulated in the metabolites,while glucose(0.15%)was very low.The metabolic pathway of tannin was predicted by analyzing the metabolites.Strain C12 can degrade tannin by continuously catalyzing tannin to produce gallic acid and glucose,and glucose is finally used as carbon source,and gallic acid decarboxylation is used to generate pyrogallic acid... |
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