| Tannin is a kind of phenolic substance produced by plant secondary metabolism,which has the characteristics of bitter and astringent taste,astringency,complexing with protein,inhibiting rumen microbial activity and anti-nutrition.When the tannin content in feed is higher than 3%,it will affect the rumen homeostasis,and when it is higher than4%,it will damage the animal body.Caragana korshinskii is a windbreak and sand-fixing shrub in Inner Mongolia,which has high yield and high protein content,but its tannin content is more than 6%,which limits its use as feed.In this study,on the basis of exploring the variation of anti-nutritional secondary metabolites in Caragana korshinskii in different periods,the recombinant strains producing tannase with high efficiency were isolated,screened and constructed,and the growth characteristics,expression stability,enzymatic characteristics and safety of the recombinant strains were evaluated;The effects of the recombinant strain on the conventional nutrients,secondary metabolites and microbial flora of the fermented Caragana korshinskii were analyzed,and the in vitro rumen fermentation characteristics of the recombinant strain fermented Caragana korshinskii were evaluated,which laid the foundation for the feed utilization of Caragana korshinskii.This study is divided into 8 experiments,and the contents and results of each experiment are as follows:Experiment1:Analysis of nutritional value and main bioactive components of Caragana korshinskii in different growth stagesThe composition of conventional nutrients in young,vigorous and withered grass stages was analyzed,and the differences of main active components such as phenols in Caragana korshinskii in different stages were accurately compared by targeted metabolomics.The results showed that the contents of crude protein,true protein and soluble carbohydrate decreased significantly(P<0.05),while the contents of neutral detergent fiber,acid detergent fiber,lignin and crude ash increased significantly(P<0.05),and the contents of polyphenols,hydrolysable tannin and condensed tannin in the withered period were significantly higher than those in other periods(P<0.05);A total of 439 phenolic compounds were identified in Caragana korshinskii in three periods.1-O-p-hydroxycinnamoyl-3-O-caffeoyl glycerol,chrysoeriolin-8-C-glucoside-7-O-(6’-feruloyl)glucoside,quercetin-3-O-(2’-acetyl)glucuronide,isorhamnetin-3-O-(6’-acetyl)glucoside,isorhamnetin-3-O-(6’-malonyl)glucoside,Hydroxy-(4-hydroxyphenyl)propionic acid,arbutin,piceoside,grevilleoside C,and D-threo-guaiacylglycero-7-O-β-D-glucoside were significantly enriched in Caragana korshinskii in the withered period,while hydrolytic tannins and condensed tannins were the least and the highest in the withered period.Experiment 2:Isolation,screening and identification of tannase-producing strains and selection of tannase gene fragment18 Tannin-degrading bacteria strains were isolated from the root soil of Caragana korshinskii,fresh animal feces,rumen fluid and natural fermentation materials,and identified by morphology,biochemistry and molecular biology.4 strains of Enterococcus faecium,1strain of Bacillus tequila,3 strains of Bacillus subtilis,3 strains of Pichia pastoris,2 strains of Aspergillus piper,2 strains of Aspergillus niger and 3 strains of Aspergillus tabinensis.The tannase activity of the seven Aspergillus strains was the highest,with an average of 239 U/m L.The tannase activity of Aspergillus tabinensis P10-2 was the highest,with an average of 309.16 U/m L.The tannase activity of Pichia pastoris was slightly higher than that of Bacillus,and the tannase activity of Enterococcus was the lowest,with an average of less than 50 U/m L.Experiment 3:Construction of tannase-producing recombinant bacteria and evaluation of its expression characteristicsThe tannase sequence Tan An from Aspergillus niger P10-2 and the tannase sequence Tan Lpl from ATCC14917T was selected and integrated into Bacillus subtilis168(BS168)and Bacillus subtilis WB600(WB600)expression lines by plasmids Tan Lpl-p43NMK and Tan Lpl-p HT43,respectively.Three strains capable of producing tannase were successfully constructed.Tan Lpl-p43NMK-Bacillus subtilis 168(BS168(p43NMK)),Tan Lpl-p HT43-Bacillus subtilis 168(BS168(p HT43))and Tan Lpl-p HT43-Bacillus subtilis WB600(WB600(p HT43)).By evaluating the growth characteristics,expression stability and enzymatic properties of the recombinant strains,it was found that the three recombinant strains reached the stationary phase after 18h of growth,and there was no significant change in the growth rate compared with the parent strain;The plasmid loss rate of BS168(p43NMK)was significantly higher than that of BS168(p HT43)and WB600(p HT43)at the 10th passage(P<0.05);The optimal temperature and p H for the enzymatic reaction of tannase production by the three recombinant strains were 30℃and 5.0,under which the tannase production activities were 68.81,397.36,and 461.12 U/m L,respectively.Recombinant strain WB600(p HT43)was superior to the other two strains in expression stability and enzyme production ability.Experiment 4:Safety evaluation of recombinant strain producing tannase with high efficiencyThe sensitivity of the three recombinant strains to 28 commonly used antibiotics was detected by K-B disk diffusion method.The results showed that BS168 transformed with Tan Lpl-p43NMK plasmid was resistant to neomycin and Tan Lpl-p HT43 plasmid was resistant to piperacillin;After WB600 was transferred into Tan Lpl-p HT43 plasmid,there was no change in antibiotic sensitivity.Sixty Healthy Kunming mice,45±2 days old and weighing 39.40±1.38 G,were randomly divided into 6 groups,and the control group was fed with the same dose of saline.BS168,BS168(p43NMK),BS168(p HT43),WB600,WB600(p HT43)were administered by gavage 2.0×109 CFU/rat in the other 5groups,respectively.The experiment lasted for 15 days.On the 15th day of the experiment,the mice were weighed and blood was collected to analyze the body weight,visceral coefficient,blood immunity and antioxidant indicators.The results showed that the weight gain of mice in WB600 and WB600(p HT43)groups was significantly higher than that in the control group(P<0.05),and the coefficients of heart,liver,spleen,lung,kidney,stomach,small intestine and large intestine in the experimental groups were not significantly different from those in the control group;In BS168,BS168(p43NMK)and BS168(p HT43)groups,the contents of blood Ig M,Ig G,Ig A,IL-2,IL-10 and TNF-αwere significantly increased(P<0.05);The blood MDA level of mice in BS168(p43NMK)group was significantly increased(P<0.05),and the blood SOD activity of mice in BS168,BS168(p43NMK)and BS168(pht43)groups was significantly decreased(P<0.05).Combined with the results of drug sensitivity test and mouse pathogenicity test,the recombinant strain WB600(p HT43)was more safe and effective.Experiment 5:The effect of tannase-producing recombinant strain on the nutritional value of fermented Caragana korshinskiiCaragana korshinskii in withered grass period is used as a fermentation substrate,and five treatments are set;A raw material before fermentation group(Ci K),a natural fermentation group(CK),a group inoculated with 10~5CFU/g empty host WB600(WB600),a group inoculated with 10~5CFU/g recombinant strain WB600(p HT43)(WB600(p HT43)),Lactobacillus plantarum ATCC14917 groups(LP)of 10~5CFU/g were inoculated with 6 replicates per group to analyze the effects on p H value,fermentation indexes,nutritional components and tannin content of fermented Caragana korshinskii.The results showed that the p H value of LP group was significantly lower than that of other groups at 3,7,14,21 and 30 days(P<0.05),the ammonia nitrogen content of WB600(p HT43)group was significantly higher than that of other groups,and there was no significant difference in the content of acetic acid and lactic acid among the fermentation groups(P>0.05);Compared with the Ci K group,the contents of crude fat,true protein and soluble carbohydrate of Caragana korshinskii in all fermentation groups decreased significantly(P<0.05);Compared with Ci K group,the contents of total phenols and hydrolysable tannin in all fermentation groups decreased significantly(P<0.05),and the contents of condensed tannin in LP group and WB600(p HT43)group decreased significantly(P<0.05),and the degradation effect of recombinant strain WB600(p HT43)on condensed tannin decreased by 35.42%.Experiment 6:The effect of tannase-producing recombinant strain on the main bioactive components of Caragana korshinskiiCaragana korshinskii in the withered period was used as the fermentation substrate,and three treatments were set up,namely,the raw material group before fermentation(Ci K),the group inoculated with 10~5CFU/g empty host WB600(WB600),and the group inoculated with 10~5CFU/g recombinant strain WB600(p HT43)(WB600(p HT43)),with6 replicates in each group.Effects of targeted metabonomics analysis on the main bioactive components of fermented Caragana korshinskii.Results a total of 3114metabolites were detected,including 1328 and 1173 up-regulated metabolites and 768and 763 down-regulated metabolites in WB600 and WB600(p HT43)groups,respectively.Bioactive compounds with higher relative abundance in Ci K group were mainly flavonoids,alkaloids and heterocyclic compounds with larger molecular weight;Bioactive substance with high relative abundance in WB600 and WB600(p HT43)groups were mainly amino acid and their derivatives,free fatty acids and phenolic acids,and small molecular substance increased in WB600 and WB600(p HT43)groups,and WB600(p HT43)group accumulated more small molecular substances.Five hydrolytic tannins and their derivatives and seven condensed tannins and their derivatives were not detected in the fermentation group,while four monomers,three dimers and three gallosides were enriched in each fermentation group.The relative abundance of three monomers(3,3’-O-dimethylellagic acid,4-O-methylgallic acid,and gallic acid)and two dimers(6-gallic acid-ascorbic acid,and gallacetophenone)was higher in the WB600(p HT43)group.Experiment 7:Effect of recombinant strain with high tannase activity on microbial flora of Caragana korshinskiiTwo treatments were set up with 10~5CFU/g empty host WB600 group(WB600)and recombinant strain WB600(p HT43)group(WB600(p HT43)),respectively,with 6replicates in each group,and the effects on the microbial flora of fermented Caragana korshinskii were studied by high-throughput sequencing.A joint analysis was perform with differential metabolites relate to tannin anabolism.The results showed that there were no significant differences in observed OTUs,chao1,Shannon,Simpson,pileous and dominance indices between the two groups;The relative abundance of Proteobacteria in WB600(p HT43)group was significantly higher than that in WB600 group(P<0.05).Weissella,Sporichthya,Nakamurella,Psychroglaciecola,Ulvibacter,Curtobacterium,OM190,The relative abundance of SCGC＿AAA164-E04,Acidiphilium,Staphylococcus and K189A-clade increased significantly(P<0.05),while the relative abundance of RF39 decreased significantly(P<0.05);Proteobacteria,Weissella and Staphylococcus of Firmicutes,Psychroglaciecola of Proteobacteria,Sporichthya and Nakamurella of Actinobacteriota,and OM190 of Planctomycetota were significantly positively correlated with gallic acid(P<0.05).The RF39 of Firmicutes had a significant negative correlation with gallic acid(P<0.05).Experiment 8:Study on rumen fermentation characteristics of Caragana korshinskii in vitro by recombinant strain with high tannase activityThe rumen fermentation characteristics(gas production,fermentation parameters and nutrient degradation rate)of Caragana korshinskii before and after fermentation by recombinant strain WB600(p HT43)were evaluated in vitro.The results showed that the gas production and VFA concentration of fermented Caragana korshinskii were higher than those of Caragana korshinskii raw material,while the p H value and NH3-N concentration were lower,but the differences were not significant;The degradation rate of DM was significantly higher(P<0.05)and the degradation rate of CP was not significantly higher(P>0.05)than those of Caragana korshinskii;The degradation rates of NDF and ADF of Caragana korshinskii were not significantly different from those of Caragana korshinskii at 4,8,12 H(P>0.05),but were significantly higher than those of Caragana korshinskii at 24 and 48 H(P<0.05).In conclusion,the recombinant strain WB600(p HT43)is used as a fermentation strain,Proteobacteria,Weissella,Sporichthya,Nakamurella,Psychroglaciecola,OM190,The method reduce that content of total phenols,hydrolytic tannin and condense tannin in the Caragana korshinskii,improves the species and the relative abundance of amino acids,amino acid derivative and free fatty acids,enriches bioactive substances such as organic acids,terpenoids,alkaloids,coumarins,phenolic acids and the like,improves the degradation rate of nutrient substances,and has application value in the aspect of feeding utilization of the Caragana korshinskii. |
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