Screening Of Glucoraphanin-transformed Strains And Regulation Of Sulforaphane Concentration To Alleviate Colitis

Glucoraphanin（GRP）is one of the most abundant glucosinolates in broccoli,which can be hydrolyzed into more bioactive sulforaphane（SFN）by myrosinase or gut bacteria.Some studies have shown that SFN can play a role in anti-inflammatory and antioxidant by inhibiting the NF-κB signaling pathway and activating the Nrf2 signaling pathway.the intake of cruciferous plants such as GRP-riched broccoli can make human produce SFN.However,some researches indicated that human with ability of highly transformation GRP into SFN is always uncommon.Therefore,it is necessary to supplement probiotics that can convert GRP into SFN to increase the concentration of SFN and promote human health.In this subject,the strain with high GRP-metabolizing ability was screened from human feces,and combined with Broccoli seed extract（BSE）to colitis amelioration.In addition,the effective dose of SFN in intestinal tract that can relieve colitis was determined by enema administration with different concentrations of SFN.Finally,by changing the viable count of bacteria,the strain achieved the effect of regulating intestinal SFN level to alleviate colitis.The main fundings are as follows:Firstly,using GRP as the sole carbon source,the strains were isolated from feces.The GRP comsumption of strains was determined by high performance liquid chromatography,and six strains capable of metabolizing GRP were screened,including Lactiplantibacillus plantarum CCLP1202,CCLP1203,Bifidobacterium（B.）longum CCFM1206,CCBL1207,Lacticaseibacillus paracasei CCLC1211,and B.pseudobulbus CCBP1213.Compared their levels of GRP comsuption,B.longum CCFM1206 was used as the experimental strain due to its higher GRP-metablizing ability.Using non-targeted and targeted metabolomics techniques,it was found that the GRP metabolites of B.longum CCFM1206 were mainly SFN,sulforaphane-cysteine and erucin.Then,dextran sulfate sodium（DSS）-induced colitis mice were treated with the maximum dose of BSE（370 mg/kg）.Compared with the model group,BSE significantly alleviated the apparent symptoms such as weight loss and colon shortening in mice.The colonic inflammatory factors levels including IL-6,IL-1βand TNF-αdecreased by 32.0%,16.2%and 16.1%,respectively.In addition,BSE intervention increased the activity of antioxidant enzyme SOD by 11.0%,and promoted the transcription and expression of tight junction proteins claudin-1,occludin and ZO-1.These results indicated that BSE could alleviate colitis.The combination of B.longum CCFM1206 and BSE could promote the production of SFN in mice,the level of wihch in the intestine is about 1.4 times that of BSE intervention alone.And the effect of reducing the level of inflammatory factors and promoting the transcription and expression of tight junction proteins in combination is better than intervention alone,indicating that promoting the transformation of SFN in vivo contributes to the colitis amelioration.In order to determine the effective dose of SFN in the intestine,different concentrations of SFN（2,10 and 25 mg/kg）were set up to explore the remission effect on colitis.The results suggested that SFN SFN alleviated colitis in dose-dependent way.2～25 mg/kg SFN could effectively improve DSS-induced colitis,and 25 mg/kg SFN had the best effect on reducing the levels of colonic inflammatory factors and promoting the transcription levels of tight junction proteins occludin and ZO-1.2 mg/kg SFN released from colon could improve colitis in mice,and the total content of intestinal SFN and its derivatives was about 21.58μmol/g feces.Finally,the intestinal SFN level was regulated to alleviate DSS-induced colitis by changing the viable count of B.longum CCFM1206(10⁸,10⁹,10¹⁰ CFU).The results showed that the total level of SFN produced by mice with only 80 mg/kg BSE intake was 16.66μmol/g feces.There was no significant difference in body weight loss,colon length,colon inflammatory factors,oxidative stress and tight junction proteins between 80 mg/kg BSE group mice and model group.After supplementation of 10⁸ CFU B.longum CCFM1206,the total SFN level in the intestine of mice was 16.14μmol/g feces,and there was no significant improvement in colitis.Supplementation with 10⁹ and 10¹⁰ CFU of B.longum CCFM1206 significantly increased the total content of SFN（27.95 and 28.98μmol/g feces）and effectively alleviated the symptoms of colitis.Therefore,when the host’s own metabolic capacity is limited,it is necessary to supplement more than 10⁹ CFU B.longum CCFM1206 to promote the conversion of GPR into SFN in vivo,thereby effectively upregulating the level of SFN to alleviate colitis.