纳豆菌液态发酵荞麦产物中抗氧化活性物质的分离鉴定

本实验通过超高效液相色谱-串联质谱（ultra performance liquid chromatography-tandem mass spectrum，UPLC-MS/MS）技术鉴定纳豆菌液态发酵荞麦的发酵产物多酚提取物中多酚组成与结构，且通过超滤、乙醇分级沉淀、大孔树脂柱层析定向分离抗氧化肽，并采用UPLC-MS/MS鉴定多肽组成与结构。从发酵产物中共分离鉴定出8种多酚类化合物：咖啡酸、原儿茶酸、丁香酸、绿原酸、儿茶素、芦丁、牡荆素、金丝桃苷。采用超滤、乙醇分级沉淀及XAD-16大孔树脂对发酵产物进行分离纯化，获得富含抗氧化肽的体积分数40%乙醇溶液洗脱组分（蛋白质量分数为66.51%、氧化自由基吸收能力为1 731.52 μmol/g）。从发酵产物中共分离鉴定出27 条多肽，搜索Mascot数据库得到23 条均为七肽以上多肽，在BIOPEP数据库中搜索均未经报道，且所有肽段都包含疏水性氨基酸，其中有11 条肽段含有至少一个强抗氧化氨基酸。GDKIYNVF中的IY片段、CRNLLLYPAGA中的LY片段、PWFFTST中的PW片段以及QGSGGPPIV中的GPP片段等在BIOPEP数据库中均被报道具有抗氧化活性。所鉴定的多酚、多肽是发酵产物发挥抗氧化活性重要的物质基础。

Identification of Antioxidants Produced by Bacillus subtilis natto in Liquid-State Fermentation of Buckwheat

The composition and structure of phenolics produced during the liquid-state fermentation of buckwheat using Bacillus subtilis natto were identified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In addition, antioxidant peptides were isolated from the fermentation broth by ultrafiltration, alcohol fractionation, and macroporous resin column chromatography. The peptide profile was identified by UPLC-MS/MS. Eight phenolic compounds including caffeic acid, protocatechuic acid, syringic acid, chlorogenic acid, catechin, rutin, vitexin and hyperin were identified. The elution fraction rich in antioxidant peptides with protein content of 66.51% and oxygen radical absorbance capacity of 1 731.52 μmol/g was obtained by ultrafiltration, ethanol fractionation and chromatography on XAD-16 macroporous resin. Twenty seven peptides were identified from the fermentation products. Twenty three peptides longer than 7 amino acids were obtained through database search against Swissprot using Mascot, which have not been reported in the BIOPEP database. All these peptides contained hydrophobic amino acids and eleven of them contained at least one amino acid with strong antioxidant activity. The IY dipeptide fragment in GDKIYNVF, the LY dipeptide fragment in CRNLLLYPAGA, the PW dipeptide fragment in PWFFTST, and the GPP tripeptide fragment in QGSGGPPIV were reported as antioxidants according to the BIOPEP database. The identified phenolics and peptides contributed largely to the antioxidant activity of the fermentation product.