纳豆菌液态发酵荞麦产物中抗氧化活性物质的分离鉴定
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摘要
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1 731.52μmol/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。
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.
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.
引文
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[18]SAFAEIAN L,EMAMI R,HAJHASHEMI V,et al.Antihypertensive and antioxidant effects of protocatechuic acid in deoxycorticosterone acetate-salt hypertensive rats[J].Biomedicine&Pharmacotherapy,2018,100:147-155.DOI:10.1016/j.biopha.2018.01.107.
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[24]SHEN S W,CHAHAL B,MAJUMDER K,et al.Identification of novel antioxidative peptides derived from a thermolytic hydrolysate of ovotransferrin by LC-MS/MS[J].Journal of Agricultural and Food Chemistry,2010,58(13):7664-7672.DOI:10.1021/jf101323y.
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[26]LIU Y F,LIU J X,CHEN X F,et al.Preparative separation and purification of lycopene from tomato skins extracts by macroporous adsorption resins[J].Food Chemistry,2010,123(4):1027-1034.DOI:10.1016/j.foodchem.2010.05.055.
[27]任尧.鸭蛋清蛋白肽的酶法制备及其抗氧化活性研究[D].广州:华南理工大学,2011:46-71.
[28]ALEMáN A,GIMéNEZ B,PéREZ-SANTIN E,et al.Contribution of Leu and Hyp residues to antioxidant and ACE-inhibitory activities of peptide sequences isolated from squid gelatin hydrolysate[J].Food Chemistry,2011,125(2):334-341.DOI:10.1016/j.foodchem.2010.08.058.
[29]ZHENG L,DONG H,SU G,et al.Radical scavenging activities of Tyr-,Trp-,Cys-and Met-Gly and their protective effects against AAPH-induced oxidative damage in human erythrocytes[J].Food Chemistry,2016,197:807-813.DOI:10.1016/j.foodchem.2015.11.012.
[30]TSOPMO A,ROMANOWSKI A,BANDA L,et al.Novel antioxidative peptides from enzymatic digestion of human milk[J].Food Chemistry,2011,126(3):1138-1143.DOI:10.1016/j.foodchem.2010.11.146.
[31]HERNáNDEZLEDESMA B,DáVALOS A,BARTOLOMéB,et al.Preparation of antioxidant enzymatic hydrolysates from alphalactalbumin and beta-lactoglobulin.identification of active peptides by HPLC-MS/MS[J].Journal of Agricultural and Food Chemistry,2005,53(3):588-593.DOI:10.1021/jf048626m.
[32]CHEN H M,MURAMOTO K,YAMAUCHI F.Structural analysis of antioxidative peptides from soybeanβ-conglycinin[J].Journal of Agricultural and Food Chemistry,1995,43(3):574-578.DOI:10.1021/jf00051a004.
[33]RAJAPAKSE N,MENDIS E,BYUN H G,et al.Purification and in vitro antioxidative effects of giant squid muscle peptides on free radical-mediatedo xidativesy stems[J].Journal of Nutritional Biochemistry,2005,16(9):562-569.DOI:10.1016/j.jnutbio.2005.02.005.
[34]SAIGA A,TANABE S,NISHIMURA T.Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment[J].Journal of Agricultural and Food Chemistry,2003,51(12):3661-3667.DOI:10.1021/jf021156g.
[35]DAI C H,ZHANG W W,HE R H,et al.Protein breakdown and release of antioxidant peptides during simulated gastrointestinal digestion and the absorption by everted intestinal sac of rapeseed proteins[J].LWT-Food Science and Technology,2017,86:424-429.DOI:10.1016/j.lwt.2017.08.026.
[2]柳杰.花生粕抗氧化肽的研究[D].无锡:江南大学,2010:1-2.
[3]LEE I H,HUNG Y H,CHOU C C.Solid-state fermentation with fungi to enhance the antioxidative activity,total phenolic and anthocyanin contents of black bean[J].International Journal of Food Microbiology,2008,121(2):150-156.DOI:10.1016/j.ijfoodmicro.2007.09.008.
[4]赵谋明,邹颖,林恋竹,等.纳豆菌液态发酵荞麦产纳豆激酶及其代谢特性分析[J].食品科学,2019,40(4):178-185.DOI:10.7506/spkx1002-6630-20180120-285.
[5]赵谋明,邹颖,林恋竹,等.纳豆菌液态发酵谷物产纳豆激酶及发酵产物抗氧化活性研究[J].现代食品科技,2018,34(2):75-81.DOI:10.13982/j.mfst.1673-9078.2018.2.013.
[6]DIAO Z J,LI J,LIU Q,et al.In vivo metabolite profiling of chicoric acid in rat plasma,urine and feces after oral administration using liquid chromatography quadrupole time of flight mass spectrometry[J].Journal of Chromatography B,2018,1081/1082:8-14.DOI:10.1016/j.jchromb.2018.02.016.
[7]SHAO Y P,ZHANG W,TONG L,et al.Simultaneous determination of eight bioactive components of Qishen Yiqi Dripping Pills in rat plasma using UFLC-MS/MS and its application to a pharmacokinetic study[J].Biomedical Chromatography,2017,31(8):1-13.DOI:10.1002/bmc.3941.
[8]KUMAR A,KUMAR P,KOUNDAL R,et al.Antioxidant properties and UPLC-MS/MS profiling of phenolics in jacquemont’s hazelnut kernels(Corylus jacquemontii)and its byproducts from western Himalaya[J].Journal of Food Science&Technology,2016,53(9):3522-3531.DOI:10.1007/s13197-016-2329-2.
[9]ZHANG X F,CHEN J,YANG J L,et al.UPLC-MS/MS analysis for antioxidant components of Lycii Fructus based on spectrumeffect relationship[J].Talanta,2018,180:389-395.DOI:10.1016/j.talanta.2017.12.078.
[10]JAKOPIC J,PETKOVSEK M M,LIKOZAR A,et al.HPLC-MS identification of phenols in hazelnut(Corylus avellana L.)kernels[J].Food Chemistry,2010,124(3):1100-1106.DOI:10.1016/j.foodchem.2010.06.011.
[11]任强.苦荞麦化学成分及其代谢产物分析和鉴定方法研究[D].北京:北京协和医学院,2013:72-75.
[12]BHATT V,SHARMA S,KUMAR N,et al.Simultaneous quantification and identification of flavonoids,lignans,coumarin and amides in leaves of Zanthoxylum armatum,using UPLC-DAD-ESI-QTOF-MS/MS[J].Journal of Pharmaceutical&Biomedical Analysis,2016,132:46-55.DOI:10.1016/j.jpba.2016.09.035.
[13]JIA W N,WANG C H,WANG Y F,et al.Qualitative and quantitative analysis of the major constituents in chinese medical preparation Lianhua-Qingwen capsule by UPLC-DAD-QTOF-MS[J].The Scientific World Journal,2015,2015:731765.DOI:10.1016/j.jpba.2011.10.007.
[14]WICZKOWSKI W,SZAWARANOWAK D,MITRUS J,et al.Profile of phenolic acids and antioxidant capacity in organs of common buckwheat sprout[J].Acta Alimentaria,2016,45(2):250-257.DOI:10.1556/066.2016.45.2.12.
[15]GUO Xudan,WU Chunsen,MA Yujie,et al.Comparison of milling fractions of tartary buckwheat for their phenolics and antioxidant properties[J].Food Research International,2012,49(1):53-59.DOI:10.1016/j.foodres.2012.07.019.
[16]SEO J M,DA B L,ARASU M V,et al.Quantitative differentiation of phenolic compounds in different varieties of buckwheat cultivars from China,Japan and Korea[J].Journal of Agricultural Chemistry&Environment,2013,2(4):109-116.DOI:10.4236/jacen.2013.24016.
[17]ZHAO M M,YANG Q Y,LIN L Z,et al.Intracellular antioxidant activities of selected cereal phenolic extracts and mechanisms underlying the protective effects of adlay phenolic extracts on H2O2-induced oxidative stress in human erythrocytes[J].Journal of Functional Foods,2017,31:160-171.DOI:10.1016/j.jff.2017.01.046.
[18]SAFAEIAN L,EMAMI R,HAJHASHEMI V,et al.Antihypertensive and antioxidant effects of protocatechuic acid in deoxycorticosterone acetate-salt hypertensive rats[J].Biomedicine&Pharmacotherapy,2018,100:147-155.DOI:10.1016/j.biopha.2018.01.107.
[19]SANCAK E B,AKBAS A,SILAN C,et al.Protective effect of syringic acid on kidney ischemia-reperfusion injury[J].Renal Failure,2016,38(4):629-635.DOI:10.3109/0886022X.2016.1149868.
[20]KIM K H,TSAO R,YANG R,et al.Phenolic acid profiles and antioxidant activities of wheat bran extracts and the effect of hydrolysis conditions[J].Food Chemistry,2006,95(3):466-473.DOI:10.1016/j.foodchem.2005.01.032.
[21]MORISHITA T,YAMAGUCHI H,DEGI K.The contribution of polyphenols to antioxidative activity in common buckwheat and tartary buckwheat grain[J].Plant Production Science,2007,10(1):99-104.DOI:10.1626/pps.10.99.
[22]KIM J H,LEE B C,KIM J H,et al.The isolation and antioxidative effects of vitexin from Acer palmatum[J].Archives of Pharmacal Research,2005,28(2):195-202.DOI:10.1007/bf02977715.
[23]THAIPONG K,BOONPRAKOB U,CROSBY K,et al.Comparison of ABTS,DPPH,FRAP,and ORAC assays for estimating antioxidant activity from guava fruit extracts[J].Journal of Food Composition&Analysis,2006,19(6):669-675.DOI:10.1016/j.jfca.2006.01.003.
[24]SHEN S W,CHAHAL B,MAJUMDER K,et al.Identification of novel antioxidative peptides derived from a thermolytic hydrolysate of ovotransferrin by LC-MS/MS[J].Journal of Agricultural and Food Chemistry,2010,58(13):7664-7672.DOI:10.1021/jf101323y.
[25]RANI W,BOWDEN N A,KOLFSCHOTEN R C,et al.Fractional precipitation of amino acids from agro-industrial residues using ethanol[J].Industrial&Engineering Chemistry Research,2016,55(27):7462-7472.DOI:10.1021/acs.iecr.6b00054.
[26]LIU Y F,LIU J X,CHEN X F,et al.Preparative separation and purification of lycopene from tomato skins extracts by macroporous adsorption resins[J].Food Chemistry,2010,123(4):1027-1034.DOI:10.1016/j.foodchem.2010.05.055.
[27]任尧.鸭蛋清蛋白肽的酶法制备及其抗氧化活性研究[D].广州:华南理工大学,2011:46-71.
[28]ALEMáN A,GIMéNEZ B,PéREZ-SANTIN E,et al.Contribution of Leu and Hyp residues to antioxidant and ACE-inhibitory activities of peptide sequences isolated from squid gelatin hydrolysate[J].Food Chemistry,2011,125(2):334-341.DOI:10.1016/j.foodchem.2010.08.058.
[29]ZHENG L,DONG H,SU G,et al.Radical scavenging activities of Tyr-,Trp-,Cys-and Met-Gly and their protective effects against AAPH-induced oxidative damage in human erythrocytes[J].Food Chemistry,2016,197:807-813.DOI:10.1016/j.foodchem.2015.11.012.
[30]TSOPMO A,ROMANOWSKI A,BANDA L,et al.Novel antioxidative peptides from enzymatic digestion of human milk[J].Food Chemistry,2011,126(3):1138-1143.DOI:10.1016/j.foodchem.2010.11.146.
[31]HERNáNDEZLEDESMA B,DáVALOS A,BARTOLOMéB,et al.Preparation of antioxidant enzymatic hydrolysates from alphalactalbumin and beta-lactoglobulin.identification of active peptides by HPLC-MS/MS[J].Journal of Agricultural and Food Chemistry,2005,53(3):588-593.DOI:10.1021/jf048626m.
[32]CHEN H M,MURAMOTO K,YAMAUCHI F.Structural analysis of antioxidative peptides from soybeanβ-conglycinin[J].Journal of Agricultural and Food Chemistry,1995,43(3):574-578.DOI:10.1021/jf00051a004.
[33]RAJAPAKSE N,MENDIS E,BYUN H G,et al.Purification and in vitro antioxidative effects of giant squid muscle peptides on free radical-mediatedo xidativesy stems[J].Journal of Nutritional Biochemistry,2005,16(9):562-569.DOI:10.1016/j.jnutbio.2005.02.005.
[34]SAIGA A,TANABE S,NISHIMURA T.Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment[J].Journal of Agricultural and Food Chemistry,2003,51(12):3661-3667.DOI:10.1021/jf021156g.
[35]DAI C H,ZHANG W W,HE R H,et al.Protein breakdown and release of antioxidant peptides during simulated gastrointestinal digestion and the absorption by everted intestinal sac of rapeseed proteins[J].LWT-Food Science and Technology,2017,86:424-429.DOI:10.1016/j.lwt.2017.08.026.