低酯果胶-咖啡酸交联物的构成机理及与抗氧化活性的构效关系  

Formation Mechanism and Structure-Antioxidant Activity Relationship of Low-Methoxyl Pectin-Caffeic Acid Conjugate

作　　者：高凡 艾连中[1] 吴艳[2] 赖凤羲 张汇[1] 谢凡 宋子波 GAO Fan;AI Lianzhong;WU Yan;LAI Phoency;ZHANG Hui;XIE Fan;SONG Zibo(Shanghai Engineering Research Center of Food Microbiology,School of Health Science and Engineering,University of Shanghai for Science and Technology,Shanghai 200093,China;School of Agriculture and Biology,Shanghai Jiao Tong University,Shanghai 200240,China;Yunnan Mouthdoleak Group Food Co.,Ltd.,Yuxi 653100,China)

机构地区：[1]上海理工大学健康科学与工程学院,上海食品微生物工程技术研究中心,上海200093 [2]上海交通大学农业与生物学院,上海200240 [3]云南猫哆哩集团食品有限责任公司,云南玉溪653100

出　　处：《食品科学》2022年第9期19-29,共11页Food Science

基　　金：上海市科技兴农项目(2019-02-08-00-07-F01152);上海食品微生物工程技术研究中心项目(19DZ2281100)。

摘　　要：本研究采用绿色安全的漆酶催化法制备柑橘低酯果胶(citrus low-methoxyl pectin,CLP)和咖啡酸(caffeic acid,CaA)交联物,解析交联物的关键组成、光谱特征、核磁共振图谱特征以及体外抗氧化活性,以明确其交联机理、分子间键结模式,并归纳其贡献抗氧化活性的结构因素。化学成分组成与离子色谱分析结果显示,CLP含有51.99%(摩尔百分比,下同)半乳糖醛酸(galacturonic acid,GalA)、30.95%半乳糖(galactose,Gal)、7.44%鼠李糖(rhamnose,Rha)和9.02%葡萄糖(glucose,Glc)及微量总酚(1.20 mg/g),酯化度为38.33%,CLP主要分子链段由同型半乳糖醛酸聚糖(homogalacturonan,HG)和第一型鼠李糖半乳糖醛酸聚糖(rhamnogalacturonan,RG-I)所组成,其中RG-I的中性糖侧链为聚半乳糖。CLP的重均分子质量(m_(w))为226.1 kDa。相较于CLP,CLP-CaA交联物的酯化度和总酚含量分别显著提高到46.93%和9.64 mg/g(P<0.05);m_(w)增加到271.4 kDa;紫外吸收光谱和傅里叶变换红外光谱都呈现与CaA有关的特征吸收峰(分别在288 nm和1519 cm^(-1))。700 MHz ^(1)H和176 MHz ^(13)C核磁共振图谱清楚地显示了CLP的α-D-GalA、β-D-Gal和部分α-L-Rha糖基的共振尖峰,可推断其主要结构;而交联物CLP-CaA中CLP的糖基碳都有新共振尖峰,位移最大的是α-D-GalA的C1和C4,伴随C6羧基碳的尖峰位移到δ_(C) 175.12,且氢谱上出现CaA的特征尖峰(δ_(H) 6~8)。综上可知,CLP-CaA的交联机理为:漆酶催化CaA作用于CLP的α-D-GalA糖基C6羧基上形成酯基,且1分子CaA与1分子自由羧基结合。此交联作用促使CLP-CaA具有优越且多元的体外抗氧化活性,对1,1-二苯基-2-三硝基苯肼自由基、2,2’-联氮-双-3-乙基苯并噻唑啉-6-磺酸阳离子自由基、·OH和O_(2)^(-)·的半抑制浓度(half maximal inhibitory concentration,IC_(50))分别为0.82、2.47、0.92 mg/mL和0.72 mg/mL。本研究发现了果胶与CaA的交联机理及其结构对抗氧化活性的贡献,可为新型改良果胶在食品The purpose of this study was to investigate the preparation of citrus low-methoxyl pectin(CLP)and caffeic acid(CaA)conjugate by a green and safe method using laccase.The conjugate was examined for its key composition,ultraviolet(UV)-visible,Fourier transform infrared(FTIR),nuclear magnetic resonance(NMR)spectroscopic characteristics and in vitro antioxidant activity,in order to clarify its formation mechanism and intermolecular bonding mode,and summarize the structural factors contributing to its antioxidant activity.The results of chemical composition and anion-exchange chromatograms showed that CLP contained 51.99%(mol%)galacturonic acid(GalA),30.95%galactose(Gal),7.44%rhamnose(Rha)and 9.02%glucose(Glc)as well as a trace amount of total phenolics(1.20 mg/g),with a methoxylation(DE)degree of 38.33%,suggesting that the major molecular fragments of CLP were homogalacturonan(HG)and rhamnogalacturonan type I(RG-I),whose neutral saccharide side chain was galactan.The weight-averaged molecular mass(m_(w))of CLP was 226.1 kDa.In contrast to CLP,CLP-CaA conjugate showed significantly increased DE and total phenolic content of 46.93%and 9.64 mg/g(P<0.05),respectively,and an increased m_(w) of 271.4 kDa.Moreover,CLP exhibited UV and FTIR absorption peaks characteristic of CaA at 288 nm and 1519 cm^(-1),respectively.The 700 MHz ^(1)H and 176 MHz ^(13)C NMR spectra indicated evidently resonant peaks forα-D-GalA andβ-D-Gal moieties,and partialα-L-Rha moieties of CLP,which confirmed its major structure.New resonant peaks were observed for all glucosyl carbons of CLP in CLP-CaA conjugate.The largest chemical shift occurred on the C1 and C4 atoms of theα-D-GalA moieties,the C6(carboxyl carbon)peak was shifted to δ_(C) 175.12,and peaks characteristic of CaA appeared at δ_(H) 6-8 in the 1 H spectra.To sum up,the conjugation mechanism of CLP with CaA could be laccase-mediated binding of CaA to the C6 carboxyl group of theα-D-GalA moieties in CLP at a 1:1 molar ratio,resulting in the formation of ester groups.As a result,th

关 键 词：低酯果胶 咖啡酸 漆酶 交联机理 结构表征 抗氧化活性 

分 类 号：TS201.2[轻工技术与工程—食品科学]