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中国精品科技期刊2020

基于UPLC-QTOF-MS的NFC与FC杨梅汁差异性代谢产物分析

孙瑞洋, 宣晓婷, 崔燕, 林旭东, 邵兴锋, 邓文艺, 凌建刚

孙瑞洋,宣晓婷,崔燕,等. 基于UPLC-QTOF-MS的NFC与FC杨梅汁差异性代谢产物分析[J]. 食品工业科技,2023,44(15):275−282. doi: 10.13386/j.issn1002-0306.2022080132.
引用本文: 孙瑞洋,宣晓婷,崔燕,等. 基于UPLC-QTOF-MS的NFC与FC杨梅汁差异性代谢产物分析[J]. 食品工业科技,2023,44(15):275−282. doi: 10.13386/j.issn1002-0306.2022080132.
SUN Ruiyang, XUAN Xiaoting, CUI Yan, et al. Analysis of Differential Metabolites between NFC and FC Bayberry Juice Based on UPLC-QTOF-MS[J]. Science and Technology of Food Industry, 2023, 44(15): 275−282. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022080132.
Citation: SUN Ruiyang, XUAN Xiaoting, CUI Yan, et al. Analysis of Differential Metabolites between NFC and FC Bayberry Juice Based on UPLC-QTOF-MS[J]. Science and Technology of Food Industry, 2023, 44(15): 275−282. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022080132.

基于UPLC-QTOF-MS的NFC与FC杨梅汁差异性代谢产物分析

基金项目: 宁波市公益类科技计划项目(2019C10033 & 2019C10104);国家重点研发计划项目(2017YFD0400703)。
详细信息
    作者简介:

    孙瑞洋(1996−),女,硕士研究生,研究方向:非热加工,E-mail:1830899541@qq.com

    通讯作者:

    凌建刚(1973−),男,博士,研究员,研究方向:农产品保鲜与非热加工,E-mail:nbnjg@163.com

  • 中图分类号: TS255.44;O657.63

Analysis of Differential Metabolites between NFC and FC Bayberry Juice Based on UPLC-QTOF-MS

  • 摘要: 为探究浓缩还原(From concentrate,FC)和非浓缩还原(Not from concentrate,NFC)杨梅汁成分的差异,本文借助超高效液相色谱-四极杆飞行时间质谱的代谢组学技术分析二者的代谢产物。通过正交偏最小二乘-判别分析实现了对NFC和FC杨梅汁的区分,根据变量投影重要度和差异倍数筛选出9个差异较大的代谢物,经对比发现,FC杨梅汁中对磺基苯甲酸、5-甲基四氢叶酸、矢车菊素、西瑞香素、花椒毒酚和异黄蝶呤等抗氧化活性成分的含量相对较低,而苦味物质(新橙皮苷、橙皮苷和槲皮素-3-O-新橙皮苷)含量相对较高。通过对差异代谢物的代谢途径分析,筛选出5条差异较大的代谢通路,其中苯丙氨酸代谢、黄酮和黄酮醇的生物合成是NFC与FC杨梅汁中差异代谢物的关键代谢途径。本研究揭示了NFC与FC杨梅汁代谢产物的差异性,为NFC杨梅汁的加工与鉴别提供理论参考。
    Abstract: This study analyzed the differential composition between from concentrate (FC) and not from concentrate (NFC) bayberry juices by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). NFC and FC bayberry juice were distinguished from each other by orthogonal partial least squares discriminant analysis, and 9 compounds that significantly differed in abundance between them were selected and identified according to the variable importance in the projection and the fold-change. By comparison, the content of antioxidant active components such as 4-sulfobenzoate, 5-methyltetrahydrofolic acid, cyanidin, daphnoretin, xanthotoxol and isoxanthopterin in FC bayberry juice were relatively low, whereas the bitter substances (neohesperidin, hesperidin and quercetin-3-O-neohesperidin) were relatively high. Metabolic pathways were analyzed on account of differential metabolites and five metabolic pathways with great differences were identified, among which phenylalanine metabolism, biosynthesis of flavonoids and flavonols were the key metabolic pathways of different metabolites in NFC and FC bayberry juice. This study revealed the difference of metabolites between NFC and FC bayberry juice, which provided theoretical reference for the processing and identification of NFC bayberry juice.
  • 杨梅(Myrica rubra Sieb. et Zucc)是中国江南地区的珍贵水果之一,其营养功效丰富、品质风味独特,含有多种对人体有益的生物活性成分[1]。但其成熟于江南梅雨季节,采后极不耐储,通常被加工成杨梅干和杨梅汁等[2]。非浓缩还原果汁(NFC)是鲜榨果汁经巴氏杀菌后直接灌装而成的,既能保持果实原有风味,又能最大限度的保留果实中的各种功能性成分和营养物质,但价格偏高[3-4]。而浓缩还原果汁(FC)是由NFC果汁经浓缩、还原和杀菌等复杂过程制备而成且价格偏低,但该过程易导致品质风味物质劣变[5]。对于NFC与FC果汁成分的差异,研究学者针对化合物、同位素比例等做了一定的研究。Sun等[6]通过HS-SPME-GC-MS结合化学计量学对NFC与FC橙汁鉴别,筛选出25种差异化合物。有研究学者对NFC与FC橙汁中稳定同位素比率δD、δ18O进行分析,发现NFC橙汁的同位素含量明显高于FC橙汁[7]。但由于检测限、检测准确性等问题限制了上述技术在果汁行业的广泛应用。

    代谢组学技术作为继基因组学、转录组学和蛋白组学后的新兴组学,因其灵敏度、分辨率高等优势,在食品的品质评估与溯源中得到广泛应用[8-9]。杨天铭等[10]基于UPLC-QTOF-MS技术在黄龙病脐橙与正常脐橙中鉴定出28个差异化合物。屠燕等[11]基于UPLC-QTOF-MS技术在不同产地丹参药材中鉴定出22个差异化学成分。刘晗璐等[12]基于代谢组学技术在NFC和FC橙汁中筛选并鉴定出16种差异化合物。Xu等[13]利用非靶向代谢组学技术对NFC橙汁和FC橙汁进行了鉴别,鉴定出13个潜在标记物,其中7个三肽标记物首次被报道。目前对于NFC/FC果汁的鉴别主要以橙汁为主,对于杨梅汁的研究主要集中在不同品种、贮藏期间的品质及风味变化,而对NFC/FC杨梅汁的差异性成分研究较少。因此本研究借助UPLC-QTOF-MS代谢组学技术结合多元统计分析等方法,根据代谢通路途径从植物代谢组学角度初步揭示NFC杨梅汁和FC杨梅汁差异代谢产物,旨在为NFC与FC杨梅汁成分差异分析提供理论和数据参考。

    实验原料为八成熟的仙居东魁杨梅(Myrica rubra Sieb. et Zucc)  采集自中国浙江省台州市。由于杨梅果实的收获季节为5月至7月,本研究以每半月取一批次样品,并将采摘后挑选新鲜无伤害的置于−80 ℃冰箱保存,直至使用;甲醇、乙腈、甲酸 质谱级,购自Sigma-Aldrich。

    MJ-JS2018A榨汁机 广东美的集团股份有限公司;DK-S12电热恒温水浴锅 上海森信实验仪器有限公司;ExionLC AD型超高效液相色谱 日本岛津公司;QTrap 6500型四极杆串联飞行时间质谱仪 美国AB Sciex公司;Heraeus Fresco17离心机 德国Eppendorf公司;MS 3 digital涡旋振荡器 德国IKA公司;ACQUITY UPLC HSS T3(1.8 μm 2.1 mm×100 mm)色谱柱 上海拜力生物科技有限公司;R3088真空旋转蒸发仪 美国Senco公司。

    参照刘晗璐等[12]的方法并略作修改。NFC杨梅汁的制备:将冷冻的杨梅果在室温下解冻后榨汁,使用4层纱布过滤除去果渣,将过滤的果汁置于耐高压聚乙烯塑料瓶中进行巴氏杀菌(80 ℃,10 min),冷却至室温置于−l80 ℃储存。

    FC杨梅汁的制备:将NFC杨梅汁置于真空旋转蒸发仪中,60 ℃水浴下浓缩1 h左右直至糖度为60±1ºBrix,再使用纯净水复配至原始糖度值,置于耐高压聚乙烯塑料瓶中并进行巴氏杀菌(80 ℃,10 min),杀菌后迅速冷却至室温并置于−80 ℃储存。

    将NFC和FC杨梅汁样品在4 ℃条件下以12000 r/min离心15 min,取上清液,使用提取液(甲醇:水=3:1)稀释5倍,涡旋30 s将其充分混合,经0.22 μm微孔滤膜过滤,转移至1.5 mL进样小瓶中,等待上机检测。每个样品进行6次平行测定。

    参照Yang等[14]的方法并略作修改。色谱条件:色谱柱为ACQUITY UPLC BEH(150 mm×2.1 mm,1.7 μm;Waters Co),柱温箱温度为40 ℃,自动进样器温度为4 ℃,进样体积为2 μL,流动相A为含0.1%甲酸水溶液,流动相B为乙腈,洗脱梯度:0.0~0.5 min,98% A、2% B;0.5~10.0 min,98%~50% A、2%~50% B;10.0~13.0 min,5% A、95% B;13.0~15.0 min,98% A、2% B。

    质谱条件:使用装备IonDrive Turbo V ESI离子源的SCIEX 6500 QTRAP+三重四极杆质谱仪,以多反应监测(MRM)模式进行质谱分析。电喷雾离子源(Electrospray ionization,ESI)进行海量数据采集,操作参数如下:离子喷雾电压:+5500/−4500 V,帘式气体:35 psi,温度:400 ℃,离子源气体1:60 psi,离子源气体2:60 psi,DP:±100 V。

    试验数据使用Excel进行整理,结果用平均数表示,通过SCIEX分析软件(1.6.3版)进行MRM数据的采集和处理。采用SIMCA 16.0软件,进行无监督的主成分分析和有监督的正交偏最小二乘判别分析,利用Origin 2021软件绘制代谢物的柱状图,同时利用差异代谢物的KEGG(Kyoto encyclopedia of genes and genomes)进行通路富集分析,获得代谢通路富集结果。

    生物代谢组易受外界因素干扰,尤其是在物质提取、检测分析过程中存在误差,从而导致样品间产生差异,如果差异越小说明整个方法稳定性越好数据质量越高。为了考察数据的质量性,数据质量控制(Quality control,QC)是获得可重复性和准确性代谢组结果的必要步骤,因此对QC样本的相关性及内标的响应稳定性进行分析。QC样本相关性越接近于1(至少应≥0.7),说明整个方法稳定性越好数据质量越高;内标为外源引入的物质,QC样本内标浓度相同,所以内标的响应差异越小(RSD≤20%),说明系统越稳定,数据质量越高。从图1可以看出,在整个实验分析过程中QC样本相关性达0.95,RSD为5.66%,说明本次实验数据质量很高。基于迈维自建数据库MWDB及代谢物信息公共数据库,在NFC和FC杨梅汁中共鉴定出208个代谢产物,其中主要包括24个黄酮类物质、23个生物碱类物质、17个氨基酸及其衍生物、17个酚类物质等,虽然黄酮和生物碱类物质数量较多,但是氨基酸中脯氨酸的相对含量最高,氨基酸是果汁中重要的生物活性物质之一,对果汁的口感、气味和色泽具有重要影响[15]。所以根据这些代谢物的性质进一步说明杨梅汁具有独特的营养与风味。

    图  1  QC样本相关性分析
    Figure  1.  QC sample correlation analysis

    为了解NFC和FC杨梅汁中代谢物的积累情况,对两种杨梅汁进行主成分分析(PCA)。代谢组学分析中,PCA主要用于观察实验模型中的组间分离是否有异常点出现,以及反映组间和组内变异度的分析技术。主成分分析结果表明,R2X代表模型的拟合能力,累积为0.839大于0.5,Q2代表了模型的预测能力为0.451,结果表明PCA的模型拟合度较好。从得分图可以看出,样本全部处于95%置信区间内,NFC杨梅汁样品点和FC杨梅汁样品点分布离散,分别位于PC1的正负半轴,说明两者的代谢成分存在一定差异。PCA作为一种反映原始数据状态的无监督方法,环境等其他因素以及系统错误都会影响实验结果。从图2中可以看出前两个主成分分析的贡献率分别为15.3%和10.3%,总贡献率之和较低,无法反应整体分散程度。为排除因与实验无关的某些因素引起的代谢变化,应用有监督的正交偏最小二乘判别分析(Orthogonal partial least squares discriminant analysis,OPLS-DA)进一步区分两组之间代谢物的差异数据,并依据该模型对数据进行分析,从而准确地鉴别出具有差异的代谢物[16]

    图  2  NFC和FC杨梅汁样品的PCA得分图
    Figure  2.  PCA score plot of NFC and FC bayberry juice samples

    图3a为OPLS-DA得分图,从图中可以看出两组样本区分非常显著,说明两种果汁样品的代谢物组成差异较大。为了避免数据出现过拟合现象,从而确保模型的有效性,对模型进行置换检验以获取随机模型的R2和Q2,以此评估OPLS-DA得分图是否存在过拟合现象。图3b为OPLS-DA置换检验图,横坐标表示置换保留度,纵坐标表示R2Y或Q2值,绿色圆点表示R2Y值,蓝色方点表示Q2值,两条虚线分别表示R2Y和Q2的回归线。一般认为,当R2Y和Q2参数值越接近于1,说明模型解释或预测的能力越强,而该OPLS-DA模型的R2Y为1,Q2为0.744,说明建立的模型符合样本数据的真实情况。且从图中可以看出Q2值均小于R2Y值,Q2的回归线与纵坐标的截距小于零,一般认为截距为负值时,说明统计模型有效,没有出现过拟合[17],另外随着置换保留度的降低,随机模型Q2值逐渐减小,说明原模型具有良好的稳健性,不存在过拟合现象。

    图  3  NFC和FC杨梅汁样品的OPLS-DA得分图(a)和置换模型(b)
    Figure  3.  OPLS-DA score map of NFC and FC bayberry juice samples (a) and displacement model (b)

    变量投影重要度(Variable Importance in the Projection,VIP)是OPLS-DA模型变量的权重值,可用于衡量各组分积累差异对各组样本分类判别的影响强度,通常认为数值大于1时,变量对组间分离具有显著贡献作用[18]。为了进一步确定二者产生差异的代谢物,本研究以差异倍数(Fold-Change)小于0.5或大于2,同时OPLS-DA模型第一主成分的VIP大于1为标准,一共筛选到40个差异代谢物(如表1),其中主要以黄酮类物质为主。

    表  1  NFC和FC杨梅汁差异代谢物数量统计表
    Table  1.  Statistical table of the number of different metabolites in NFC and FC bayberry juice
    序号化合物名称CAS一级分类保留时间
    (min)
    VIPNFCFCFold-ChangeLog-Fold
    Change
    13-Methylxanthine
    3-甲基黄嘌呤
    1076228核苷酸及其衍生物2.71021.65672.21394E-054.80232E-052.16911.1171
    24-Sulfobenzoate
    对磺基苯甲酸
    636782有机酸4.68271.97570.0005515720.0002261580.4100−1.2862
    35-Heneicosylresorcinol
    5-二十一烷基间苯二酚
    70110597酚类12.05041.72221.38433E-054.2671E-053.08241.6241
    45-Methyltetrahydrofolic acid
    5-甲基四氢叶酸
    134350叶酸及其衍生物3.28002.28114.25081E-050.0011825260.0359−4.7980
    55-Tricosyl-1,3-benzenediol
    5-二十三烷基间苯二酚
    70110600酚类13.84611.32389.40658E-062.35345E-052.50191.3230
    65'-S-Methyl-5'-thioadenosine
    5'-脱氧-5'-甲硫腺苷
    2457809核苷酸及其衍生物3.99432.24880.0228694820.0625742562.73611.4521
    7Acanthoside B
    刺五加甙B
    7374790木脂素6.51402.25350.0209663360.0529944482.52761.3378
    8Agomelatine
    阿戈美拉汀
    138112762其他类型10.00291.40597.11534E-061.45277E-052.04171.0298
    9Spathulenol
    桉油烯醇
    6750-60-3孕烯醇酮脂类10.89011.12337.36955E-050.0001879332.55011.3506
    10alpha-Hexylcinnamaldehyde
    α-己基肉桂醛
    101-86-0酚类8.70532.07644.9007E-050.0001048072.13861.0967
    11Colchicine
    秋水仙碱
    64868生物碱5.09241.45472.11974E-054.46753E-052.10761.0756
    12Convolvine
    旋花碱
    537304生物碱5.06871.63793.90251E-059.97865E-052.55701.3544
    13Cyanidin
    矢车菊素
    528585黄酮5.89811.84380.0190355890.001231410.0647−3.9503
    14Daphnoretin
    西瑞香素
    2034697香豆素11.44621.94100.0001618414.41912E-050.2731−1.8727
    15Deguelin
    鱼藤素
    522178黄酮12.92371.71511.66812E-054.13666E-052.47981.3102
    16Ellagic acid
    鞣花酸
    476664酚类6.12301.58960.0009410280.0024922962.64851.4052
    17Glucoliquiritin
    葡萄糖基甘草苷
    93446185黄酮5.06621.92484.86499E-059.83577E-052.02171.0156
    18Guanine
    鸟嘌呤
    73405核苷酸及其衍生物2.62791.19790.0389492320.0389492322.15311.1064
    19Hesperidin
    橙皮苷
    520263黄酮6.65001.54640.0001177431.95571E-056.02052.5899
    20Isovitexin
    异牡荆黄素
    38953854黄酮5.85501.36410.0003346468.11445E-054.12412.0441
    21Isoxanthopterin
    异黄蝶呤
    529691蝶呤类化合物3.25612.27690.0001587770.0014397960.1103−3.1808
    22Kaurenoic acid
    贝壳杉烯酸
    6730832二萜5.90231.28830.0661522560.0305269022.16701.1157
    23Lithospermic acid
    紫草酸
    28831654苯丙素7.11621.95512.98492E-058.34576E-063.57661.8386
    24Lucidin
    芦西定
    478080蒽醌8.89471.73152.56859E-051.106E-052.32241.2156
    25Malvidin-3-O-galactoside
    锦葵色素-3-O-半乳糖苷
    30113372黄酮7.75402.00371.60009E-053.94272E-064.05832.0209
    26Neohesperidin
    新橙皮苷
    13241333黄酮6.77402.03160.000205612.79295E-057.36182.8801
    27Norlichexanthone
    3,6,8-三羟基-1-甲基呫吨酮
    20716987氧杂蒽酮4.48131.97520.0003572990.0001653512.16091.1116
    28Octadecyl p-coumarate
    对羟基桂皮酸十八酯
    72943885苯丙素12.06721.30267.70192E-052.68342E-052.87021.5211
    29Petasitenine
    蜂斗菜烯碱
    60102376生物碱4.68271.49594.41133E-051.81308E-052.43311.2828
    30Phillyrin 连翘苷487412苯丙素7.76101.47730.0002046519.99422E-052.04771.0340
    31Porphobilinogen
    胆色素原
    487901有机氮化合物1.58741.79383.13247E-051.07281E-052.91991.5459
    32Practolol 心得宁6673354芳香类化合物2.73591.75996.55189E-053.18083E-052.05981.0425
    33Quercetin 槲皮素117395黄酮8.07002.02630.0005924310.0002019972.93291.5523
    34Quercetin 3-O-neohesperidoside
    槲皮素-3-O-新橙皮苷
    32453364黄酮6.58592.08210.0002208334.41036E-055.00722.3240
    35Quercetin-3-O-glucuronide
    槲皮素3-O-葡萄糖酸苷
    22688795黄酮5.92461.45640.0073050160.0026380312.76911.4694
    36S-Lactoylglutathione
    s-乳酰谷胱甘肽
    41656568羧酸类及其衍生物4.09221.74504.38305E-051.80098E-052.43371.2832
    37Sesamolin
    芝麻林素
    526078木脂素7.55261.91638.92372E-053.21499E-052.77571.4728
    38Tricetin
    三粒小麦黄酮
    520310黄酮8.04611.69500.0078110740.0033125992.35801.2376
    39Vitexin 2''-O-beta-L-rhamnoside
    牡荆素-2-O-鼠李糖苷
    64820991黄酮12.11371.10122.31267E-056.31288E-063.66341.8732
    40Xanthotoxol
    花椒毒酚
    2009247香豆素8.74291.71632.09788E-054.27557E-050.4907−1.0272
    下载: 导出CSV 
    | 显示表格

    为了更清楚的了解杨梅汁浓缩还原前后代谢物差异倍数的变化情况,制作了如图4所示的代谢物差异倍数柱状图,并将排名靠前(上调和下调)的20位代谢物进行展示,一般认为,当Fold-Change>2时,值越大差异越明显;当Fold-Change<0.5时,值越小差异越明显,所以结合Fold-Change大小筛选出9个差异最大的物质,分别是下调物质中的5-甲基四氢叶酸、矢车菊素、异黄蝶呤、西瑞香素、对磺基苯甲酸和花椒毒酚,上调物质中的新橙皮苷、橙皮苷、槲皮素-3-O-新橙皮苷。而它们所对应的Log-Fold change分别为−4.7980、−3.9503、−3.1808、−1.8727、−1.2862、−1.0272、2.8801、2.5899、2.3240。

    图  4  NFC和FC杨梅汁代谢物差异倍数柱状图
    Figure  4.  Histogram of the metabolite fold difference between NFC and FC bayberry juice

    箱形图可以直观表达每种化合物在NFC和FC杨梅汁中的含量变化(图5)。从图中可以看出,与NFC杨梅汁相比,FC杨梅汁中的对磺基苯甲酸、5-甲基四氢叶酸、矢车菊素、西瑞香素、花椒毒酚和异黄蝶呤物质的含量相对较低,而新橙皮苷、橙皮苷和槲皮素-3-O-新橙皮苷物质的含量相对较高。西瑞香素、花椒毒酚属于香豆素类化合物,是生药中的一类重要的活性成分,具有抗菌、抗氧化等多种生物活性功能[19],但其不溶于冷水,易溶于热水,浓缩过程中随着温度的升高,水分不断蒸发,导致其含量有所损失。5-甲基四氢叶酸是合成叶酸活性化代谢的关键产物,在天然叶酸中活性最强,且在人体生命活动中起重要作用[20],但其稳定性较差,受温度、pH等因素影响, Islam[21]研究发现蒸煮和微波加热都使叶酸含量有所损失,与本研究结果类似,说明浓缩过程中的热处理会造成FC杨梅汁中的营养物质发生损失。矢车菊色素是花色苷中常见的一种化合物,而花色苷是黄酮物质中呈现红色的一族化合物,广泛存在于果蔬、花卉的根茎、花、果实中,能有效去除自由基,显示强烈的抗氧化性[22]。花色苷的稳定性受多种因素的影响,其中温度就是一个及其重要的因素,它对花色苷的降解具有显著影响作用,加热或高温都可加变色反应,尤其是在加热至沸腾时极易氧化褪色[23]。张燕等[24]的研究发现,当红莓的受热温度在45~75℃时,花色苷会发生降解,导致其含量及红值均发生显著下降。FC杨梅汁中这些物质含量减少,可能就是由于浓缩过程中水分的蒸发及加热处理。

    图  5  NFC和FC杨梅汁的差异标记化合物比较
    Figure  5.  Comparison of differentially labeled compounds in NFC and FC bayberry juice

    新橙皮苷、橙皮苷和槲皮素-3-O-新橙皮苷属于黄酮物质,作为水果中主要的生物活性成分之一,具有较强的抗氧化活性,但受温度影响较大[25]。槲皮素-3-O-新橙皮苷是槲皮素和一种天然类黄酮的代谢产物,而槲皮素是大量存在的一种类黄酮化合物,常以苷类形式存在[26]。但橙皮苷、新橙皮苷、柠檬苦素等是柑橘类水果中常见的苦味代谢产物[27],其含量的高低对水果及果汁的感官品质具有较大影响[28]。橙皮苷是柑橘类水果白色内果皮中发现的主要的水溶性苦味成分[29],在加热过程中,随着时间的延长逐渐被析出,导致果汁的苦味增加。丁胜华等研究发现,高温处理导致橙皮中橙皮苷、没食子酸等类黄酮物质含量显著增加[30]。所以综上,浓缩过程的水分蒸发和加热可能就是导致NFC/FC杨梅汁代谢物产生差异的主要原因。

    不同的代谢产物在生物体中相互作用,形成不同的代谢通路[31]。为深入了解差异代谢物涉及的代谢路径,通过KEGG数据库进一步对差异代谢物进行显著性富集分析,从而筛选出二者的差异代谢途径,并对其进行相关性分析[32]。根据鉴定出的代谢物共筛选出48条代谢通路,将其绘制成气泡图,如图6所示。图中每一个气泡代表一个代谢通路,而横坐标和气泡大小表示该通路在拓扑分析中的影响因子大小(Impact值),Impact值越大气泡越大,Impact值越小气泡越小;另外纵坐标和气泡颜色表示富集分析的P值(取负自然对数,即-ln(p)),红色越深代表-ln(p)的值越大,蓝色越深表示-ln(p)的值越小,而颜色越深表示富集程度越显著。

    图  6  NFC和FC杨梅汁差异代谢物富集通路气泡图
    Figure  6.  Bubble diagram of differential metabolite enrichment pathways in NFC and FC bayberry juice

    通过Impact值和-ln(p)值综合分析得到与代谢物差异相关的代谢途径,从图6中可以看到,NFC与FC杨梅汁中的代谢物共参与48条代谢途径,其中富集程度较高的代谢通路共有5条,分别为苯丙氨酸代谢、黄酮和黄酮醇的生物合成、单萜生物合成、异喹啉生物碱生物合成和β-丙氨酸代谢。其中苯丙氨酸代谢通路最为显著,P值为0.19867,-ln(p)为1.6161,富集在此通路上的L-苯丙氨酸在苯丙氨酸脱氨酶(Phenylalanine ammonia-lyase,PAL)的作用下生成苯丙素类代谢物(香豆素、萜类、木质素、花青素等)[33],而在苯丙氨酸羟化酶(Phenylalanine hydroxylase,PAH)的催化作用下则会生成L-酪氨酸。但L-苯丙氨酸稳定性受到温度的影响,受热时会发生部分降解[34],导致FC杨梅汁中香豆素(西瑞香素、花椒毒酚)、矢车菊素、L-酪氨酸含量减少。另外,黄酮和黄酮醇的生物合成这条代谢途径,P值为0.23889,-ln(p)为1.6161,被检测到的代谢物为槲皮素和芦丁。槲皮素是杨梅中含量较丰富的黄酮醇化合物,其在二氢黄酮醇4-还原酶(Dihydroflavonol 4-reductase,DFR)作用下生成矢车菊素[35]。由于温度对黄酮类物质稳定性有较大影响[36],所以导致FC杨梅汁中由槲皮素转化生成的矢车菊素含量降低。所以综上说明苯丙氨酸代谢、黄酮和黄酮醇的生物合成是NFC与FC杨梅汁的关键差异代谢途径。

    本研究借助代谢组学技术,利用PCA和OPLS-DA分析了NFC和FC杨梅汁代谢产物的差异,并进一步识别潜在的标记化合物,为分析NFC与FC杨梅汁间差异成分提供了一种有效方法。根据VIP>1和Fold-Change>2或Fold-Change<0.5筛选出40个差异代谢物,基于差异倍数大小从40种代谢物中筛选出9个差异较大的代谢物。经分析发现,FC杨梅汁中对磺基苯甲酸、5-甲基四氢叶酸、矢车菊素、西瑞香素、花椒毒酚和异黄蝶呤等抗氧化活性成分的含量相对较低,而苦味代谢物(新橙皮苷、橙皮苷和槲皮素-3-O-新橙皮苷)的含量相对较高。通过对代谢通路分析,筛选出5条差异较大的代谢途径,分别为苯丙氨酸代谢、黄酮和黄酮醇的生物合成、单萜生物合成、异喹啉生物碱生物合成和β-丙氨酸代谢。NFC与FC杨梅汁中筛选出的9个差异代谢物是以富集在通路上的L-苯丙氨酸和槲皮素在不同酶的作用转化生成,由于两种物质受温度的影响,使得转化生成的代谢物含量减少,而L-苯丙氨酸和槲皮素富集所在的通路为苯丙氨酸代谢、黄酮和黄酮醇的生物合成。所以综上结果显示,9个代谢产物和苯丙氨酸代谢、黄酮和黄酮醇的生物合成是NFC与FC杨梅汁形成差异的主要代谢物和代谢途径。本研究说明代谢组学可用于阐明NFC和FC杨梅汁间的成分差异,为二者差异代谢物富集的代谢通路提供研究方向。

  • 图  1   QC样本相关性分析

    Figure  1.   QC sample correlation analysis

    图  2   NFC和FC杨梅汁样品的PCA得分图

    Figure  2.   PCA score plot of NFC and FC bayberry juice samples

    图  3   NFC和FC杨梅汁样品的OPLS-DA得分图(a)和置换模型(b)

    Figure  3.   OPLS-DA score map of NFC and FC bayberry juice samples (a) and displacement model (b)

    图  4   NFC和FC杨梅汁代谢物差异倍数柱状图

    Figure  4.   Histogram of the metabolite fold difference between NFC and FC bayberry juice

    图  5   NFC和FC杨梅汁的差异标记化合物比较

    Figure  5.   Comparison of differentially labeled compounds in NFC and FC bayberry juice

    图  6   NFC和FC杨梅汁差异代谢物富集通路气泡图

    Figure  6.   Bubble diagram of differential metabolite enrichment pathways in NFC and FC bayberry juice

    表  1   NFC和FC杨梅汁差异代谢物数量统计表

    Table  1   Statistical table of the number of different metabolites in NFC and FC bayberry juice

    序号化合物名称CAS一级分类保留时间
    (min)
    VIPNFCFCFold-ChangeLog-Fold
    Change
    13-Methylxanthine
    3-甲基黄嘌呤
    1076228核苷酸及其衍生物2.71021.65672.21394E-054.80232E-052.16911.1171
    24-Sulfobenzoate
    对磺基苯甲酸
    636782有机酸4.68271.97570.0005515720.0002261580.4100−1.2862
    35-Heneicosylresorcinol
    5-二十一烷基间苯二酚
    70110597酚类12.05041.72221.38433E-054.2671E-053.08241.6241
    45-Methyltetrahydrofolic acid
    5-甲基四氢叶酸
    134350叶酸及其衍生物3.28002.28114.25081E-050.0011825260.0359−4.7980
    55-Tricosyl-1,3-benzenediol
    5-二十三烷基间苯二酚
    70110600酚类13.84611.32389.40658E-062.35345E-052.50191.3230
    65'-S-Methyl-5'-thioadenosine
    5'-脱氧-5'-甲硫腺苷
    2457809核苷酸及其衍生物3.99432.24880.0228694820.0625742562.73611.4521
    7Acanthoside B
    刺五加甙B
    7374790木脂素6.51402.25350.0209663360.0529944482.52761.3378
    8Agomelatine
    阿戈美拉汀
    138112762其他类型10.00291.40597.11534E-061.45277E-052.04171.0298
    9Spathulenol
    桉油烯醇
    6750-60-3孕烯醇酮脂类10.89011.12337.36955E-050.0001879332.55011.3506
    10alpha-Hexylcinnamaldehyde
    α-己基肉桂醛
    101-86-0酚类8.70532.07644.9007E-050.0001048072.13861.0967
    11Colchicine
    秋水仙碱
    64868生物碱5.09241.45472.11974E-054.46753E-052.10761.0756
    12Convolvine
    旋花碱
    537304生物碱5.06871.63793.90251E-059.97865E-052.55701.3544
    13Cyanidin
    矢车菊素
    528585黄酮5.89811.84380.0190355890.001231410.0647−3.9503
    14Daphnoretin
    西瑞香素
    2034697香豆素11.44621.94100.0001618414.41912E-050.2731−1.8727
    15Deguelin
    鱼藤素
    522178黄酮12.92371.71511.66812E-054.13666E-052.47981.3102
    16Ellagic acid
    鞣花酸
    476664酚类6.12301.58960.0009410280.0024922962.64851.4052
    17Glucoliquiritin
    葡萄糖基甘草苷
    93446185黄酮5.06621.92484.86499E-059.83577E-052.02171.0156
    18Guanine
    鸟嘌呤
    73405核苷酸及其衍生物2.62791.19790.0389492320.0389492322.15311.1064
    19Hesperidin
    橙皮苷
    520263黄酮6.65001.54640.0001177431.95571E-056.02052.5899
    20Isovitexin
    异牡荆黄素
    38953854黄酮5.85501.36410.0003346468.11445E-054.12412.0441
    21Isoxanthopterin
    异黄蝶呤
    529691蝶呤类化合物3.25612.27690.0001587770.0014397960.1103−3.1808
    22Kaurenoic acid
    贝壳杉烯酸
    6730832二萜5.90231.28830.0661522560.0305269022.16701.1157
    23Lithospermic acid
    紫草酸
    28831654苯丙素7.11621.95512.98492E-058.34576E-063.57661.8386
    24Lucidin
    芦西定
    478080蒽醌8.89471.73152.56859E-051.106E-052.32241.2156
    25Malvidin-3-O-galactoside
    锦葵色素-3-O-半乳糖苷
    30113372黄酮7.75402.00371.60009E-053.94272E-064.05832.0209
    26Neohesperidin
    新橙皮苷
    13241333黄酮6.77402.03160.000205612.79295E-057.36182.8801
    27Norlichexanthone
    3,6,8-三羟基-1-甲基呫吨酮
    20716987氧杂蒽酮4.48131.97520.0003572990.0001653512.16091.1116
    28Octadecyl p-coumarate
    对羟基桂皮酸十八酯
    72943885苯丙素12.06721.30267.70192E-052.68342E-052.87021.5211
    29Petasitenine
    蜂斗菜烯碱
    60102376生物碱4.68271.49594.41133E-051.81308E-052.43311.2828
    30Phillyrin 连翘苷487412苯丙素7.76101.47730.0002046519.99422E-052.04771.0340
    31Porphobilinogen
    胆色素原
    487901有机氮化合物1.58741.79383.13247E-051.07281E-052.91991.5459
    32Practolol 心得宁6673354芳香类化合物2.73591.75996.55189E-053.18083E-052.05981.0425
    33Quercetin 槲皮素117395黄酮8.07002.02630.0005924310.0002019972.93291.5523
    34Quercetin 3-O-neohesperidoside
    槲皮素-3-O-新橙皮苷
    32453364黄酮6.58592.08210.0002208334.41036E-055.00722.3240
    35Quercetin-3-O-glucuronide
    槲皮素3-O-葡萄糖酸苷
    22688795黄酮5.92461.45640.0073050160.0026380312.76911.4694
    36S-Lactoylglutathione
    s-乳酰谷胱甘肽
    41656568羧酸类及其衍生物4.09221.74504.38305E-051.80098E-052.43371.2832
    37Sesamolin
    芝麻林素
    526078木脂素7.55261.91638.92372E-053.21499E-052.77571.4728
    38Tricetin
    三粒小麦黄酮
    520310黄酮8.04611.69500.0078110740.0033125992.35801.2376
    39Vitexin 2''-O-beta-L-rhamnoside
    牡荆素-2-O-鼠李糖苷
    64820991黄酮12.11371.10122.31267E-056.31288E-063.66341.8732
    40Xanthotoxol
    花椒毒酚
    2009247香豆素8.74291.71632.09788E-054.27557E-050.4907−1.0272
    下载: 导出CSV
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