基于UPLC-MS_MS方法分析绿茶非挥发性代谢物在常温贮藏中的动态变化.pdf

安全检测 食品科学 2023,Vol.44,No.16 301Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry Analysis of Dynamic Changes in Non-volatile Compounds in Green Tea during Storage at Ambient TemperatureZHOU Hanchen,WANG Hui,LIU Yaqin,LEI Pandeng*(Tea Research Institute,Anhui Academy of Agricultural Sciences,Huangshan 245000,China)Abstract:Herein,we used an ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)system to investigate changes in the profile of non-volatile metabolites in green tea after different periods(15,40,80 and 120 days)of storage at 37.The color of green tea and tea infusion changed after 40 days of storage at 37 when compared with the control(stored at 80).During 40 days of storage,the content of catechins did not significantly change,but the content of free amino acids significantly decreased.A total of 684 non-volatile components were identified,77 of which were found to be characteristic differential metabolites,the major ones being polyphenols,lipids and organic acids.The content of octadecadien-6-ynoic acid,an umami-related compound,significantly decreased after 40 days and was undetected after 80 days.The content of theaflavine-3-catechin,related to the color and taste of tea infusion,significantly increased with storage time.Similarly,the content of flavone glycoside increased with storage time.Moreover,the content of lipids such as triacylglycerols(TAGs)and diacylglycerol(DAGs)changed significantly after 15 days,and the content of free fatty acids increased with storage time.The findings from this study showed that the contents of lipids and flavonoids in green tea significantly changed during storage,which played an important role in the quality deterioration of green tea.Keywords:green tea;storage;non-volatile compounds;ultra-high performance liquid chromatography-tandem mass spectrometry;flavonoids;lipids基于UPLC-MS/MS方法分析绿茶非挥发性代谢物在常温贮藏中的动态变化周汉琛，王 辉，刘亚芹，雷攀登*（安徽省农业科学院茶叶研究所，安徽 黄山 245000）摘 要：为探究非挥发性代谢物在绿茶贮藏中的动态变化，本研究将贮藏温度设定为37 以处理绿茶样，后通过超高效液相色谱-三重四极杆离子阱串联质谱联用技术分析在不同贮藏时间条件下（15、40、80、120 d）非挥发性代谢物的变化过程。结果表明，以80 贮藏绿茶为对照，37 贮藏40 d后绿茶干茶、茶汤色泽开始转变。儿茶素类物质在37 贮藏40 d内，变化不显著，而游离氨基酸含量则显著降低。超高效液相色谱-串联质谱方法共鉴定出684 种化合物，其中77 种化合物在绿茶贮藏中有显著变化，为特征性差异化合物，且主要是多酚类、脂类及有机酸类物质。与鲜味相关的化合物十八二烯-6-炔酸在贮藏40 d后极显著降低；在贮藏80 d的绿茶中已经无法检测到该化合物。与茶汤色泽、滋味相关的茶黄素-3-儿茶素随着贮藏时间的延长而显著上升。类黄酮代谢分析显示，黄酮糖苷类物质在贮藏过程中呈现上升趋势。脂质代谢分析表明，脂类物质中的三酰基甘油类和二酰基甘油类物质在贮藏15 d即发生显著变化；游离脂肪酸类物质随着贮藏时间延长而上升。本研究结果表明黄酮类和脂质类化合物在绿茶贮藏进程中变化较为显著，对绿茶的品质下降起重要作用。关键词：绿茶；贮藏；非挥发性代谢物；超高效液相色谱-串联质谱联用技术；类黄酮代谢；脂质代谢DOI:10.7506/spkx1002-6630-20220914-126中图分类号：S571.1；O657.63 文献标志码：A 文章编号：1002-6630（2023）16-0301-11收稿日期：2022-09-14基金项目：国家自然科学基金青年科学基金项目（32002096）；安徽省农业科学院青年英才计划项目（QNYC-202119）第一作者简介：周汉琛（1989）（ORCID:0000-0002-9844-5029），女，博士，研究方向为茶叶生物化学及分子生物学。E-mail:T*通信作者简介：雷攀登（1983）（ORCID:0000-0002-5140-0675），男，副研究员，硕士，研究方向为茶叶加工与品质调控。E-mail: 302 2023,Vol.44,No.16 食品科学 安全检测引文格式：ZHOU Hanchen,WANG Hui,LIU Yaqin,et al.Ultra-high performance liquid chromatography-tandem mass spectrometry analysis of dynamic changes in non-volatile compounds in green tea during storage at ambient temperatureJ.食品科学,2023,44(16):301-311.DOI:10.7506/spkx1002-6630-20220914-126.http:/ZHOU Hanchen,WANG Hui,LIU Yaqin,et al.Ultra-high performance liquid chromatography-tandem mass spectrometry analysis of dynamic changes in non-volatile compounds in green tea during storage at ambient temperatureJ.Food Science,2023,44(16):301-311.DOI:10.7506/spkx1002-6630-20220914-126.http:/Tea,as beverage popular worldwide,includes many types of products obtained from the leaves of Camellia sinensis by different manufacturing processes1.Normally,teas can be classified into two families,namely non-fermented tea(e.g.,green tea)and fermented tea(e.g.,black tea,dark tea and oolong tea)2-4.Green tea products without the fermented stage during processing tend to maintain the original properties of metabolites,whereas fully-or post-fermented tea products undergo the fermentation stage at suitable humidity and temperature or in the presence of various microorganisms,in which the metabolites either have stable structures or transform into smaller compounds5-7.Green tea products contain amounts of original metabolites such as flavonoids,flavonoid glycosides,and free amino acids.Epigallocatechin gallate(EGCG)is the most abundant catechins in green teas and gives bitterness and astringency to tea infusion,with about 90%of the total EGCG in fresh leaves being transferred into green tea samples2.In addition,the compounds with high antioxidant potential are extracted from green tea products and applied to other foods,such as fruit and yogurt,to extend their storage time8-10.However,these compounds are sensitive to oxygen,moisture and temperature during prolonged storage,leading to quality deterioration and reduced marketability of green tea products.Non-volatile compounds,such as flavonoids,amino acids,carbohydrates,organic acids,nucleotides,and alkaloids,have an important influence on the taste and color of tea infusion11-12.For example,flavonoids and flavonol glycosides play important roles in the astringent taste and the color of tea infusions13-15.Lipids that contribute to tea color and aroma quality undergo degradation during the green tea production16 and also show significant changes during tea storage17.It was reported that procyanidins,alkaloids,flavonol/flavone glycosides,amino acids,organic acids,lipids,and carbohydrates endured significant changes during storage of tea18.However,the appropriate storage improves the quality of white tea or dark tea,and the tea products that undergo the post-fermented stage develop a mellow taste.It is well-known that aged green tea products are not good for selling.Many studies on the storage of green tea have focused mostly on the changes in catechins or amino acids or sensory quality11,19.However,the comprehensive metabolite profiles during the storage of green tea still need to be elucidated.In the present study,a widely targeted metabolomic technique with ultra-high performance liquid chromatography connected to an ESI-triple quadrupole linear ion trap mass spectrometry(UPLC-QTRAP-MS)system was applied.The non-volatile metabolite profiles developed during green tea storage were investigated.1 Materials and Methods1.1 Raw material and reagentsFresh leaves(one bud and two leaves)of Camellia sinensis var.sinensis cv.“Fuzao#2”were plucked in late April and then processed into green tea according to the traditional practice(i.e.spreading,fixation,rolling,and drying).Briefly,fresh leaves were spread indoor for 4-5 h,followed by pan-firing procedure(Shangyang company,Quzhou,China)at 350 (30 r/min)for 90 s.After cooled to room temperature,the tea shoots were rolled at 45 r/min for 10 min and further dried via three steps:110 for 1015 min,room temperature for 30 min,and 80 for 30 min.Tea samples were placed into the foil bag and immediately sealed using a capper.The tea samples stored at cold temperature(-80)were used as control,and others were stored at 37 for 15,40,80,and 120 days.After treatments,all tea samples were stored at -80 before further analysis.Standards including caffeine,gallic acid(GA),gallocatechin(GC),epigallocatechin(EGC),catechin,epicatechin(EC),EGCG,gallocatechin gallate(GCG),epicatechin gallate(ECG),catechin gallate(CG)were purchased from Sigma-Aldrich(Darmstadt,Germany).Standard of amino acids mixture was purchased from SYKAM company(Munich,Germany).Flavonoids and lipids used in this study were purchased from Sigma-Aldrich(Darmstadt,Germany).安全检测 食品科学 2023,Vol.44,No.16 3031.2 Instruments and equipmentHigh performance liquid chromatography(HPLC)(Waters,Massachusetts,USA);auto amino acid analyzer S-433D(SYKAM,Munich,Germany);UPLC(CBM30A,SHIMADZU,Japan);QTRAP-MS system(6500QTRAP,Applied Biosystems,USA);C18 column(4.6 mm 250 mm,5 m;Phenomenex,Tor-rance,CA,USA);LCA K07/Li column (4.6 mm 150 mm,SYKAM,Munich,Germany);C18 column(2.1 mm 100 mm,1.8 m;Waters,Milford,MA);C18 column(2.1 mm 150 mm,1.7 m;Waters,Milford,MA).1.3 Methods1.3.1 Sensory evaluationThe sensory evaluation was conducted according to the national criterion(GB/T 23776-2018).A 3 g green tea sample was placed in a teapot and infused with 150 mL boiling water.The tea infusion was poured out after brewing for 4 min.Six skilled experts were employed to assess the taste and color of tea infusions.1.3.2 Catechins analysis by HPLCCatechins and caffeine in tea samples were quantified according to the previous study20.HPLC with a C18 column(4.6 mm 250 mm,5 m)was used.Identification and quantitation of catechins and caffeine were performed via comparison with the authentic standards and the calibration curves;data were ex-pressed as mg/g(dry mass).Three biological replicates were measured.1.3.3 Free amino acids analysisAn automatic amino acid analyzer S-433D coupled to an LCA K07/Li column was used to detect amino acids in tea samples.The rate of elution flow was maintained at 0.45 mL/min.The identification of amino acids was conducted via comparison with authentic standards;data were expressed as mg/g(dry mass).Three biological replicates were measured.1.3.4 Sample preparation and extractionTo investigate the key components of non-volatiles during green tea storage,three tea samples(the control,40-day and 80-day storage at 37)were crushed using a mixer mill(MM 400,Retsch),and 100 mg of powder was extracted at 4 with 1.0 mL 70%(V/V)aqueous methanol.The internal standard of 0.1 mg/L lidocaine was added to the samples as the internal standard.Then,the extract was centrifuged at 12 000 r/min for 10 min and the supernatant was filtered(SCAA-104,0.22 m,ANPEL,Shanghai,China)before ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)analysis.1.3.5 UPLC-MS/MS analysisAn UPLC system connected to an MS/MS system was used for analyzing non-volatiles.Two microliters of sample were injected in a C18 column(2.1 mm 100 mm,1.8 m),and the flow rate was kept at 0.4 mL/min.Solvents A(water with 0.04%(V/V)acetic acid)and B(acetonitrile with 0.04%(V/V)acetic acid)were run in a linear gradient with B increasing from 5%to 95%within 11 min,then maintained for 1 min,and decreasing from 95%to 5%within 3 min.The UPLC-MS/MS was operated in the positive and negative ion modes,and the set-up was:ESI source temperature 500;ion spray voltage 5 500 V;curtain gas 25 psi;m/z 50-1 000.1.3.6 Data pre-processing and metabolites identificationData filtering,peak detection,alignment,and calculations were performed by Analyst(1.6.1)software.To eliminate the biased and redundant data,peaks were checked manually for signal/noise(RSN)10,and in-house software written in Perl was used to remove redundant signals.The area of each chromatographic peak was calculated based on the spectral pattern and retention time(RT).Metabolites were identified by searching the internal and public databases(MassBank,KNApSAcK,HMDB,MoTo DB,and METLIN)21-22 and comparing the m/z values,RT and fragmentation patterns with the standards.1.3.7 Identification of differential metabolitesThe orthogonal partial least-squares discrimination analysis model(OPLS-DA)and the t-test were used to screen differential metabolites.Compounds with the P value of t-test less than 0.05 and the variable importance for the projection(VIP)value above 1 was considered as the differential metabolites among the treatments.1.3.8 Flavonoids extraction and analysis by UPLC-MS/MSTo further analyze the changes in flavonoids during green tea storage,100 mg of powder of five tea samples including the control and four tea samples stored at 37 for 15,40,80,and 120 days was extracted with 3 mL of 75%(V/V)methanol containing 1%(V/V)acetic acid.The samples were vortexed for 30 s,sonicated for 30 min in an ice-water bath and then centrifuged at 12 000 r/min at 4 for 15 min.The supernatants were dried under a gentle nitrogen flow.The pellets were diluted with 600 L of 50%(V/V)methanol containing 0.1%formic acid.After 30 s of vortexing and 304 2023,Vol.44,No.16 食品科学 安全检测15 min of ultrasonication,the samples were centrifuged again at 12 000 r/min at 4 for 15 min.The supernatants were filtered through a 0.22 m filter for UPLC-MS/MS analysis.Six replicates were measured.The UPLC-MS/MS system was the same as specified above.Two microliters of sample were injected in a C18 column(2.1 mm 150 mm,1.7 m)and the flow rate was set at 0.3 mL/min with solvents A(water with 0.1%(V/V)acetic acid)and B(acetonitrile with 0.1%(V/V)acetic acid).The elution gradient of B was maintained at 10%for 0.5 min,increased to 60%at 15 min,to 98%at 16 min and maintained at 98%for 2 min,to 10%at 20 min.Mass acquisition was operated in the positive and negative ionization modes.The parameters were set as follows:ion spray voltage+5 000/-4 500 V,curtain gas 35 psi,temperature 500,ion source gas 1 at 55 psi,and ion source gas 2 at 60 psi.The identification of flavonoids was based on their authentic standards.1.3.9 Lipid extraction and analysis by UPLC-MS/MSTo further analyze the changes in lipids during green tea storage,20 mg of powder of each sample was homogenized in 800 L water for 60 s.The 100 L of homogenate was mixed with 190 L of distilled water and 480 L of extractant(MTBE:methanol,5:1(V/V)containing the internal standard.After 60 s of vortexing and 10 min of ultrasonication,the solution was centrifuged at 3 000 r/min at 4 for 15 min.The supernatant(250 L)and 250 L MTBE were mixed.After 60 s of vortexing and 10 min of ultrasonication,the solution was centrifuged at 3 000 r/min at 4 for 15 min.This step was repeated twice.The triple supernatants were combined and dried in a vacuum concentrator at 37.The dried samples were diluted with 200 L of buffer(DCM:methanol:water,60:30:4.5(V/V).The solution was centrifuged at 12 000 r/min at 4 for 15 min,and the supernatant was used for UPLC-MS/MS analysis.Six replicates were measured.The UPLC-MS/MS system was the same as specified above.Two microliters of the solution was injected in a C18 column(2.1 mm 100 mm,1.8 m),and the flow rate was kept at 0.3 mL/min with solvents A(water:acetonitrile,40:60(V/V)and B(acetonitrile:isopropanol,10:90(V/V)with 10 mmol/L ammonium formate).The elution gradient of B was maintained at 20%for 1 min,increased to 60%at 4 min,to 98%at 15 min and maintained at 98%for 1 min,to 20%at 20 min.Mass acquisition was done in the positive and negative ionization modes.The parameters were set as follows:ion spray voltage +5 500/-4 500 V,curtain gas 40 psi,temperature 350,ion source gas 1 at 50 psi,ion source gas 2 at 50 psi,and DP 80 V.The identification of lipids was conducted according to the m/z values,RT,and fragmentation patterns using the internal and public databases.1.4 Statistical analysisPrincipal component analysis(PCA)and OPLS-DA were conducted using SIMCA 13.0 software.The significant differences among the treatments were determined by the least significant differences(LSD)test using the SPSS 19.0 software package.2 Results and Analysis2.1 The