Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry Analysis of Dynamic Changes in Non-volatile Compounds in Green Tea during Storage at Ambient Temperature

ZHOU 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

Tea,as beverage popular worldwide,includes many types of products obtained from the leaves ofCamellia sinensisby different manufacturing processes[1].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 fullyor 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 compounds[5-7].

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 samples[2].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 time[8-10].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 infusion[11-12].For example,flavonoids and flavonol glycosides play important roles in the astringent taste and the color of tea infusions[13-15].Lipids that contribute to tea color and aroma quality undergo degradation during the green tea production[16]and also show significant changes during tea storage[17].It was reported that procyanidins,alkaloids,flavonol/flavone glycosides,amino acids,organic acids,lipids,and carbohydrates endured significant changes during storage of tea[18].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 quality[11,19].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 Methods

1.1 Raw material and reagents

Fresh leaves (one bud and two leaves) ofCamellia sinensisvar.sinensiscv.“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 10-15 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).

1.2 Instruments and equipment

High 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);C18column (4.6 mm × 250 mm,5 μm;Phenomenex,Tor-rance,CA,USA);LCA K07/Li column(4.6 mm × 150 mm,SYKAM,Munich,Germany);C18column(2.1 mm × 100 mm,1.8 μm;Waters,Milford,MA);C18column(2.1 mm × 150 mm,1.7 μm;Waters,Milford,MA).

1.3 Methods

1.3.1 Sensory evaluation

The 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 HPLC

Catechins and caffeine in tea samples were quantified according to the previous study[20].HPLC with a C18column(4.6 mm × 250 mm,5 μm) 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 analysis

An 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 extraction

To 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 12000 r/min for 10 min and the supernatant was filtered (SCAA-104,0.22 μm,ANPEL,Shanghai,China)before ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis.

1.3.5 UPLC-MS/MS analysis

An UPLC system connected to an MS/MS system was used for analyzing non-volatiles.Two microliters of sample were injected in a C18column (2.1 mm × 100 mm,1.8 μm),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 5500 V;curtain gas 25 psi;m/z50-1000.

1.3.6 Data pre-processing and metabolites identification

Data 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 inhouse 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 them/zvalues,RT and fragmentation patterns with the standards.

1.3.7 Identification of differential metabolites

The orthogonal partial least-squares discrimination analysis model (OPLS-DA) and thet-test were used to screen differential metabolites.Compounds with thePvalue oft-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/MS

To 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 12000 r/min at 4 ℃ for 15 min.The supernatants were dried under a gentle nitrogen flow.The pellets were diluted with 600 μL of 50% (V/V) methanol containing 0.1% formic acid.After 30 s of vortexing and 15 min of ultrasonication,the samples were centrifuged again at 12000 r/min at 4 ℃ for 15 min.The supernatants were filtered through a 0.22 μm 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 C18column (2.1 mm × 150 mm,1.7 μm) 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 +5000/-4500 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/MS

To further analyze the changes in lipids during green tea storage,20 mg of powder of each sample was homogenized in 800 μL water for 60 s.The 100 μL of homogenate was mixed with 190 μL of distilled water and 480 μL 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 3000 r/min at 4 ℃ for 15 min.The supernatant (250 μL) and 250 μL MTBE were mixed.After 60 s of vortexing and 10 min of ultrasonication,the solution was centrifuged at 3000 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 μL of buffer (DCM:methanol:water,60:30:4.5 (V/V)).The solution was centrifuged at 12000 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 C18column (2.1 mm × 100 mm,1.8 μm),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+5500/-4500 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 them/zvalues,RT,and fragmentation patterns using the internal and public databases.

1.4 Statistical analysis

Principal 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 Analysis

2.1 The change in green tea quality during storage

The sensory results showed that the appearance and flavor of green tea samples after 40 and 80 days of storage at 37 ℃ were significantly different from those stored at-80 ℃ (Fig.1).Dry tea became yellow after 40-day storage and darker yellow after 80 days of storage (Fig.1a).The color of tea infusion after storage was also yellow,darkening with prolonged storage (Fig.1b).The astringency,bitterness and taste intensity of green tea infusions after 40 and 80 days of storage weakened compared to the control (Table 1).In the early storage (40 days),the tea infusions remained green and had fresh taste,but these flavor properties were lost after 80 days of storage at 37 ℃.Hence,the results indicated the metabolites involved in the color and taste of tea products changed with duration of storage.

Table 1 Sensory evaluation of green tea infusion after storage

Fig.1 Color changes in green tea (a) and tea infusion (b) after storage

2.2 Changes in catechins and amino acids during storage

Catechins and amino acids were investigated as the major components in tea products (Table 2).After 40 days of storage at 37 ℃,there was little fluctuation in the concentrations of catechins,most of which decreased with storage,except for GA,GC and EC.With increasing storage time,EGCG,EGC,GCG,ECG,and CG gradually decreased,especially galloyl catechins EGCG and ECG whose concentrations changed from (48.81 ± 6.23) to(45.58 ± 2.13) mg/g and from (10.93 ± 1.50) to (5.60 ± 0.69) mg/g,respectively (Table 2).A previous study showed that ECG was more susceptible to degradation than EGCG during tea storage for 19 days.It was evident that the amount of GA significantly increased after 80-day storage,from(0.59 ± 0.05) to (0.71 ± 0.01) mg/g (Table 2),consistent with a previous study that showed that catechins could be degraded to GA[23].

Table 2 Contents of major taste compounds in green tea with different storage times

A total of 20 amino acids were identified and quantified(Table 2).There was no doubt that theanine,the most abundant amino acid in tea,sharply decreased after 80-day storage at 37 ℃.Of the detected amino acids,glutamine(Glu),theanine (Thea),glycine (Gly),alanine (Ala),cysteine(Cys),tyrosine (Tyr),alanine (Ala),γ-aminobutyric acid(GABA),and ornithine (Orn) decreased significantly(P＜ 0.05) with storage,whereas the concentration of arginine(Arg) increased during the storage.Previous studies showed that the concentrations of amino acids decreased during short storage but increased with prolonged storage,which was attributed to the degradation of soluble proteins[23-25].It was reported that the total concentration of free amino acids in green teas was reduced by 40% after 150-day of storage at room temperature[11].In the present study,the amounts of free amino acids decreased by approximately 17% after 80-day of storage.Thea,Glu and aspartic acid (Asp) with umami traits influence the taste strongly[26],and other amino acids,such as Tyr,Gly and Ala,give bitter and sweet tastes.Thus,a decrease in concentration of these amino acids was an important factor in the deterioration of green tea products during prolonged storage.

PCA of catechins clearly showed that the control was grouped with the 40-day tea samples,whereas the 80-day tea samples were far away (Fig.2a).The PCA of amino acids indicated that the 40-day and 80-day tea samples were grouped together and far away from the control (Fig.2b).It was suggested that catechins had a slower transformation than amino acids during storage,perhaps attributed to the different structures of these two types of compounds.The abundance of metabolites was set as the variable in the PCA,which separated green tea samples into three groups (Figs.2c,d).

Fig.2 PCA plots of non-volatile compounds in tea samples stored under different conditions

2.3 The profiling of non-volatile metabolites

Tea tastes,including astringency,bitterness,umami,and sweetness,rely on the contributions of complex non-volatile compounds[27],such as flavonoids,their glycosides and organic acids.Hence,to characterize further the changes in non-volatile compounds during storage,UPLC-QTRAP-MS was used to obtain the metabolite profiling.After data filtering,peak detecting,peak alignment,684 compounds were identified with high confidence by searching databases.Organic acids and their derivatives were the most abundant classes of compounds in tea samples,accounting for 15.79%of the total,followed by amino acids and their derivatives(11.40%),flavone (10.09%),and phenylpropanoids (8.63%).

Variable importance in the projection (VIP) score of the OPLS model was applied to rank the metabolites among groups,and metabolites with aPvalue ＜ 0.05 and VIP ≥ 1 were considered differential metabolites.A total of 77 differential compounds were identified (Fig.3).Polyphenols and lipids were the main differential metabolites(Fig.3).Polyphenols (flavones,flavanol,flavanone,etc.)are an important class of anti-oxidative compounds in tea that are beneficial to our health.Catechins,as the major polyphenols in tea,are flavanols,that are easily oxidized and dehydrogenated,resulting in the formation of quinones.Coniferin and esculin,as phenolic glycosides,are excellent natural antioxidants,influenced by temperature,oxygen,and other factors during storage[28-29].Hence,a decrease in concentration of these compounds with prolonged storage might result in the weakened taste of tea infusions.

Fig.3 Differential metabolites in green tea samples compared with the control

It was evident that dry tea and tea infusion of green tea samples after storage were yellowed and darkened.Theaflavins are an important class of compounds influencing the color and taste of black tea products.In the present study,with prolonged storage,theaflavin-3-gallate increased significantly (Fig.3).It was suggested that the accumulation of these compounds contributed to the changes in the color and taste of green tea.

Lipids were reported to undergo peroxidation and lipolysis during storage;unsaturated fatty acids first transform to nonvolatile hydroperoxides as primary oxides and then decompose to volatile compounds[30].It was proven that a lower storage temperature enhanced the stability of lipids,for example,when stored at 20 ℃,lipids remained constant but were altered significantly under 30 and 40 ℃ storage conditions[31].Of the differential lipids in green tea samples,mono-acylglycerol decreased with storage (Fig.3).In plants,lipoxygenase and hydroperoxide lyase are responsible for the oxidation and conversion of polyunsaturated fatty acids such as C18:2and C18:3fatty acids into short-chain volatiles[32],and these volatiles are unstable during storage.It was reported that C18:1and C18:2fatty acids were easily oxidized and decomposed,leading to off-flavor development in rice with storage[33].In the present study,sensory evaluation showed that a strong oily flavor was found in the green tea samples after 80-days of storage at 37 ℃,suggesting that decomposition of lipids was responsible for this change.Interestingly,octadeca-dien-6-ynoic acid significantly decreased with storage,reduced by 10%after 40-day storage and almost disappeared after 80-day storage (Fig.3).Octadecadien-12-ynoic acid was proven to enhance kokumi activity[34].We would suggest that decomposition of octadecadien-6-ynoic acid had a negative influence on the taste of green tea.

Organic acids and derivatives were important classes of compounds in tea leaves.Among the differential metabolites,most organic acids significantly increased,especially azelaic acid,gallic acid and dihydroxybenzoic acids (Fig.3).It was considered that these compounds were mainly from the degradation and transformation of catechins and other polyphenols[23,35].A study on tea presented that approximately 31 compounds might be derived from decomposition of catechins after post-fermented procedure,first into nongalloyl catechins and GA,and then forming some simple phenols such as 2,5-dihydroxybenzoic acid[36].In the present study,2,5-dihydroxybenzoic acid,2,3-dihydroxybenzoic acid,and 2,4-dihydroxybenzoic acid gradually increased with storage,approximately 1.5-2-fold (Fig.3),likely due to the C-ring degradation of galloyl catechins such as EGCG,ECG,GCG and CG or other polyphenols[36].Thep-coumaroylquinic acid has effects on inflammation,hypertension,cardiovascular disease,and arteriosclerosis[37]and gives a strong astringent taste to tea infusions[27],which is diminished with storage.It was reported that the degradation of this type of compounds was attributed to the change in pH value[38].We thus assessed the relative pH value of the tea infusions (1.00 g in 50 mL of water),and the result showed that the pH value decreased with storage.The pH value was 6.12 ± 0.03 in the control sample,which decreased to 6.04 ± 0.02 after 40-day storage at 37 ℃,and to 5.92 ± 0.02 after 80-day storage.It was suggested that increased concentrations of organic acids exacerbated the decrease in the pH value,which might have a negative influence on this type of compounds.

2.4 The changes in flavonoids during tea storage

According to the above analyses,the most differential metabolites during the storage of green tea were polyphenols.Among polyphenols,flavonoids have an important influence on the color,bitterness and astringent taste of tea infusions[13,27].Hence,comprehensive profiling of flavonoids was performed using a targeted metabolomics approach.A total of 49 flavonoids were identified,including 21 flavonoid glycosides and 28 flavonoids.

Of the 21 flavonoid glycosides,most showed an increasing trend during storage,especially glycitin,vitexin rhamnoside,vitexin,isorhamnetin 3-O-nehesperidine,schaftoside and apigenin-7-glucoside,which increased during early storage.A recent study showed that quercetin 3-glucuronide and quercetin 3-O-rutinoside had a positive correlation with tea storage time,while quercetin had a negative correlation with storage time[39].In our study,the abundance of quercitrin (quercetin 3-O-rutinoside) did not change after 15-days of storage but increased 1.15-fold after 40-day storage compared with the control (Fig.4).Quercetin glycosides,myricetin glycosides and kaempferol glycosides were more abundant than other flavonoid glycosides in tea[14].In the present study,rutin increased with storage,which might have given a strong bitter taste to the tea infusion[15].The flavonoid glycosides were associated with the mouthdrying and velvety-like astringency taste,suggesting that increasing flavonoid glycosides had a negative influence on the taste of green tea infusion.In addition,flavonoids are closely associated with color of tea infusions[39].Compared to the control (-80 ℃),high temperature (37 ℃) accelerated the changes of flavonoid and its glycosides.It is suggested that relative low temperature benefit to maintain the quality of green tea during storage.Recent study showed that condition of storage with 25 ℃,55% humidity and 4.5%moisture content of dry tea was beneficial to shelf quality of green tea[40].

Fig.4 Changes in contents of flavonoids and flavonoid glycosides during the storage of green tea

2.5 The changes in lipids during tea storage

Lipids are an important class of differential metabolites during tea storage.Targeted metabolomics was used to identify the lipid profile.A total of 216 lipids were identified and quantified based on the internal standards(each type of lipid had an internal standard),including 111 triacylglycerols (TAGs),31 free fatty acid (FFAs),24 phosphatidylcholines (PCs),24 phosphatidylethanolamines(PEs),13 diacylglycerols (DAGs),4 ceramides,6 l y s o-phosphatidylcholines (LysoPCs),and 3 lyso-phosphatidylethanolamines (LysoPEs).Among them,33 differential lipids were identified based on VIP value ＞1,including 11 TAGs,7 FFAs,9 PCs,4 PEs,and 2 DAGs (Fig.5).

Fig.5 Differential lipids in green tea samples stored at 37℃versus the control

A previous study showed that lipid derivatives increased during storage due to the autooxidation of lipids[17].TAGs are the predominant lipids accumulating in some plants[41].In the present study,TAGs decreased after 15-day storage.However,several TAGs increased after 40-and 80-days of storage and decreased after longer storage,such as TAG 54:2 (FA 18:1),TAG 52:2 (FA 18:1),and TAG 52:3 (FA 18:2) (Fig.5).As non-aqueous compounds,the changes in TAGs might have a negative influence on the appearance of dye tea samples.Linoleic acid (C18:2),linolenic acid (C18:3)and linoleic acid (C18:1) increased with prolonged storage,especially FA 16:1 (2.28-fold increase after storage).Palmitic acid (C16:0) showed an increasing trend but decreased after 40-day storage.The PCs and PEs declined significantly after 40 days of storage.The DAGs decreased significantly with increasing storage time.

PCA of flavonoids showed that 120-day tea samples were far away from the other groups (Fig.6a).It was notable that flavonoids had a dramatic change between 80-and 120-day tea samples.The OPLS-DA was conducted to identify the key metabolites.Cianidanol had the highest VIP(4.54),followed by astragalin (3.11),gallocatechin (2.32),kaempferol-3-O-rutinoside (1.39),isoquercitrin (1.22),rutin(1.22),and hyperoside (1.22) (Fig.6b).In addition,a highly correlation was obtained between quercetin (0.894) and myricetin (0.929) with storage time (Figs.6c,d).These two compounds with astringent traits increased with storage,possibly having a negative influence on tea quality.

Fig.6 PCA plots of flavonoids and lipids in tea samples stored under different conditions and correlation analyses between contents of flavonoids and free fatty acids and storage time

The PCA on the lipids indicated that 15-day tea samples and the control were grouped together and were far away the groups including 40-,80-,and 120-day tea samples(Fig.6e).It was evident that lipids already had changed in the early storage.The orthogonal partial least squares discrimination analysis was conducted to identify the key lipids among15-and 40-day tea samples.PC (16:0/18:2) had the highest VIP (6.48),followed by PC (16:0/18:3) (6.47),PC (18:2/18:2) (5.44),palmitic acid (2.48),PE (16:0/18:2)(2.46),PE (18:2/18:2) (2.28),and PC (18:1/18:2) (2.17)(Fig.6f),strongly supporting that PCs and PEs are sensitive with tea storage.The correlations between FFA 24:1 and FFA 26:1 with storage time were 0.667 and 0.624 (P＜0.01)(Fig.6g,h).It was worth noting that free fatty acids had a dramatic change with storage and influenced tea quality.The autooxidation of lipids needs the participation of oxygen[17].To avoid the rapid variation of lipids during the storage of green tea,the packaging of green tea should be trying to isolate oxygen.Previous study showed that the packaging material with polyethylene terephthalate/aluminum/polythene was beneficial to keep quality of green tea[42].

3 Conclusions

We characterized the changes in active compounds in tea.Catechins,as natural antioxidants,inhibit the transformation of other compounds,but undergo oxidation and degradation to reduce taste quality.During the early storage,lipids such as unsaturated fatty acids,flavanones whose structures were unstable or polyhydroxy that were easily transformed into small molecules such as alcohols,contributed to the changes in the color and taste of dry tea and tea infusion.Increasing concentrations of organic acids decreased the pH value of dry tea.In summary,the storage of green tea samples should be done at the suitable conditions to minimize alterations of the original properties of the compounds with unstable structures.