Keywords:

Instant black tea

Liquid-state fermentation

Enzymatic-enhanced extraction

Quality characteristics

A B S T R A C T

Repeated thermal treatment could impair the flavor of instant black tea (IBT) as the volatile compounds and characteristic constituents dramatically vary during the manufacturing process.Utilization of fresh leaves as raw material in IBT manufacture at low temperature may become an alternative to conventional extraction methods.This study aimed to improve the quality of IBT using fresh tea leaves by a two-stage enzymatic processing.The influence of combined enzymatic oxidation and extraction conditions for maximal extractable flavour (taste and aroma) characteristics from fresh tea leaves were investigated.The changes of aroma profiles and chemical constituents including theaflavins (TFs), thearubigins (TRs), theabrownines (TBs)and TRs/TFs ratio in converted tea brews under two-stage enzyme catalyzed fermentation and extraction conditions were further compared between different treatments, based on which the optimised conditions were determined.During the two-stage enzymatic processing, 75 kinds of volatile compounds were identified in all processed samples, and the contents of TFs, TRs, TBs and TRs/TFs ratio could be optimized by adjusting different procedures and parameters.The overall properties offinal product were compared with two types of commercial IBT.High quality IBT with desired properties and antioxidant activity can be manufactured using fresh tea leaves by two-stage enzymatic processing approach.

1.Introduction

Tea (Camellia sinensis), the second most popular type beverage after water, has been demonstrated to have health-promoting properties associated with the presence of bioactive components such as polyphenols, pigments and theasinensins, which may help preventing a wide variety of diseases [1-3].And about 78% of the worldwide tea production is allotted to black tea, due to its unique quality (taste and aroma) [4,5].The major quality attributes of black tea are flavor, aroma, color, and strength.Out of these,flavor and aroma which is almost solely responsible for the quality of black tea particularly depend on the so-called “fermentation”(oxidation) process.During the process polyphenols are oxidized by the polyphenol oxidase (PPO) and peroxidase (POD), resulting in the formation of two major groups of pigments, the orangered theaflavins (TFs) and the reddish-brown thearubigins (TRs),which determine the taste and color of the black tea infusions [6,7].Meanwhile, the aromatic precursors are converted into characteristic aromatic volatiles of black tea due to enzymatic oxidation [8-10].To date, several hundreds of volatiles have been identified in black tea.With the evolution of commercial tea products, instant black tea(IBT) is currently becoming popular due to its ready to have and the convenience to be mixed in ready-to-drink (RTD) tea, which is also applied widely in food industry as the additives.Conventional IBT is manufactured through thermal extraction of the brew using processed leaves, low grade black tea [11], and then subjected to concentration and drying to get instant tea powder [12-14].

However, hot water extraction could impair the quality of IBT by a substantial loss of tea aroma and chemical constituents, which causes undesirable alteration in the characteristic flavor of black tea such as low aroma, poor taste, and dull color [15].It is desired to produce IBT with equivalent flavor characteristics of black tea at lower temperature.Comparatively, innovative techniques such as various enzymes-aided extraction methods were applied alternatively to improve the quality of IBT during the manufacturing procedures [16,17].Recently, enzymes, such asβ-glucosidase,β-xylosidase and tannase have been employed mainly to improve the flavor and tasty quality of green tea brew or RTD beverages [18,19].In addition, several cellwall-digesting enzymes (such as pectinase, cellulase) were utilized to maximize extract yield of tea soluble solids [20,21].However,scientific reports on how to prepare IBT using fresh tea leaves by endogenous and exogenous simultaneously are rather scarce.

Though procedures have been established for IBT manufacture by using fresh tea leaves via liquid-state fermentation (LSF) approach in our previous study [22], it is essential to investigate the synergistic effects endogenous and exogenous enzymes on the transformation of aroma and chemicals related to overall quality of IBT.In comparison to the traditional fermentation, the reaction between substrate and enzyme in LSF system proved to be more efficient and more environment-friendly [22].Furthermore, attempts were made to optimize the process parameters involved for maximizing the flavor and tasty quality during both the LSF and enzymatic-enhanced extraction (EAE) stages.It is expected that the outcome of this study may provide a desirable technology for the preparation of high quality IBT, by which the autumn fresh tea leaves could be converted into value added products.

2.Materials and methods

2.1 Material and chemicals

Shoots with three leaves and a bud from tea plant (C.sinensiscv.Longjing 43 varieties) was plucked from Hangzhou Tea Experimental Plantation in September.Fresh tea leaves were withered for 12 h in an air-conditioned room (25 °C), and then stored at -20 °C for the subsequent use.

Market brand IBTs were provided by China National Center of Quality Supervision and Inspection of Tea.Enzymes were provided by Novozymes (Wuxi, China), and all other chemicals purchased were of analytical grade (East China pharmaceutical Co., Ltd.,Hangzhou, China).

2.2 Sample preparation

Fresh tea leaves were withered for 18 h in an air-conditioned room (25 °C, 65% RH), and then stored at -20 °C for future use.Subsequently, the frozen tea leaves were then treated with LSF and EAE systems.The schematic of different sample preparation is presented in Fig.1.

Fig.1 Schematic for sample preparation.

2.3 Liquid-state fermentation systems

Experiments were performed to investigate the influence of LSF conditions including fermentation temperature, time, pH and endogenous enzyme on the quality of fermentation infusion.Sequential steps involved in the process are given below.

The 100 g frozen tea leaves were divided into two parts, being used as resource of oxidation enzyme and substrate in subsequent experiments, respectively.Heating fixing (hf) specifically refers to heating in microwave oven at power P100 for 1.5 min in this study,by which part of the oxidase of fresh leaves was inactive to regulate the oxidation of tea polyphenols in fresh leaves, and promote the formation of aroma [23].And then the hf leaves were blended with the rest of unheated frozen tea leaves, following with being crushed by homogenizer with 300 mL citrate phosphate buffer (0.1 mol/L,pH 4.8).The ratio of enzyme/substrate was 1:3, 1:1, 3:1 (m/m),corresponding to hf 25%, 50% and 75% tea leaves, respectively.

All the homogenate was subjected to fermentation in a fermenter with 1 L/min aeration flow at 380 r/min.Concretely, the reaction was carried out at different temperature (33, 35 and 37 °C), time(45, 55 and 65 min), and pH (4.2, 4.8 and 5.6) to obtain optimized fermentation conditions.Once fermentation was complete, the infusion was heated to 95 °C for 5 min and centrifuged (5 500 r/min,10 min）before subsequent analysis (Table 1).

Table 1LSF and EAE treatment conditions of different samples.

2.4 Enzymatic-enhanced extraction systems

The post-transformation of fermented infusion was another key process to obtain a superior quality IBT.Similar to the procedures adopted in LSF, 100 g frozen tea leaves were crushed to homogenate with tea leaves/buffer ratio of 1/3 (g/mL,m/V).In addition, all the homogenate were subjected to fermentation in a reaction kettle with 1 L/min ventilation flow rate at 380 r/min stirring, and fermentation at 35 °C for 65 min.Subsequently, EAE systems were carried out at different temperature (45, 50 and 55 °C), time (50, 60 and 75 min)scales and pectinase/cellulose (0, 0.25%, 0.5%, 1.0%) in a 500 mL tank.Furthermore, the effect of adding pectinase (1.0%) and cellulose(1.0%) prior or posterior to fermentation was also investigated.The extraction infusion was heated 95 °C for 5 min and centrifuged(5 500 r/min, 10 min）before subsequent analysis .Afterward, the clear extraction infusion was concentrated to 30% of its initial volume by a rotary vacuum evaporator (RV 3 V-C rotary evaporator, IKA,Germany) while maintaining the vacuum pressure at 0.1 MPa and the exhaust temperature at 60 °C respectively.Finally, the concentrate was freeze-dried to IBT powder (with about 5.0 g of moisture per 100 g)using freeze dryer (ALPHA 1-2 LD plus freeze dryer, Marin Christ Co., Ltd.Germany), which was programmed at 0.01 MPa, -40 °C for 36 h (Table 1).

2.5 Determination of TFs, TRs and theabrownines (TBs)

TFs, TRs and TBs in the fermentation infusion or different IBT were analyzed by Roberts method as previously reported with slight modifications [24].Absorbance of test solutions was measured at 380 nm by a spectrophotometer (PerseeTU-1901, China).The concrete assay was calculated according to system approach [25].

2.6 Catechins, tea polyphenols and amino acid analysis of IBT by high-performance liquid chromatography (HPLC)

The different IBT samples were analyzed for tea polyphenols and catechins according to the method described in International Standard“Determination of substances characteristic of green and black tea-Part 1 Content of total polyphenols in tea - Colorimetric method using Folin-Ciocalteu reagent” and “Determination of substances characteristic of green and black tea - Part 2 Content of catechins in green tea - Method using high-performance liquid chromatography”(ISO14502—1:2005; ISO 14502—2:2005) [26,27]with appropriate modification, respectively.In detail, powdered sample (200 mg) was extracted twice with 70% MeOH (5 mL) at 70 °C for 10 min.The extracts were combined and diluted to 10 mL with the extraction solvent.The extract was used to analyze the contents of total polyphenols and free amino acids [28].

The tea polyphenols were determined using Folin-Ciocalteu reagent with gallic acid as the standard [29].Before reaction, the extract was diluted 125-fold with distilled H2O.Diluted extract or standard gallice acid solution (2 mL) was decanted into a 10 mL centrifuge tube.Then, 10% Folin-Ciocalteu reagent (5 mL) was added to the tube and the mixture was shaken.After 5 min, 7.5%Na2CO3solution (4 mL) was added to the mixture.After incubating for 60 min at room temperature, the absorbance against the prepared reagent blank (distilled deionized H2O) was measured at 765 nm(ISO 14502—1:2005/COR 1:2006) [30,31].

For determining the free amino acids, the sample was prepared the same way as for tea polyphenols measurement (ISO 19563—2017) [32].In detail, the extract was diluted 10-fold with distilled H2O.Diluted extract or distilled H2O (1 mL) was decanted into a 25 mL graduated tube.Then, 2.5% ninhydrin solution (0.5 mL) and pH = 8.0 phosphate buffer (0.5 mL) was added to the tube and the mixture was shaken.The mixture was heated in a boiling water bath for 15 min, which was cooled and diluted to 25 mL with distilled water and incubated for 20 min at room temperature.The absorbance (A)was measured by a spectrophotometer (UV-2102PC UNICO(Shanghai) Instruments Co., Ltd.) at a wavelength of 570 nm.

The content of catechins was determined by Waters 1525 HPLC(1525 pump, 2487 UV-visible dual-channel detector, C18reverse phase 250 mm × 4.6 mm, 5 μm column) according to the method of International Standard (ISO 14502—2:2005) [27,33].1 mL of the extract was taken to a volume of 10 mL with 70% methanol, and filtered through a 0.45 μm membrane, and the injection amount was 10 μg.The mobile phase A consisted of 90 mL acetonitrile, 20 mL acetic acid and 2 mL EDTA was added to a 1 000 mL volumetric flask, and diluted with distilled water to volume, shaken and passed through a 0.45 μm membrane filter.The mobile phase B consisted of 800 mL acetonitrile, 20 mL acetic acid, and 2 mL EDTA were respectively added to a 1 000 mL volumetric flask, and diluted with distilled water to volume, shaken and passed through membranefilters (HAWP; 0.45 μm pore size).The chromatographic conditions were set as follow, mobile phase flow rate of 1 mL/min, column temperature 35 °C, and UV detectorλ= 278 nm.In detail, the mobile phases were mixed from 100% mobile phase A maintained for 10 min,then converted to 68% phase A and 32% mobile phase B in 15 min and held for 10 min, finally converted to 100% phase A [34,35].

2.7 Gas chromatography-mass spectrometer (GC-MS)analysis

The aroma components were isolated by simultaneous distillation extraction (SDE) existed [36]and analyzed by 7890A GC system(Agilent Technologies, CA, USA) [37,38].Tea infusion was prepared by adding 20 g of powder sample to 300 g boiling distilled water in a 500 mL round bottom flask, which was heated and kept boiling.Then,50 mL of dichloromethane was added and the mixture was re fluxed for 2 h in a 60 °C water bath.Finally, the mixture was concentrated by rotary evaporation in a water bath at 35 °C to 1.5 mL for GC-MS analysis [39].

Furthermore, the system combined with 5975C MSD (Agilent Technologies) was used for GC-MS analysis [40,41].The chromatographic column was a DB-5MS column (30 m × 0.25 mm ×0.25 μm film thickness), with high-purity helium as the gas carrier, at a flow rate of 1.2 mL/min.The injector temperature was 250 °C and the column temperature was set initially to 40 °C (5 min), increased to 160 °C at 5 °C/min, and then to 250 °C at 10 °C/min, finally to 280 °C (20 min).The injection mode was at the split ratio of 5:1.The MS ion source temperature was 230 °C and electron energy 70 eV.The scan range and the solvent delay time were 20-300 Da and 2 min,respectively [42].

2.8 Volatiles identification and relative amounts

The volatile compounds were identified by comparing their GC retention, and GC/MS spectra with those of the reference substances,based on the spectral library search (NIST08.L)[43].The relative content of each volatile was determined as follows.

2.9 Sensory evaluation

Color (i), aroma (ii), and taste (iii) were selected as quality attributes for sensory evaluation [44].The sensory evaluation was obtained by a panel composed of six tea experts according to procedure of GB/T 23776—2018 for evaluating tea leaves [30].In detail, 0.45 g of instant tea powder was infused with 80 mL of boiling water (100 °C), and the tea slurry was poured out after brewing for 5 min.Participants were carefully instructed about the test protocol before the evaluation began [45].

2.10 Anti-oxidative activity measurement

The anti-oxidative activity of the sample was measured according to the flow injection analysis method [46,47].Briefly, the reaction mixture which was diluted to 25 mL, contained 5 mL of FeSO4-EDTA solution (0.3 mol/L), 0.2 mL luminol solution (2.0 × 10-4mol/L) and 50 μL of the sample (1.0 mg/mL).The phosphate buffer (0.05 mol/L,pH 7.4) used as a carrier, and H2O2(8.4%,m/V) as mobile phase,luminescence intensity analyzed via flow injection luminescence analyzer (IFFM-E, Xi’an Ruimai Analytical Instrument Co., Ltd.).

WhereFarepresents the luminescence value of all the reaction reagents without the samples andFbis the luminescence value of the reaction mixture after adding tea infusion.

2.11 Statistical analysis

One-way analysis of variance (ANOVA) was conducted to determine the difference among various treatments over three replications.Duncan’s multiple-range test with 95% confidence limit(P＜ 0.05) was applied to determine the significance of difference by SPSS 9.0 software.All of the experiments were conducted in triplicate with data reported as the mean ± standard deviation.

3.Results and discussion

3.1 Effect of LSF process on TFs, TRs and TBs

The infusions produced at the stage of the fermentation (oxidation)were analyzed for various chemical constituents in Fig.2.It was found that differences in quantities of major groups of pigments TFs, TRs and TBs at different LSF conditions (Fig.2).Due to fermentation, the content of TFs, TRs and TBs increased significantly in all treated samples as compared with fresh leaves.TFs content under LSF conditions varied between 1.07% and 2.71%, TRs varied between 12.12% and 20.55%, TRs/TFs ratio varied between 7.08 and 13.54, and TBs varied between 6.29% and 19.76%.It has been reported that the TRs/TFs ratio should be within the range of 10 to 15 for an advantageous quality tea [48].Previous research has found the enzyme PPO is responsible for oxidizing the catechins to TFs and TRs, the tea pigments, which are responsible for the color and taste of black teas [49].At hf 75%, the content of TFs and TRs were the highest compared to other samples, but the TRs/TFs ratio(7.59) was not in the idea quality range.At hf 25%, the content of TRs was higher than that of hf 50%, TRs/TFs ratio (12.28) was in the acceptable range, thus hf 25% was facilitated for the high quality products.In addition, the contents of TFs (1.03%) and TRs (13.91%)decreased at 37 °C, which reached the highest value at 35 °C.Similar results were found that optimum temperature in CTC black tea production was in the range of 30-35 °C [50].The pH value also showed significant effects on tea pigments.The level of TRs at pH 4.8 was notably higher than those at pH 4.2 and pH 5.6.Despite the TRs/TFs ratio (10.85) dropping in the acceptable range at pH 5.6,the content of TFs and TRs were lower, while TBs value (19.76%)was maximum.Furthermore, the suitable duration of oxidation stages regarding to the formation of TFs and TRs could be favored at 65 min.To sum up, the optimized conditions in the LSF processing were 35oC, 65 min, pH 4.8 and hf 25%.

Fig.2 Effects of different LSF process parameters on the content of tea pigments and TRs/TFs.(a, Temperature; b, hf; c, pH; d, Time.)

3.2 Effect of EAE process on TFs, TRs and TBs

The effects of EAE process on TFs, TRs and TBs were obtained under the optimization condition for fermentation.TFs and TRs contents in the EAE process were relatively stable for the deactivation of the endogenous enzyme activities, which was slightly different in LSF stage (Fig.3).While, TBs decreased due to the further polymerization.The TFs content under EAE conditions varied between 0.64% and 2.25%, TRs varied between 9.67% and 16.09%,TBs varied between 5.63% and 7.89% and the TRs/TFs ratio varied between 6.75 and 15.02.

Fig.3 Effects of different EAE process parameters on the content of tea pigments and TFs/TRs ratio.(a, Temperature; b, Time; c, Added amount; d, Enzyme adding time points.)

The effect of temperature on TFs, TRs and TBs was studied and the optimized value was attained at 50oC.At 45oC/60 min, the content of TRs and TBs were 15.86% and 7.63%, and TFs (2.25%)was the highest, while the TRs/TFs ratio (7.05) was out of the favored range.The highest TRs (16.09%) were observed at 50oC/60 min,and TRs/TFs ratio (10.76) was in the acceptable range.However,TRs/TFs ratios were not acceptable with addition of 0, 0.25% and 0.50% pectinase or cellulose posterior to fermentation, until the amount of both exogenous enzymes increased to 1.0%.The TFs,TRs, and TFs/TRs ratio varied with the changes in extraction times.With the extraction time extending from 50 to 75 min, the infusions were observed a decline in the levels of TFs and TFs/TRs ratio.Furthermore, the addition of 1.0% pectinase and cellulose prior to the fermentation process, TFs value was found to be lowest compared with other groups, which was still higher than that of the control.This may be related to the broken of the cell wall during over fermentation,which needs further investigation.While the TRs/TFs ratio was higher than that in the case with addition of the enzymes posterior fermentation, which implied a degradation of TFs.

3.3 Identification of volatile compounds under LSF and EAE conditions

The influences of different LSF and EAE conditions on the aroma profiles were evaluated.The results of volatile constituents were summarized in Table S1.Totally 75 volatile compounds were identified by SDE/GC-MS analysis and classified by kinds of substance in all set of treatments, including 24 alcohols, 16 aldehydes, 7 ketones, 2 esters, 12 hydrocarbons, 1 acid, 2 phenolic compounds, 3 oxygen compounds, 7 nitrogenous compounds and 1 sulfur-compound.Aroma constituents by percentage were shown in Fig.4.As can be seen, aldehydes and esters were the major constituents increased in the treated samples compared with fresh tea leaves.By comparison, the content of some volatiles (particularly ketones, hydrocarbons and nitrogenous compounds) was observed with significant decrease during the two-step enzymatic processing.

Fig.4 Aroma compositions under different (a) LSF and (b) EAE conditions.

The contribution of volatiles to the overall aroma depends upon compounds quantitative abundance and odor thresholds [51].It was reported that methyl salicylate and benzothiazole in the volatile compounds contribute the peppermint and rubber type aromas to the final tea infusion [52].Heat map results of volatile alcohols compounds in the present study were shown in Fig.5.It was notable alcohols accounted for approximately 67% of the total aroma constituents, among which, linalool (31.39%) was detected as the highest level, followed by geraniol (8.81%), 3-hexen-1-ol(6.58%), 2-phenylethyl alcohol (4.34%) and linalool oxide (4.17%)under the LSF process.Similar results were observed in EAE process, linalool contributed the highest percentage (31.00%),followed by geraniol (8.07%), 3-hexen-1-ol (6.48%), 2-phenylethyl alcohol (5.35%) and linalool oxide (3.86%).It has been reported that alcohols were the most important compounds of aroma constituents in black teas [43].Linalool was responsible for providing a floral and sweet scent, and might affect the perception of astringent in black tea [17,53], especially in large leaf varieties, such as Assam tea.(Z)-3-hexen-1-ol contributes the grass type aromas to the final normal fragrance [54].It is interesting that the (Z)-3-hexen-1-ol content is very close and almost consistent in the two-stage enzymatic processing.While, 2-phenylethyl alcohol and geraniol’s aroma was defined as being honey-like or rose odor notes, which contributed to a significant improvement of product quality [55].On the other hand,during the EAE process of novel instant black tea (NIBT)，the liking of the floral aroma and geranium was positively associated with the intensity of the linalool and geraniol [52].Probably the formation of IBT aroma molecules was promoted by glycosidase hydrolysis during the LSF and EAE process [56].

Fig.5 Heat map result of relative amount percent of volatile alcohols compounds with different samples (blank area notated the results ‘‘not detected’’).

Heat map results of volatile aldehydes compounds were shown in Fig.6.It was found that the (E)-2-hexenal increased apparently up to 12.28% compared with fresh leaves (5.19%).In addition, there was a considerable increase in hexanal (0.96%) and (Z)-3-hexenal (0.60%).Furthermore, the level of 3-methylbutanal was almost doubled in the treated samples (0.47), along with a minor increase in benzaldehyde(0.69%).(E)-2-hexenal, hexanal, (Z)-3-hexenal, 3-methylbutanal and benzaldehyde were identified as the main aldehydes.These compounds have been reported to be responsible for a sweet and fruity flavor, green, grassy, malty, fragrant odor, respectively, which has also been recognized as important odorants contributing to the aroma of various teas [10].Furfural was not detected in the treated samples, and a relatively low proportion of phenyl acetaldehyde(0.93%) was found.During LSF process, the heptanal and nonanal found in samples were considered to be representatives of the active compounds enhancing the almond and sweet aroma of NIBT [57].In contrast, other typical volatile fraction components, such as hexanal,(E)-2-hexenal, benzaldehyde, (E)-2-octenal were detected during the LSF and EAE process, which contributed to the aroma profile of the NIBT with almond, burnt or nut floral [52,54].

Fig.6 Heat map result of relative amount percent of volatile aldehydes compounds with different samples (blank area notated the results ‘‘not detected’’).

Furthermore, compared with fresh leaves, it was interesting to find that the amount of esters increased sharply to a level of 11.05%after the enzyme treatments.The predominant compound of esters,methyl salicylate, contributed the highest average percentage(10.97%) among the tested esters.Methyl salicylate was previously recognized as one of the key flavor compounds contributing to sweet note of Dianhong black tea [58].Therefore it can be inferred that fermentation process was beneficial for the formation of IBT flavor profiles.Moreover, heat map result of volatile ketones compounds with difference samples were shown in Fig.7.

Fig.7 Heat map result of relative amount percent of volatile ketones compounds with different samples (blank area notated the results ‘‘not detected’’).

Regarding other identified compounds, the levels of nitrogenous compounds and hydrocarbons decreased apparently during the LSF and EAE process.The nitrogenous compounds such as 2,5-dimethylpyrazine, 2-ethyl-5-methylpyrazine, 2,5-dimethyl-3-ethylpyrazine, 2-ethylpyrrole, 1-ethyl-2-acetylpyrrole, 2-acetyl pyrrole were not detected in both two stages, whereas the content of indole was stable.The hydrocarbons compounds such as(1S)-(-)-β-pinene, limonene, (E)-β-ocimene, (Z)-β-ocimene were also not detected in the two processes, along with varieties mentioned above indicated the significant changes of volatiles during fermentation process.Recent researches have confirmed (Z)-βionone as the main volatile aroma components of tea, which made contributions to the violet, and flower odor [57-59].

Regarding to the rest of the identified components, ketones and oxygen compounds have a minor decrease.Moreover, benzothiazole and geranic acid were the only sulfur compound and acid detected in treatment samples, respectively.

3.4 Characteristics analysis of the NIBT

To further evaluate the quality characteristics of final products processed from fresh tea leaves, a verified experiment was performed under the optimum conditions, and the predominant constituents and related characteristics of the NIBT were analyzed.Market brand IBT was taken as control for comparison among the quality attributes.Various kinds of IBT differentiated in content of TFs, catechin,total polyphenol and amino acids, which might be due to the fact of cultivars or processing method.And these commercial IBT were classified into high grade and low grade according to International Standard, Instant tea in solid form-specification [60,61].TFs content in the NIBT was similar to high grade IBT produced from Assam cultivars, anti-oxidative activities as well.As can be seen from Table 2, the amount of TFs and the hydroxyl radical scavenging activities did not show significant variations (P＜ 0.05).Generally,strong antioxidant activity of green tea is ascribed to catechins [62].Although catechins are converted into polymerized derivatives (TFs,TRs and TBs) during the fermentation process, TFs also possess a strong antioxidant activity [63].It was interesting that the NIBT in this study showed higher antioxidant activity, which was similar with the high quality ones.The amino acid content of the NIBT was found to be highest as compared to some special grade commercial NIBT,probably attributing to cell-wall matrix disassembly by the pectin degradation [64].As for sensory evaluation, the NIBT had distinctive feature as described in Table 2.

Table 2Quality characteristics for different grade IBT.

Additionally, Fig.8 shows that the infusion color of three kind IBT products, the appearance of the NIBT were red and bright, which seems more appreciated in young consumers.

Fig.8 Color appearance of different grade instant tea samples (a, high grade IBT; b, low grade IBT; c, NIBT).

4.Conclusions

The key technology of preparing NIBT with excellent sensory quality and high TFs content was established by the combination of enzymatic fermentation, and enzyme-assistant extraction of the fermentation brew in this study.The results suggested that LSF conditions with 35oC/65 min/pH 4.8/hf 25% and EAE systems at 50oC/60 min, addition of 1.0% pectinase and cellulose posterior to fermentation were beneficial to high quality formation of NIBT.A total of 75 kinds of volatile compounds were identified, among which alcohols, aldehydes and esters were the major groups.Furthermore,the different profiles of TFs, TRs, TBs and TRs/TFs ratio in various processing conditions were evaluated and compared.

The NIBT produced via the novel submerged fermentation was comparatively assessed relative to commercial IBT.The results showed that the product was comparable to high grade IBT in respect to main chemical composition (P＜ 0.05), sensory properties (P＜ 0.05)and anti-oxidative activity.Accordingly, it can be concluded that choosing appropriate technical measures is efficacious to improve IBT quality by co-treatment of endogenous and exogenous enzymes via two-stage submerged processing.This research provides new clues for a high quality IBT production, which may lead to specified products development and promote consumption of such products.

Conflict of interest

No potential conflict of interest was reported by the authors.

Acknowledgment

This work was financially supported by the National Key R&D Program of China (2017YFD0400804), Public Technology Research Project of Zhejiang Province (LGN18C160001), and Special Fund for Quality Research in the Public Welfare (20110210) to X.Yang.

Appendix A.Supplementary data

Supplementary data associated with this article can be found in the online version, at http://doi.org/10.1016/j.fshw.2021.12.025.