A synergistic flower and fruit fragrance fermented tea based on pre-harvest adversity regulation and a preparation method thereof

Abstract

The application belongs to the technical field of tea processing, and particularly relates to a synergistic type of flower and fruit aroma fermented tea based on pre-harvest adversity regulation and a preparation method thereof. The preparation method combines a directional processing process to improve the flower and fruit aroma and taste quality of the fermented tea. Summer and autumn tea tree fresh leaves subjected to pre-harvest drought-shading combined treatment (D+S group) are used as raw materials, including one bud and two leaves standard tender shoots. Through regulation and control of wilting, rolling, fermentation and other process parameters, black tea or white tea tea bases are prepared. In the tea bases, the total content of chlorophyll reaches 1.078 mg / g, the content of free amino acids reaches a maximum of 54.10 mg / g at the wilting stage of 40 h, and the total content of theaflavins reaches 8.19 mg / g.

Description

Technical Field

[0001] This invention belongs to the field of agricultural planting and tea processing technology, specifically involving a tea garden management method that regulates the formation of flavor and aroma substances during the processing of fermented tea (black tea, white tea) through the synergistic treatment of pre-harvest drought and shading. It is particularly suitable for alleviating the quality degradation problem caused by drought stress in summer and autumn teas and optimizing tea garden adversity management strategies. Background Technology

[0002] As consumers increasingly demand more diverse flavors and health benefits from tea, fermented tea products that combine floral and fruity aromas with functional properties have become a new market favorite. While traditional scenting methods can impart aroma to tea leaves through the absorption of fresh flowers, they suffer from issues such as limited aroma diversity and poor stability. This study innovatively proposes a three-pronged technology system integrating pre-harvest stress regulation, metabolic induction, and synergistic fermentation. Using Baojing Golden Tea No. 1 as raw material, this system precisely regulates pre-harvest stress conditions (such as periodic drought and UV-B radiation) to activate phenylpropanone metabolism and terpene synthesis pathways, promoting the accumulation of floral and fruity aroma precursors such as phenylethanol and linalool in the leaves. Simultaneously, leveraging the high polyphenol content of Golden Tea's summer and autumn harvests, functional strains producing β-glucosidase (such as *Aspergillus cristatus* TD-8) are screened. During fermentation, these strains directionally hydrolyze bound aroma aglycones and co-ferment with extracts from flowers and fruits such as jasmine and grapefruit, achieving a synergistic effect of endogenous aroma release and exogenous aromatic substance incorporation. The prepared fermented tea combines the sweetness and mellowness of spring tea with the floral and fruity aroma of autumn tea. The content of characteristic aroma compounds such as ionone and nerolidol in its volatile components is increased by 2.3-3.5 times, and the ester / aldehyde ratio is optimized to over 1.8, effectively masking the bitterness of summer and autumn teas. This technology provides an innovative path for the high-value utilization of summer and autumn tea resources and the development of novel flavored fermented teas, promoting the upgrading of tea processing towards flavor customization and functional integration. Summary of the Invention

[0003] The purpose of this invention is to provide a synergistic effect of pre-harvest stress regulation in the fermentation of floral and fruity aroma tea and its preparation method.

[0004] To solve the above technical problems, the technical solution of the present invention is as follows:

[0005] This invention provides a method for synergistically managing pre-harvest drought and shading in tea gardens, achieved through the following steps:

[0006] Treatment combination: A "drought + shade (D+S)" treatment group was set up with the following parameters: Shading degree: 80% (covered with black high-density polyethylene netting); Drought period: watering once every 8 days (soil moisture ≤18.23%); Treatment time: continuous treatment for 80 days in summer (July 5 to September harvest season). Microenvironment regulation: The D+S group reduced the air temperature in the tea garden by 2.64℃, increased humidity by 6.52%, and stabilized soil moisture at 18.23%. Molecular mechanism activation: By upregulating JA pathway genes (AOX gene expression increased 65-fold) and ABA pathway genes (NCED gene upregulated 300-fold), the synthesis of umami substances such as theanine and aspartic acid, as well as theaflavins (8.19 mg / g in black tea) was driven. Processing technology adaptation: Combining the processes of black tea (withering-rolling-fermentation) and white tea (withering for 40 hours), key aroma substances such as esters (71.7 μg / kg for black tea) and alcohols (190.6 μg / kg for white tea) are selectively enriched.

[0007] The beneficial effects of this invention are as follows:

[0008] Synergistic Effect Design: For the first time, pre-harvest drought and shading were combined, leveraging their synergistic effects on the tea garden microenvironment (temperature and humidity) and metabolic pathways (JA / ABA) to overcome the limitations of single treatments. Spatiotemporal Metabolic Regulation: Pre-harvest treatment activated metabolic precursors in fresh leaves, combined with enzymatic reactions during processing (such as withering-induced β-glucosidase activity), achieving dynamic enrichment of flavor and aroma substances. Targeted Enrichment of Key Substances: 18 aroma substances with OAV > 1 (such as phenylethanol and linalool oxide) were screened, and their positive correlation with theanine and theaflavins was clearly established (R > 0.75, p < 0.05). 3. Improved Quality and Technical Effects: The sensory score of black tea in the D+S group reached 92.1 ± 0.2, and the free amino acid content of white tea reached 54.10 mg / g after 40 hours of withering, significantly higher than the control group. Enhanced Stress Resistance: Total chlorophyll increased to 1.078 mg / g, and the chlorophyll A / B value was optimized to 2.85, enhancing the photosynthetic capacity of tea trees. Wide adaptability: Applicable to medium-leaf tea varieties such as "Zhu Ye Qi", it can be promoted to summer and autumn tea producing areas to reduce yield losses caused by high temperature and drought. 4. Claim relevance: The core protection scope of this invention covers "D+S treatment parameters", "JA / ABA gene regulatory network", and "key aroma substance enrichment process", providing technical support for the subsequent development of tea garden management equipment (such as intelligent shading irrigation system) and the optimization of processing technology. Attached Figure Description

[0009] Figure 1 Environmental indicators, SPAD value of tea trees, and determination of photosynthetic pigments under shading and drought treatments.

[0010] Figure 2 Quality analysis of tea leaves processed into black and white tea after shading and drought treatment;

[0011] Figure 3 Changes in nonvolatile substances during the processing of black and white tea after drought and shade treatment;

[0012] Figure 4 Multivariate statistical analysis of volatile components in black tea and white tea in four groups and RT-qPCR of key aroma-forming enzyme genes;

[0013] Figure 5 Correlation network analysis of taste (electronic tongue), aroma (OAV screening) and major quality compounds in four groups after drought and shade treatments. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of this invention are only used to explain this invention and are not intended to limit this invention.

[0015] Example 1

[0016] The perennial tea cultivar 'Zhuyeqi' was planted at Yunyou Tea Industry Co., Ltd. in Changsha City, Hunan Province (112.69°E, 28.44°N). The experiment was divided into four groups: control group (CK), drought group (D), shade group (S), and drought + shade group (D+S). Each treatment used four rows of healthy tea trees (15m long, planted in single rows with two trees each), with four rows spaced between treatments. The CK group received normal watering management, watering once every four days. The D group received watering once every eight days. The S group received normal watering, and the tea trees were covered with black high-density polyethylene netting, providing 80% shade, starting on July 5, 2023, and lasting for 80 days. The D+S group received the same watering management as the D group, and the shading treatment was the same as the S group. Fresh leaves were harvested starting in September 2023, with a standard of one bud and two leaves. The harvested tea leaves were divided into two parts: one part was used as a test sample, and the other part was processed. The processing of black tea includes fresh leaves, withering for 6 hours, rolling, fermentation, and drying. The processing of white tea includes fresh leaves, withering for 6 hours, 20 hours, 40 hours, and 70 hours, and drying. All samples were stored at -80℃ for subsequent analysis. The effects of environmental changes on tea tree growth and tea quality were investigated through regular watering and shading treatments. Significant differences were found in air and soil temperature and humidity under different treatments.

[0017] like Figure 1As shown, the average temperature of group D was 32.79℃, while that of group D+S was 30.15℃, with most temperatures exceeding 40℃ falling into group D. Simultaneously, the air humidity of group D+S was 82.51%, while that of group D was 75.99%, with most cases below 50% belonging to group D. Shading significantly affected soil temperature and humidity. From mid-August to September, under the high summer temperatures, the soil humidity of group D+S was consistently lower than that of group D. The difference in soil humidity was even more clearly demonstrated, with the average humidity of group D+S being 18.23%, while group D only had 14.94%. These results indicate that shading has a significant promoting effect on cooling and water retention in the tea tree microenvironment. SPAD values ​​and chlorophyll content effectively reflect the color changes of tea tree leaves. Under shading treatment, the SPAD values, chlorophyll A, B, and A+B contents of groups S and D+S were significantly higher than those of groups CK and D (no shading) (p < 0.05), especially the total chlorophyll content in group D+S, which reached a maximum of 1.078 mg / g ± 0.012. However, the chlorophyll A / B ratio showed the opposite trend, with group D reaching a maximum of 2.850 ± 0.015. In conclusion, shading can improve the environment, promoting photosynthetic pigments and some important metabolites in tea under arid conditions, thereby enhancing the flavor and quality of tea.

[0018] Example 2

[0019] Sensory evaluations were conducted by a seven-person panel of trained reviewers, comprising three men and four women aged 25 to 30. All sensory evaluation experiments received formal approval and ethical permission from the Ethics Committee of Hunan Agricultural University (Approval No.: 2023-138). All participants were informed of the details of the experiment before it began and participation was voluntary. Traditional sensory evaluations were conducted according to the Chinese standard method (GB / T 23776-2018). In short, 3 grams of black or white tea were steeped in 150 ml of boiling water for 5 minutes, then the tea leaves were filtered out. The panel members evaluated the quality of the tea leaves and assigned corresponding scores. After applying four treatments—drought and shade—in the tea garden, we found that shading significantly improved the unfavorable microenvironment caused by drought, thereby promoting tea growth and development. Therefore, to further investigate the impact of this pre-harvest treatment on tea quality, we collected fresh leaves for processing into black and white tea and conducted taste evaluations.

[0020] like Figure 2As shown, the quality evaluation results indicate that the D+S group of black tea had significantly higher scores in terms of liquor color, aroma, and taste than the D group, with a total score of 92.1±0.2. For white tea, the S group had the highest scores in appearance, liquor color, and taste, while the D+S group had a significantly higher aroma score than other groups (p<0.05). Electronic tongue verification was performed for the taste scores. Analysis of the electronic tongue results for black tea showed that the D+S group had the highest values ​​in aftertaste, freshness, and intensity, significantly higher than other groups (p<0.05), while the D group had the lowest values. This suggests that the D group's tea liquor was weak, possibly due to a reduced content of internal substances. Simultaneously, the D group had significantly higher acidity than other groups, while the D+S group had the lowest value (p<0.05). For white tea, the D+S group had significantly higher sweetness than other groups, reaching 11.28±0.01. However, the freshness, aftertaste, and intensity were all the lowest in the D group, suggesting that shading significantly improved this situation. Studies have shown that the growing environment and processing techniques of tea trees have a significant impact on the chemical composition and sensory characteristics of tea. For example, studies on black tea produced from tea trees of different ages have found that black tea from older tea trees has a stronger sweetness and sourness, and a weaker bitterness and astringency. This is related to changes in the content of specific metabolites in tea, such as L-tartaric acid and trans-citric acid.

[0021] Example 3

[0022] After tea infusion extraction, six catechin components (DL-C, EC, EGC, ECG, EGCG, GCG), three alkaloid components (caffeine, theobromine, theophylline), and gallic acid were determined by high-performance liquid chromatography (HPLC). Chromatographic parameters: C18 column (4.6 mm × 150 mm, 5 μm), detection wavelength 278 nm, injection volume 10 μL, column temperature 30 °C, flow rate 1.0 mL / min. Mobile phase A was ultrapure water, and mobile phase B was a mixture of N,N-dimethylformamide, methanol, and glacial acetic acid (N,N-dimethylformamide:methanol:glacial acetic acid = 39.5:2:1.5). The separation process included gradient elution. The initial concentration of mobile phase B was 9%, increased to 14% after 10 min, further increased to 36% after 27 min, maintained at this concentration for 4 min, decreased to 9% at 32 min, and terminated at 37 min.

[0023] like Figure 3As shown, to further investigate the impact of metabolite changes on tea quality, we detected non-volatile components, including catechins, amino acids, and theaflavins, in samples from different processing stages after drought and shade treatments. We performed peak quantification using liquid chromatography, and the results can be used for further analysis. This study determined the changes in the content of gallic acid, three alkaloids, and six catechins during the processing of black and white tea. With the progress of processing, catechins, including DL-C, EC, EGC, ECG, GCG, and EGCG, showed a decreasing trend in both black and white tea. Enzymatic oxidation during processing led to the conversion of catechins to theaflavins, which is the main reason for the decrease in catechins, consistent with our results. In white tea, catechins showed different trends in the four groups. For example, the contents of ECG, DL-C, and EGCG in the shaded groups (S and D+S groups) were higher than those in the unshaded groups (CK and D groups) at 40h and 70h withering and in the finished white tea. The content of EGC and EC in the shaded groups was lower than that in the unshaded groups from the fresh leaf stage. This may be because shading downregulates the expression of genes related to catechin biosynthesis and activates polyphenol oxidase to promote the conversion of theaflavins. Gallic acid content showed an increasing trend during the processing of both black and white tea, especially during fermentation and the 70-hour withering stage. The levels of caffeine, theobromine, and theophylline in black tea did not change significantly during processing, but the content in the D+S group was consistently significantly higher than that in the D group. In white tea, theobromine content gradually decreased under prolonged withering, while caffeine content in the shaded groups (S and D+S groups) was higher than that in the unshaded groups (CK and D groups) from fresh leaves to the prolonged withering stage. The study indicates that the expression level of the key synthetic enzyme gene N-methyltransferase (NMT) was significantly upregulated in shaded tea leaves, which may have led to caffeine accumulation.

[0024] Example 4

[0025] Volatile compounds were extracted using headspace solid-phase microextraction (HS-SPME) and identified by gas chromatography-mass spectrometry (GC-MS) (Agilent Technologies Inc.). 1 g of accurately weighed tea powder (accurate to 0.001 g) was placed in a 15 mL headspace vial, and 10 μL of ethyl decanoate (8.64 mg L⁻¹) was added and the vial was sealed. The vial was placed in an 80 °C water bath for 10 min. The HS-SPME head (65 μm DVB / PDMS, Superclo, Bellefonte, PA, USA) was inserted into the vial, with the fiber tip extended approximately 1 cm away from the tea powder. Extraction was continued at 80 °C for 40 min. The fiber tip was then inverted, and the microextraction head was immediately inserted into the gas chromatograph inlet. Analysis was performed at 250 °C for 5 min.

[0026] like Figure 4As shown, to comprehensively understand the similarities and differences between finished black and white teas after four drought and shade treatments, we conducted unsupervised principal component analysis (PCA) based on the identified volatile compound content. The results showed that the four groups were significantly separated in both black and white teas, and the models all had good explanatory and predictive capabilities. The PCA model parameters for black tea (PC1 = 39.7%, PC2 = 30.6%, totaling 70.3%), with PC1 potentially corresponding to strong changes in aroma components. In the PCA analysis of white tea, the first two principal components explained 66.1% of the original data variation (PC1: 27.2% + PC2: 38.9%), with PC2 potentially related to changes in aroma compound content related to withering. Next, we selected the representative D+S group for specific aroma compound classification analysis. Esters accounted for 28% of the content in black tea, making them the largest component, and they were highly correlated with aroma compounds produced during the fermentation stage of black tea, such as fruity and honey aromas. As a fully fermented tea, black tea's aroma is directly related to the esterification process triggered by polyphenol oxidation during processing. Alcohols (23%) and alkenes (20%) also account for a significant proportion, together with esters, making up 71% of the aroma compounds. The remaining compounds include ketones (7%), aldehydes (6%), and heterocyclic compounds (4%). In white tea, alcohols and esters each account for 29%, totaling 58% of the aroma compounds, indicating a greater contribution from these two substances. The remaining alkenes (13%) are slightly higher than in black tea. The slow water loss process caused by the prolonged withering of white tea is more conducive to the hydrolysis of bound terpenoids (such as linalool) by β-glucosidase.

[0027] Example 5

[0028] GC-MS conditions were as follows: DB-5MS capillary column (thickness: 0.25 μm, length: 60.0 m, inner diameter: 0.25 mm, Agilent Technologies Inc.) initial temperature 60 °C for 5 min, then increased to 140 °C at 3.0 °C min⁻¹ for 5 min. This was followed by increasing to 230 °C at 10 °C min⁻¹ for 1 min, finally reaching a peak at 260 °C for 5 min, then increasing at 5.0 °C min⁻¹. Helium (He, purity ≥99.999%) was used as the carrier gas at a flow rate of 1.96 mL min⁻¹, with no splitting injection. GC-MS analysis employed electron impact (EI) ionization at an electron energy of 70 eV, with a scan range of 45–500 m / z. Qualitative methods: The NIST-20 standard mass spectrometry database was used for comparison. For samples with similarity greater than 80%, component qualitative analysis was performed using characteristic ions, relative abundance, and retention time. Quantitative analysis: The relative content of volatiles was calculated by the ratio of the target peak area to the standard area of ​​ethyl decanoate.

[0029] like Figure 5As shown, to further screen for key substances that may determine the aroma of tea, we used OAV analysis based on odor thresholds to evaluate important volatile compounds. In this study, we found 18 substances with an OAV greater than 1 in black tea and 21 substances with an OAV greater than 1 in white tea. We performed commonality analysis on the substances with an OAV greater than 1 in the four groups of black and white tea after drought and shade treatments, ultimately focusing on 18 key compounds, of which 6 had an OAV value greater than 100, including Phenylethyl alcohol, Linalool, (E)-linalool oxide (pyranoid), Nonanal, Creamylactone, and Indole, indicating that they have an important influence on the formation of the basic aroma of tea. Figure 5 As shown, six substances were identified, exhibiting the highest values ​​in the D+S group of black tea: Benzyl alcohol (slightly sweet, floral), Phenylethyl alcohol (Rose, honey), Linalool (Floral, fruity), (Z)-linalool oxide (furanoid) (Floral, green), Dehydrolinalool (Green, floral, and slightly woody), and Benzaldehyde (Sweet, bitter almond-like). Previous studies have shown that linalool and its derivatives, phenylethyl alcohol, and benzaldehyde are important components of the floral and fruity aromas in black tea, consistent with the findings of this study.

Claims

1. A synergistic effect fermented tea with floral and fruity aroma based on pre-harvest stress regulation and its preparation method, characterized in that... Includes the following steps: Processing condition settings: During the tea tree growing season (summer to autumn), a pre-harvest treatment group is set up, including: shading treatment: covering the tea tree canopy with a shading net with an 80% shading rate and continuing the treatment for 60-80 days; Drought treatment: Reduce the irrigation frequency (water once every 8 days) to lower the soil moisture to 15-18%; Synergistic treatment: Shading and drought are implemented simultaneously, which reduces the microenvironment temperature of the tea garden by 2-3℃ and increases the air humidity by 5-7%. Treatment groups were set up as follows: control (CK), drought (D), shade (S), and drought + shade (D+S). Fresh leaf harvesting and processing: After processing, pick standard fresh leaves with one bud and two leaves; Fresh leaves are processed into fermented tea (black tea or white tea). The black tea process includes withering, rolling, fermentation, and drying, while the white tea process includes withering for 40-70 hours and drying. Quality control mechanism: By activating the jasmonic acid (JA) and abscisic acid (ABA) signaling pathways, the expression of AOX gene (65-fold) and NCED gene (300-fold) was upregulated; It promotes the accumulation of free amino acids (54.10 mg / g in white tea after 40 hours of withering) and theaflavins (8.19 mg / g in black tea); It induces the formation of key aroma compounds, including phenylethyl alcohol, linalool, benzaldehyde, and β-ionone. Quality Inspection: The contents of catechins, theaflavins, and amino acids were determined by HPLC. Volatile components were analyzed by HS-SPME-GC-MS to screen key aroma compounds with an OAV > 1; The enhancement of umami, sweetness, and aftertaste intensity was verified by combining electronic tongue and sensory evaluation.

2. The method according to claim 1, characterized in that: The shade net is made of black high-density polyethylene with a shading rate of 75-85%; the soil moisture gradient for drought treatment is 14-20% and the duration is 60-90 days.

3. The method according to claim 1, characterized in that: The key genes regulating the JA signaling pathway include LOX, AOS, and JAR; the key genes regulating the ABA signaling pathway include NCED and ABA8OX; the formation of the aroma substances is related to the linalool synthesis pathway activated by the CsMYB68-MYC2 complex.

4. The method according to claim 1, characterized in that: The total amount of theaflavins in black tea processing is 6.26-8.40 mg / g, while the content of free amino acids in white tea during the withering stage is 26.03-54.10 mg / g. The content of key aroma substances is as follows: esters 71.7 μg / kg and alcohols 271.1 μg / kg in black tea, and alcohols 190.6 μg / kg and aldehydes 27.47 μg / kg in white tea.

5. The method according to claim 1, characterized in that: The sensory quality improvements include: an increase of 15-20% in the umami intensity of black tea and an increase of 10-15% in the sweetness intensity of white tea; among the electronic tongue detection indicators, umami and aftertaste are significantly positively correlated with the content of theanine, aspartic acid and water extract (R>0.75).

6. A fermented tea product prepared by any one of claims 1-5, characterized in that: It contains 18 key aroma compounds with OAV > 1, of which the total proportion of phenylethyl alcohol, linalool, benzaldehyde and β-ionone is ≥ 30%; the water extract content in the tea soup is ≥ 40%; the total amount of umami amino acids (theanine and aspartic acid) is ≥ 25 mg / g; the theaflavins content in black tea is ≥ 7.5 mg / g; and the free amino acid content in white tea is ≥ 50 mg / g.

7. The product according to claim 6, characterized in that: The black tea has floral and fruity aroma characteristics, while the white tea has sweet floral aroma characteristics; among the aroma substances, phenylethanol (OAV≥200), linalool (OAV≥90), and β-ionone (OAV≥5) are the characteristic components. The application of any one of the methods in claims 1-5 in improving the quality degradation of summer and autumn tea specifically includes: alleviating the excessive accumulation of tea polyphenols and the decrease in amino acid content caused by drought stress; and improving the efficiency of metabolite conversion during the processing stage by synergistic regulation of the tea garden microenvironment through shading and drought.

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