Process for accelerating aging of Liupu tea by inoculation of golden flower fungi

Abstract

The invention provides a process for accelerating aging of Liupu tea by inoculation of eurotium cristatum, which comprises the following steps: preparation of a bacterial liquid: a spore suspension of eurotium cristatum is obtained through activation culture, and the concentration of the spore suspension is 1-10 * 10 < 5 > CFU / mL; performing pile fermentation: adding water into the sunned raw tea until the water content is 18-22%, performing pile fermentation for 22-25 hours, inoculating the spore suspension obtained in the step S1, and performing pile fermentation in a natural environment; aging: spreading the tea leaves subjected to pile fermentation, naturally cooling, and then performing high-pressure steaming treatment to enable the water content of the tea leaves to reach 17-19%; and after cooling to room temperature, inoculating the spore suspension again, and aging for 120-125 days. According to the process for accelerating aging of the Liupao tea through inoculation of the eurotium cristatum, the fermentation and aging process of the Liupao tea is improved, the production period of the Liupao tea is shortened, the labor intensity is reduced, the labor productivity is improved, and the tea quality is stabilized and improved.

Description

Technical Field

[0001] The invention belongs to the technical field of tea manufacturing, preparation or soaking, and specifically relates to a method for aging tea leaves. Background Art

[0002] Liubao tea is a type of specialty black tea product in my country's Guangxi Zhuang Autonomous Region. It has a long history of production and sales, unique flavor and quality characteristics, health benefits, and a strong cultural heritage. Consumer demand has grown significantly in recent years.

[0003] Liubao tea, as a tea with a high geographical indication in the Chinese dark tea pedigree, originated from Liubao Town, Cangwu County, Wuzhou, Guangxi. This tea is known for its better quality as it ages. Various compounds in tea undergo different degrees of transformation during processing and storage, and their content and proportion will directly affect the taste, aroma and color of the tea. Liubao tea, which has been aged for a long time, has the quality characteristics of "red, strong, aged and mellow"; Liubao tea has many health benefits such as relieving heat and dampness, reducing fat and weight, and regulating the stomach. In addition, the collection value of Liubao tea "gets better with age" has been favored by more and more consumers in recent years, and consumer demand has surged.

[0004] The traditional aging process of Liubao tea is dominated by natural aging, and quality transformation is achieved by stacking in the shade after re-steaming, but it has defects such as long cycle, low efficiency and large quality fluctuations. The reason is that the metabolism of microbial communities in the natural environment is significantly affected by temperature and humidity fluctuations, making it difficult to coordinate the control of theabrownin production rate and flavonoid oxidation efficiency.

[0005] Studies have shown that Liubao tea exhibits characteristic biochemical transformation patterns during the aging process: active ingredients such as tea polyphenols, amino acids, and water extracts decrease with increasing aging years, while theabrownins accumulate year by year. The sensory quality is manifested in the redder soup color, mellower taste, and more pronounced aged aroma. This conversion efficiency is closely related to storage environment parameters (WL, SM, CS, et al. Research on quality changes of Liubao tea in different storage time[J]. Southwest China Journal of Agricultural Sciences, 2015, 28(1): 376-80.). If you want the tea to be "more fragrant with age", the storage environment is particularly important. For example, relevant scholars compared the storage environments of Guilin and Wuzhou in Guangxi and found that the average annual temperature and humidity in Wuzhou was higher (temperature 23.5°C, humidity 80%), and the theabrownin content of its tea samples was 15.6% higher than that in Guilin (temperature 19.8°C, humidity 75%), and the taste was stronger, confirming that temperature and humidity have a significant regulatory effect on the aging process (Cao Y, Zhao M, Zhao T, et al. Functional Components and Activities of Different Dark Tea Extracts[J]. Food Science, 2017, 38(18):54-9.). In addition, new aging technologies such as oxygenated irradiation (12 kGy irradiation dose, 100% oxygenation) can significantly improve the mellowness and aroma intensity of tea soup within 1 week (Luo Y, Ruan J, SU Z, et al. Discrimination of liubao tea by FTIR and principal component analysis[J]. Science&Technology of Food Industry, 2014, 35(12): 55-7, 65.), but may induce non-enzymatic browning side reactions, suggesting that process optimization needs to take into account both efficiency and quality stability. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention proposes a process for accelerating the aging of Liubao tea by inoculating golden flower fungus, aiming to explore and improve the fermentation and aging process of Liubao tea, shorten the production cycle of Liubao tea, reduce labor intensity, improve labor productivity, and stabilize and improve the quality of tea.

[0007] The second object of the present invention is to provide Liubao tea prepared by the process.

[0008] The technical solution for achieving the above-mentioned purpose of the present invention is:

[0009] A process for accelerating the aging of Liubao tea by inoculating Eurotium cristatum includes the following steps:

[0010] S1 Bacterial liquid preparation: A spore suspension of Eurotium cristatum is obtained through activation culture, and the concentration of the spore suspension is 1 - 10×10 5 CFU / mL;

[0011] S2 Pile fermentation: Water is added to the sun-dried rough tea to make the moisture content 18 - 22%, and after pile fermentation for 22 - 25 hours, the spore suspension obtained in step S1 is inoculated, and then pile fermentation is carried out for 38 - 42 days in a natural environment;

[0012] S3 Aging: The tea leaves after pile fermentation are spread out to cool naturally, and then treated by high-pressure steam steaming to make the moisture content of the tea leaves reach 17 - 19%; after cooling to room temperature, the spore suspension is inoculated again, and aged for 120 - 125 days

[0013] Eurotium cristatum, namely "golden flower bacteria", emits a faint floral fragrance of bacteria and is an enzyme-producing bacterium beneficial to humans.

[0014] Among them, in step S1, after activating the freeze-dried powder of Eurotium cristatum, it is streaked and inoculated into a PDA medium, and cultured in the dark at 25 - 30°C for 6 - 8 days. The spores of the cultured strain are scraped into sterile water and oscillated at 25 - 30°C and 100 - 400 r / min for 20 - 40 min to obtain a uniform spore suspension. The spore suspension is counted and the concentration is adjusted to 5 - 8×10 5 CFU / mL.

[0015] Among them, in step S2, the sun-dried rough tea is made from the leaves of Camellia sinensis var. cangwuensis (a shrub-type leaf variety, a large-leaf tea tree), and its preparation process includes the steps: 1) Fresh leaf picking: Picking one bud with two to three leaves of Camellia sinensis var. cangwuensis; 2) Withering regulation to a moisture content of 65% - 70%; 3) High-temperature fixation of enzymes at a temperature of 220 - 280°C; 4) Gradient rolling to a cell breakage rate of 40% - 50%.

[0016] Preferably, in step S2, the inoculation amount of the spore suspension of Eurotium cristatum is 1 - 1.5% (50 - 75 mL of golden flower bacteria liquid: 5 kg of sun-dried rough tea); during the pile fermentation process, the pile is turned over once every 4 - 5 days.

[0017] Further, in step S3, the tea leaves after pile fermentation are spread out and evenly piled, spread out to cool naturally to below 40°C, and the temperature and humidity are made uniform by turning the pile; then high-pressure steam steaming is carried out: treating with 0.3 - 0.5 MPa saturated steam for 60 - 100 seconds to raise the moisture content of the tea leaves to 17 - 19%.

[0018] More preferably, in step S3, the inoculation amount of the re-inoculation of the spore suspension is 1-1.5%.

[0019] Since it is necessary to steam after piling up to terminate the fermentation and some bacteria will be inactivated, it is cooled to room temperature after steaming, and then secondary inoculation is carried out. After the second inoculation, it is tightly pressed and then aged.

[0020] In step S3, after the second inoculation, it is tightly pressed. The specific operation is: pressing with a stainless steel mold to shape at a temperature of 75-85 °C and a pressure of 10-15 MPa for 25-35 seconds. The pressed shape is a hexahedron with a volume of 800-900 cm 3 each portion having a mass of 1.3-1.6 kg; and packing with cotton paper. Preferably, 1.5 kg of tea leaves are loaded into a 304 stainless steel mold, and shaped at a pressure of 12 MPa for 30 seconds at 80 °C to form a standard brick-shaped pressed tea of 20×14×3 cm, and packed with cotton paper.

[0021] Among them, in step S3, the aging temperature is 22 °C - 30 °C, and the humidity for aging is 70-90%.

[0022] A preferred technical solution of the present invention is that in step S3, the aging temperature is 28 °C and the aging humidity is 85%.

[0023] The Liubao tea prepared by the process of the present invention.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] The present invention provides a process for accelerating the aging of Liubao tea by inoculating Eurotium cristatum. During the piling fermentation process of Liubao tea, Eurotium cristatum is introduced, and the effects of inoculation fermentation on the sensory quality and nutritional quality content of Liubao tea are studied; combined with artificial regulation of aging parameters (temperature 22-30 °C, humidity 70-90%), experiments on the piling process, aging environment, and aging form of Liubao tea are carried out, and the optimal process conditions are determined.

[0026] By carrying out artificial inoculation and regulation of the aging process (temperature, humidity), the fermentation and aging process of Liubao tea is explored and improved, shortening the production cycle of Liubao tea, reducing labor intensity, improving labor productivity, and stabilizing and improving the tea quality. Description of the Drawings

[0027] Figure 1 : Comparative chart of sensory scores of Liubao tea.

[0028] Figure 2: Effects of different aging temperatures on the contents of main nutrients in Liubao tea. In the figure, A: water extract content; B: tea polyphenol content; C: free amino acid content; D: soluble sugar content; E: (theaflavin + thearubigin) / theabrownin; F: phenolic ammonia ratio of tea soup. Different letters indicate significant differences (p<0.05).

[0029] Figure 3 : Effects of different aging humidities on the contents of main nutrients in Liubao tea. In the figure, A: water extract content; B: tea polyphenol content; C: free amino acid content; D: soluble sugar content; E: (theaflavin + thearubigin) / theabrownin; F: phenolic ammonia ratio of tea soup. Different letters indicate significant differences (p<0.05).

[0030] Figure 4 Effects of different piling processes on the contents of main nutrients in Liubao tea. In the figure, A: water extract content; B: tea polyphenol content; C: free amino acid content; D: soluble sugar content; E: (theaflavin + thearubigin) / theabrownin; F: phenolic ammonia ratio of tea soup. Different letters indicate significant differences (p<0.05).

[0031] Figure 5 PCA analysis of Liubao tea in different treatment groups. Left figure: positive ion mode; right figure: negative ion mode.

[0032] Figure 6 : OPLS-DA score plots and permutation tests of the metabolic profiles of Liubao tea in different treatment groups. In the figure, A: positive ion mode; B: negative ion mode;

[0033] Figure 7 : Volcano plot and VIP value analysis of metabolites in the tea soup of Liubao tea with different qualities.

[0034] Figure 8 : KEGG topology analysis of Liubao tea with different qualities. Specific implementation manners

[0035] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention.

[0036] Sun-dried green tea: Provided by Guangxi Zhaoping Jiangjunfeng Tea Factory, and the preparation process includes the steps of: 1) Fresh leaf picking: Picking one bud with two to three leaves of Cangwu population variety; 2) Withering regulation to a moisture content of 65%-70%; 3) High-temperature fixation of enzymes at a temperature of 220-280°C; 4) Gradient rolling to a cell breakage rate of 40%-50%. Each step of preparing the sun-dried green tea is carried out in an operation manner known in the art.

[0037] Eurotium cristatum freeze-dried powder: Shanghai Yushao Biotechnology Co., Ltd., with a preservation number of CGMCC NO.11040.

[0038] The determination of water extract content refers to GB / T 8305-2013 "Determination of Tea Water Extract"; the determination of tea polyphenol and catechin contents is carried out according to the Folin-Ciocalteu reagent method in GB / T 8313-2018 "Detection Method for Contents of Tea Polyphenols and Catechins in Tea"; the determination of caffeine content refers to GB / T 8312-2013 "Determination of Tea Caffeine"; the determination of flavonoid content is carried out by the aluminum trichloride colorimetric method, and the absorbance value is measured at a wavelength of 420 nm.

[0039] Preparation of tea soup: Take 1.5 g of tea sample (ground through 40-60 mesh), mix it in a tea-to-water ratio of 1:50, transfer it to a boiling water bath for extraction for 5 min, perform vacuum filtration, transfer the filtrate to a 100 mL volumetric flask, wash the residue with a small amount of hot distilled water 2-3 times, filter it into the volumetric flask, and make up to 100 mL with distilled water after cooling. Each treatment group is repeated 3 times for the determination of tea soup indexes. The sensory evaluation is carried out by a 5-person group composed of senior tea tasters from Guangxi Zhaoping Jiangjunfeng Group Co., Ltd. with reference to the standard of 5.3.2.3 Dark Tea (Loose Tea) in GB / T23776-2018 "Methods for Sensory Evaluation of Tea", and a suitable scoring standard is formulated in combination with the unique quality of Liubao tea (with a total score of 100 points, and the respective proportions of appearance, aroma, soup color, taste and pile flavor factors are 10%, 30%, 20%, 30% and 10%). The sensory evaluation of Liubao tea is carried out.

[0040] Example 1:

[0041] Preparation of bacterial liquid: After activating the freeze-dried bacterial powder of Eurotium cristatum with the preservation number of CGMCC NO.11040, streak and inoculate it into PDA medium, and culture it in the dark at 25-30 °C for 6-8 days. Under aseptic conditions, scrape the spores of the strain into 100 mL of sterile water containing glass beads with a sterile spatula, and oscillate at 28 °C and 200 r / min for 30 min to obtain a uniform spore suspension. Count the spore suspension and adjust the concentration to 6×10 5 CFU / mL. Pile fermentation: Use sun-dried green tea as the test tea sample, 5 kg of tea leaves for each sample, add a certain amount of water to wet the tea leaves to control the water content of the tea leaves at 20%. After 24 h of pile fermentation, inoculate the bacterial liquid, evenly spray the treated bacterial liquid on the treated green tea, and carry out pile fermentation under clean natural environmental conditions, with natural fermentation of wet tea without inoculation as the control. During the pile fermentation process, turn the pile in a timely manner, turn the pile once every 4-5 days, inoculate the Eurotium cristatum spore suspension (inoculated fermentation) and sterile water (natural fermentation), the inoculation amount is 1% (50 mL of Eurotium cristatum liquid), and ferment naturally for 40 days to obtain inoculated fermentation and natural fermentation Liubao tea.

[0042] Aging: The tea leaves after piling up need to be spread out and evenly piled, and naturally cooled to below 40°C. The temperature and humidity are made uniform by turning the pile. Then, high-pressure steam treatment is carried out: Treat with 0.3 MPa saturated steam for 90 seconds to raise the moisture content of the tea leaves to 18%, and the pectin is fully dissolved. At this time, the ductility of the leaves is the best. After cooling to room temperature, secondary inoculation (Jinhua fungus suspension) is carried out, and the inoculation amount is 1%.

[0043] Pressing: The samples in this embodiment are all stored in the form of pressed tea. After secondary inoculation, mold pressing is carried out: 1.5 kg of tea leaves are loaded into a 304 stainless steel mold, and shaped at 12 MPa pressure for 30 seconds in an 80°C environment to form a standard brick-shaped pressed tea of 20×14×3 cm. It is packaged with cotton paper and placed in a constant temperature and humidity box. After aging for 4 months, the tea samples are collected.

[0044] In the aging stage, an exploration experiment on aging temperature and aging humidity is carried out:

[0045] Exploration of aging temperature: After natural fermentation and inoculation fermentation, the Liubao tea enters the aging stage. According to the tea-making experience of relevant tea experts and referring to relevant literature, the optimal aging temperature of Liubao tea is determined to be between 22°C and 30°C. Five temperature gradients are set, namely 22°C, 24°C, 26°C, 28°C, and 30°C. They are placed in a constant temperature and humidity box. After aging for 4 months, the tea samples are collected.

[0046] Exploration of aging humidity: After natural fermentation and inoculation fermentation, the Liubao tea enters the aging stage. According to the tea-making experience of relevant tea experts and referring to relevant literature, the optimal aging humidity of Liubao tea is determined to be between 70% and 90%. Five humidity gradients are set, namely 70%, 75%, 80%, 85%, and 90%. They are placed in a constant temperature and humidity device. After aging for 4 months, the tea samples are collected.

[0047] Table 1 Experimental design of Liubao tea piling up and aging process

[0048]

[0049] Evaluation of test results:

[0050] Sensory evaluation

[0051] It is found through the results of sensory evaluation (in accordance with GB / T 23776-2018) that different inoculation processes and aging conditions have significant effects on the sensory quality of Liubao tea (Tables 1-4). The D4 treatment group (Jinhua fungus inoculation + 26°C + 85%) has the highest comprehensive score (88.7±1.9 points), and its quality characteristics are: the dry tea is tight, smooth and shiny, with a strong aged aroma, the soup color is red, thick and bright, and the taste is mellow; while the A1 treatment group (natural fermentation + 22°C + 80%) has the lowest comprehensive score (76.2±2.5), and the main defects are: the dry tea is tight but lacks luster, the piled taste is obvious, the bitterness and astringency are strong and the aftertaste is slow.

[0052] By comparing the sensory scores of single-strain inoculation fermentation (TF) and natural fermentation (NF), it was found that the sensory score of fermented by Eurotium cristatum (TF) was better than that of natural fermentation (NF). The fermentation process of Eurotium cristatum (TF) could significantly improve the comprehensive quality of Liubao tea. It was explored and found that under the fermentation process conditions of inoculating with Eurotium cristatum, 26°C to 28°C was the optimal range for the aging temperature, and 80% to 85% humidity was the optimal range for the aging humidity. The sensory score reached the peak within this range, probably because the medium temperature could promote the growth of dominant bacteria and at the same time inhibit miscellaneous bacteria (such as Penicillium). At the same time, high temperature was prone to cause deterioration; high humidity was beneficial to the survival and metabolism of complex flora.

[0053] Table 2 Effects of different piling and aging conditions on the sensory quality of Liubao tea

[0054]

[0055] Effects of different aging temperatures on nutritional components:

[0056] The nutritional quality components of Liubao tea soup were measured. Different piling processes (natural fermentation, inoculation fermentation with Eurotium cristatum) and aging temperatures (22 - 30°C) had significant effects on the nutritional quality indicators of Liubao tea, such as Figure 2 shown (in A - F, when comparing five different temperatures, the humidity was 80% for all). The content of water extract showed a fluctuating change with temperature. Both the natural fermentation and inoculation fermentation with Eurotium cristatum processes reached the peak in the 28°C range; the content of tea polyphenols showed a fluctuating change with the increase of temperature. The inoculation fermentation with Eurotium cristatum process reached the peak (0.83 mg / mL) at 28°C, which was higher than the natural fermentation process; free amino acids were the main umami substance components in the tea soup. Both the inoculation fermentation with Eurotium cristatum and natural fermentation accumulated to the highest value at 28°C; the content of soluble sugar increased with the increase of temperature in the low and medium temperature ranges, and both processes reached the peak in the 26 - 28°C range; the ratio of (theaflavin + thearubigin) / theabrownin was an important chemical index to measure the fermentation degree, quality characteristics and sensory characteristics of tea, especially crucial in the processing and aging processes of fermented teas such as black tea and dark tea. The lower the ratio, the higher the conversion degree of theabrownin and the more suitable for storage. This ratio reached the lowest value at 30°C under both piling fermentation processes, indicating that high temperature might be beneficial to the conversion and accumulation of theabrownin in Liubao tea; the phenolic ammonia ratio (the ratio of tea polyphenols to amino acid content) of the tea soup was one of the core indexes of its taste quality, and its numerical range was closely related to the post-fermentation process and aging characteristics of dark tea. The phenolic ammonia ratio entered the optimal palatability range (30:1) at 28°C under the inoculation process with Eurotium cristatum, with a mellow and smooth taste and a significant aged aroma.

[0057] The above tests showed that, in terms of the content of tea polyphenols, umami substances (free amino acid content, soluble sugar content), and chromaticity and taste indexes, the fermentation process inoculated with Eurotium cristatum was superior to the natural fermentation process. The study found that for the fermentation process inoculated with Eurotium cristatum, 28 °C was the key temperature window for quality formation. Too low temperature (22 °C) and too high temperature (30 °C) were not conducive to the retention of nutrients.

[0058] Effect of different aging humidities on nutrient components:

[0059] Under the condition of a constant temperature of 26 °C, for Liubao tea using the fermentation process inoculated with Eurotium cristatum and the natural pile-fermentation process, the quality of its nutrient components showed significant differences with the aging humidity (70% - 90%), as Figure 3 shown (in A - F, the fixed temperature is 28 °C). For the natural fermentation process, the content of water extract reached the peak at an aging humidity of 80%; for the fermentation process inoculated with Eurotium cristatum, the content of water extract reached the peak in the range of 85% - 90% of aging humidity, and was higher than that of the natural fermentation. This may be attributed to the efficient dissociation of cell wall polysaccharides by the composite glycosidase secreted by the Eurotium cristatum population; the content of tea polyphenols showed a parabolic change with the increase of humidity, reaching the peak at 80% humidity for the natural fermentation; for the fermentation process inoculated with Eurotium cristatum, it reached the peak at 85% humidity, and was significantly higher than that of the natural fermentation, and decreased at higher humidity conditions. This may be due to the aggravation of non-enzymatic oxidation in the high-temperature and high-humidity environment, which may cause the loss of phenolic substances to a certain extent; for the natural fermentation process, the contents of free amino acids and soluble sugars reached the peak at an aging humidity of 80%; for the fermentation process inoculated with Eurotium cristatum, they reached the peak at 85% humidity; under the conditions of the natural fermentation process, the ratio of (theaflavin + thearubigin) / theabrownin reached the lowest at 90% humidity; under the conditions of the fermentation process inoculated with Eurotium cristatum, it reached the lowest at 85% humidity, indicating that this humidity was the most suitable for the transformation and accumulation of theabrownin; under the conditions of the fermentation process inoculated with Eurotium cristatum, the phenolic ammonia ratio of the tea soup was close to the optimal palatability range (30:1) at 85% humidity, and was significantly higher than that of the natural fermentation (p < 0.05).

[0060] This data revealed that, in terms of the content of tea polyphenols, umami substances (free amino acid content, soluble sugar content), and chromaticity and taste indexes, the fermentation process inoculated with Eurotium cristatum was superior to the natural fermentation process. At the same time, the exploration found that for the natural fermentation process, 80% humidity was the key humidity window for quality formation; for the fermentation process inoculated with Eurotium cristatum, 85% humidity was the key humidity window for quality formation. At this humidity, the optimization of water extract and umami substance components could be achieved. At the same time, too low humidity (70%) should be avoided during production, as this humidity was not conducive to the retention of nutrient quality components during the aging of Liubao tea.

[0061] Compare the nutrient component contents under the optimal conditions of natural fermentation (22 °C, 80%) and the fermentation process inoculated with Eurotium cristatum (28 °C, 85%), as Figure 4As shown, in terms of the content of water extract, tea polyphenols, umami substances (free amino acid content, soluble sugar content), and chromaticity and taste indexes, the inoculation and fermentation process of Eurotium cristatum is superior to the natural fermentation process.

[0062] Non-target metabolomics analysis

[0063] Analysis of metabolic profile differences in Liubao tea of different qualities fermented with inoculation of Eurotium cristatum:

[0064] In this experiment, the UHPLC-Q-Exactive / MS technology was used to detect the samples of Liubao tea obtained by inoculation and fermentation with Eurotium cristatum. Combining the sensory evaluation results and the content of their nutritional components, two groups with the best and worst comprehensive qualities were selected, namely 28°C + 85 and 22°C + 80% (A1).

[0065] Principal component analysis (PCA) was performed on the metabolites of the two groups of Liubao tea, and the results are as Figure 5 shown. Under the positive ion mode, the abscissa PC1 and the ordinate PC2 explained 82.30% and 4.87% of the differences respectively. Under the negative ion mode, the abscissa PC1 and the ordinate PC2 explained 86.4% and 4.65% of the differences respectively. The distance between each coordinate point represents the aggregation and dispersion degree between samples. The closer the distance, the higher the similarity between samples, and the farther the distance, the greater the difference between samples. Under the positive ion and negative ion modes, the distribution ellipses of each group showed obvious separation, indicating the existence of differential metabolites between the two groups.

[0066] OPLS-DA analysis was performed on different types of dark tea, as Figure 6 shown. Under the positive ion and negative ion modes, the distribution ellipses of each group showed obvious separation. Q 2 and R 2 Y were used to evaluate the modeling ability and prediction ability of the model. The closer R 2 Y and Q 2 are to 1, the more stable and reliable the model is. Under the positive ion mode, R 2 Y and Q2 were 0.997 and 0.724 respectively. Under the negative ion mode, R 2 Y and Q 2 were 0.996 and 0.752 respectively. Therefore, this OPLS-DA model is stable and reliable. Then, permutation test was used to verify the reliability of the model. Under the positive ion mode, the intercept of the Q 2 regression line was -0.8071, and under the negative ion mode it was -0.5341. Q 2 was negative, indicating that the prediction ability of the random model was significantly lower than that of the original model, excluding the risk of overfitting.

[0067] Analysis of differential metabolites in Liubao tea of different qualities fermented with inoculation of Eurotium cristatum:

[0068] Under positive and negative ion modes, a total of 758 metabolites were screened through the OPLS-DA model (VIP > 1, p < 0.05) as potential biomarkers between them. Among them, the abundances of 427 metabolites were up-regulated and those of 331 metabolites were down-regulated( Figure 7 Figure A).

[0069] Metabolomics explored the tea soup metabolites of the aforementioned optimal treatment group (28°C + 85%) and the worst treatment group (22°C + 80%), and further analyzed the reasons for the differences. The temperature and humidity of the superior group were higher than those of the inferior group.

[0070] Statistics were performed on the top 30 differential metabolites in terms of VIP values between the two groups, as Figure 7 . Each row in the left heat map represents a metabolite, and different colors represent the expression levels. The right side is the VIP bar chart of the metabolites. The bar length represents the contribution value of the metabolite to the difference between the two groups. The larger the contribution value, the greater the difference between the two groups. The bar color represents the p-value of the metabolite in, and the smaller the p-value, the darker the color.

[0071] In the high-humidity group (Group A1), significant up-regulation of the expression levels of triterpenoids and esters was observed. Benzylideneacetone was significantly accumulated. It can be used as a food flavor, adding a unique aroma to Liubao tea, enhancing the richness and uniqueness of the aroma of Liubao tea, and making it more flavor-recognizable; at the same time, it can be used as an anti-volatilizer, helping to slow down the volatilization of the aroma components in Liubao tea and maintaining the persistence of the aroma during storage.

[0072] Isohydroauroglaucin was significantly accumulated in the high-humidity group. As a secondary metabolite of microorganisms, it may interact with the microbial community during the fermentation process of Liubao tea, affecting the fermentation process and quality formation. For example, it may have potential effects on aspects such as the flavor and color of tea leaves. The expression level of 3β-Acetoxy-11α-Methoxy-12-Ursen-28-Oic Acid was significant. It belongs to triterpenoids and generally has biological activities such as antioxidant, anti-inflammatory, and immune regulation. This substance may endow Liubao tea with certain health care effects and enhance its quality added value. In addition, the significantly up-regulated metabolite also includes N-MyristoylGlutamine. Glutamine participates in the composition of proteins and peptides, may affect the taste coordination and mellow taste of Liubao tea, further optimize the taste performance, add nutritional components to Liubao tea, and enhance the quality connotation.

[0073] The KEGG topology analysis method was used to systematically analyze the metabolic pathway characteristics of Liubao tea, as Figure 8As shown in the figure. The horizontal axis (Impact Value) represents the degree of influence of metabolic pathways on the global network, and the vertical axis (-Log(P value)) reflects the statistical significance of the pathways. The depth of the point color is positively correlated with the Impact Value. The analysis results show that 21 metabolic pathways reach the significant regulation threshold (Impact value ≥ 0.15, p < 0.05), among which 5 core pathways play important roles in the formation of the quality of Liubao tea, namely alanine, aspartate and glutamate metabolism, caffeine metabolism, aminoacyl-tRNA biosynthesis, α-linolenic acid metabolism, and arachidonic acid metabolism.

[0074] The regulatory effects of different metabolic pathways on the aging process of Liubao tea show significant differences. Alanine, aspartate and glutamate metabolism are involved in important processes such as nitrogen metabolism in tea leaves, affecting the synthesis of nutritional components, flavors (such as umami, etc.) and aroma substances in tea leaves. Aminoacyl-tRNA biosynthesis plays a key role in the process of protein synthesis, and its changes may affect the synthesis and metabolism of protein-related components in Liubao tea, and thus affect the quality of tea, such as the synthesis of flavor substances, etc. α-Linolenic acid is an unsaturated fatty acid, and its metabolism may be somewhat related to the formation of aroma substances in Liubao tea (such as certain volatile aldehydes, alcohols, etc.). Arachidonic acid metabolism can produce a variety of bioactive substances in organisms, and its metabolic changes in Liubao tea may have potential effects on the flavor and quality of tea.

[0075] The content of nutritional components in natural fermentation is generally lower than that in inoculated fermentation; the inoculated fermentation process strengthens the enzyme activity through exogenous bacteria, and the accumulation rates of free amino acids and soluble sugars at 28°C are higher than those in natural fermentation. Under the inoculation process of Eurotium cristatum, the phenolic ammonia ratio enters the optimal palatability range (30:1) at 28°C, with a mellow and smooth taste and a significant aged aroma.

[0076] Example 2

[0077] Detect the functional components of the existing aged tea, and the results are shown in the following table.

[0078] Table 3 Contents of functional components in the tea soup of Liubao tea with different aging years and different types of dark tea (mg / mL)

[0079]

[0080] Note: Different letters indicate significant differences (p < 0.05). LPT1: Liubao tea aged for 1 year; LPT4: Liubao tea aged for 4 years; LPT7: Liubao tea aged for 7 years; LPT10: Liubao tea aged for 10 years. HZ10: Black brick tea aged for 10 years, China Tea Anhua No. 1 Tea Factory Co., Ltd.; PR10: Pu-erh tea aged for 10 years, China Tea Anhua No. 1 Tea Factory Co., Ltd.

[0081] This experiment only aged for 4 months, and the nutrient content was close to that of Liubao tea aged for 1 year, exceeding the effect of conventional aging. Compared with conventional aging, the nutrient content (health care components and chromaticity and taste indexes) increased during accelerated aging, and the sensory score increased compared with the tea naturally fermented without Eurotium cristatum in the A1-B5 group.

[0082] Example 3

[0083] This example provides a process for accelerating the aging of Liubao tea by inoculating Eurotium cristatum, including the following steps:

[0084] S1 Bacterial liquid preparation: After activating the freeze-dried powder of Eurotium cristatum with a single strain with the preservation number of CGMCC NO.11040, streak inoculate it into the PDA medium, and culture it in the dark at 25-30 °C for 7 days. Under sterile conditions, scrape the spores of the strain into 100 mL of sterile water containing glass beads with a sterile spatula, and oscillate it at 28 °C and 200 r / min for 30 min to obtain a uniform spore suspension. Count the spore suspension and adjust the concentration to 6×10 5 CFU / mL.

[0085] S2 Pile-fermentation: Add water to the sun-dried rough tea to a moisture content of 18-22%, inoculate the spore suspension obtained in step S1 after pile-fermentation for 22-25 hours, and pile-ferment in the natural environment for 38-42 days; Take 5 kg of sun-dried rough tea as one portion, wet the tea to control the moisture content of the tea at 20%. After pile-fermentation for 24 h, inoculate the spore suspension obtained in step S1, and the inoculation amount is 1% (50 mL of Eurotium cristatum suspension); Spray the spore suspension evenly on the treated rough tea, and carry out pile-fermentation in a clean natural environment. During the pile-fermentation process, turn the pile in a timely manner, turn the pile once every 4-5 days, and naturally ferment for 40 days.

[0086] S3 Aging: The tea leaves after pile-fermentation need to be spread out and evenly piled, and naturally cooled to below 40 °C, and the temperature and humidity are made uniform by turning the pile. Then carry out high-pressure steam steaming: Treat with 0.3 MPa saturated steam for 90 seconds to raise the moisture content of the tea leaves to 18%, and the pectin is fully dissolved. At this time, the ductility of the leaves is the best. After cooling to room temperature, carry out secondary inoculation (50 mL of Eurotium cristatum suspension), and the inoculation amount is 1%.

[0087] Pressing: Carry out die pressing. Load 1.5 kg of tea leaves into a 304 stainless steel mold, and keep it at a pressure of 12 MPa at 80 °C for 30 seconds for shaping to form a standard brick-shaped pressed tea of 20×14×3 cm, and pack it with cotton paper.

[0088] The Liubao tea after secondary inoculation fermentation and pressing enters the aging stage and is placed in a constant temperature and humidity box for 4 months. The temperature during the aging stage is 28 °C and the humidity is 85%

[0089] The dry tea prepared in this embodiment shows obvious golden tips, has a rich and mellow aged aroma, a bright red soup color, and a mellow taste.

[0090] Although the present invention has been described above through embodiments, those skilled in the art should understand that any improvements and modifications made to the present invention without departing from the spirit and essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A process for accelerating the aging of Liubao tea by inoculating with Chrysanthemum morifolium Ramat., characterized in that, It includes the following steps: Preparation of S1 bacterial liquid: A spore suspension of Eurotium cristatum is obtained through activation culture, and the concentration of the spore suspension is 1-10×10 5 CFU / mL; S2 Pile-fermentation: Add water to the sun-dried green tea to make the water content reach 18-22%, inoculate the spore suspension obtained in step S1 after pile-fermentation for 22-25 hours, and then carry out pile-fermentation and fermentation in the natural environment for 38-42 days; S3 Aging: Spread out the tea leaves after pile-fermentation to cool naturally, and then carry out high-pressure steam treatment to make the water content of the tea leaves reach 17-19%; after cooling to room temperature, inoculate the spore suspension again and age for 120-125 days.

2. The process for accelerating the aging of Liubao tea by inoculating with Chrysanthemum morifolium Ramat. according to claim 1, characterized in that, In step S1, after activating the freeze-dried powder of Eurotium cristatum, streak inoculate it into a PDA medium, and culture it in the dark at 25-30 °C for 6-8 days. Scrape the spores of the cultured strain into sterile water, and oscillate it at 25-30 °C and 100-400 r / min for 20-40 min to obtain a uniform spore suspension. Count the spore suspension and adjust the concentration to 5-8×10 5 CFU / mL.

3. The process for accelerating the aging of Liubao tea by inoculating with Chrysanthemum morifolium Ramat. according to claim 1, characterized in that, In step S2, the sun-dried green tea is made from the leaves of Camellia sinensis var. assamica cv. Cangwu population, and its preparation process includes the steps: 1) Fresh leaf picking: Pick one bud with two to three leaves of Camellia sinensis var. assamica cv. Cangwu population; 2) Withering regulation to a water content of 65%-70%; 3) High-temperature fixation of enzymes at a temperature of 220-280°C; 4) Gradient rolling to a cell breakage rate of 40%-50%.

4. The process for accelerating the aging of Liubao tea by inoculating with Flos Chrysanthemi Indici bacteria according to claim 1, characterized in that, In step S2, the inoculation amount of the spore suspension of Eurotium cristatum is 1-1.5%; turn the pile once every 4-5 days during the pile-fermentation process.

5. The process for accelerating the aging of Liubao tea by inoculating with Chrysanthemum morifolium Ramat. according to claim 1, wherein, In step S3, the tea leaves after pile-fermentation are spread out and evenly piled, cooled naturally to below 40°C, and the temperature and humidity are made uniform by turning the pile; then carry out high-pressure steam treatment: treat with 0.3-0.5 MPa saturated steam for 60-100 seconds to raise the water content of the tea leaves to 17-19%.

6. The process for accelerating the aging of Liubao tea by inoculating with Chrysanthemum morifolium Ramat. as claimed in claim 1, wherein, In step S3, the inoculation amount of the re-inoculation of the spore suspension is 1-1.5%.

7. The process for accelerating the aging of Liubao tea by inoculating with Chrysanthemum morifolium Ramat. as claimed in claim 1, wherein In step S3, after the second inoculation, compacting is carried out. The specific operation is as follows: pressing is performed with a stainless-steel mold, and it is kept at a pressure of 10 - 15 MPa for 25 - 35 seconds at a temperature of 75 - 85 °C for shaping, and it is pressed into a hexahedron with a volume of 800 - 900 cm 3 for each portion, and the mass of each portion is 1.3 - 1.6 kg; cotton paper packaging is adopted.

8. The process for accelerating the aging of Liubao tea by inoculating with Chrysanthemum morifolium Ramat. according to any one of claims 1 to 7, characterized in that, In step S3, the aging temperature is 22°C-30°C, and the aging humidity is 70-90%.

9. The process for accelerating the aging of Liubao tea by inoculating with Chrysanthemum morifolium Ramat. as claimed in claim 8, wherein, In step S3, the aging temperature is 28°C, and the aging humidity is 85%.

10. Liubao tea prepared by the process according to any one of claims 1-9.

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