A fermentation method for increasing the soluble sugar content of ripe Pu-erh tea and its uses

Abstract

The present invention relates to the technical field of ripe tea processing, and particularly relates to a fermentation method for increasing the soluble sugar content of Pu-erh ripe tea and its uses. During the piling fermentation process of the present invention, non-toxin-producing Saccharomyces cerevisiae is inoculated for fermentation, thereby controlling the processing of Pu-erh ripe tea by microorganisms. The present invention discovers that the inoculated bacteria become the dominant bacteria and participate in the fermentation of Pu-erh ripe tea. No mycotoxins are detected in the Pu-erh ripe tea prepared by the method of the present invention. The number of Saccharomyces cerevisiae colonies in the Pu-erh tea fortified with the inoculated Saccharomyces cerevisiae fermentation is significantly increased compared with the control group, it is rich in Saccharomyces cerevisiae, and the soluble sugar content is increased. The sensory quality of the Pu-erh ripe tea prepared by the method of the present invention shows that the aroma is a special mushroom aroma, the taste shows a sense of heaviness, smoothness, sweetness and aftertaste are increased compared with the control tea, while the bitterness, astringency and sourness are significantly reduced compared with the control tea.

Description

Technical Field

[0001] The present invention relates to the technical field of ripe tea processing, and particularly relates to a fermentation method for increasing the soluble sugar content of Pu-erh ripe tea and its uses. Background Art

[0002] Post-fermented pu-erh tea is made from sun-dried green tea by using specific processing techniques, and is a tea with quality characteristics such as red and thick (brown) soup color, mellow (smooth) taste, and obvious aged fragrance. Pu-erh tea is a national geographical indication protected product, and has become the most characteristic and advantageous tea product in Yunnan. It is an important object for the construction of the plateau characteristic agricultural industry and the green food brand.

[0003] The post-fermentation of modern Pu-erh tea is a process of adding appropriate water to sun-dried green tea, mixing evenly, piling up, and turning the pile several times according to the fermentation process, which may last for 40 - 60 days. From the perspective of microbiology or food processing, Pu-erh tea is a unique microbial fermented tea, and post-fermentation is the key process for the formation of the quality characteristics and health care functions of Pu-erh tea, and is a natural inoculation food fermentation process with tea as the matrix. The natural fermentation of traditional Pu-erh ripe tea has the basic characteristics of a wide variety of microbial species and diverse functions, and is a typical complex fermentation process carried out by a combination of multiple microbial communities; it has the basic characteristics of a wide variety of microbial species and diverse functions. In addition, due to the use of the natural inoculation method for fermentation, there are problems such as uncontrollable process and being extremely susceptible to the influence of the external environment, resulting in unstable product quality and even food safety risks, which seriously restricts the development of the industry and urgently needs to be changed. Strengthening inoculation or controlled fermentation is one of the effective ways to solve the uncontrollability of the Pu-erh tea fermentation process.

[0004] Saccharomyces cerevisiae is a single-celled microorganism with a short growth cycle and strong reproductive ability. As the earliest domesticated microorganism by humans, Saccharomyces cerevisiae is widely used in different food matrices and has an important impact on the stability, sensory characteristics, nutrition and health of food. Although Saccharomyces cerevisiae has also been found in the Pu-erh tea fermentation process or products, no research has been found on using Saccharomyces cerevisiae to carry out enhanced fermentation of Pu-erh tea and increase its soluble sugar content.

[0005] Previous studies have conducted pure culture inoculation fermentation tests on the dominant yeast strains isolated from Pu-erh tea pile-fermentation samples. The results show that the tea samples fermented by pure yeast strains have a sweet aroma, a slightly bitter taste, produce saliva, and have a sweet aftertaste. They can effectively reduce the tea polyphenol value of raw tea and increase the content of tea brown pigment. The metabolites of Saccharomyces cerevisiae can increase the effective nutrients in Pu-erh tea, endow Pu-erh tea with unique quality characteristics, and are beneficial to the formation of quality styles such as sweet, mellow, and fragrant in Pu-erh tea. Strengthened fermentation with Saccharomyces cerevisiae can regulate the flora and its metabolism. The mixed fermentation of Metschnikowia pulcherrima and Saccharomyces cerevisiae has a synergistic effect on the production of flavor substances, producing higher concentrations of fatty acids, ethyl esters, and terpenoids. Saccharomyces cerevisiae causes significant changes in the volatile and non-volatile substances in tea leaves, and the antioxidant capacity remains stable after fermentation. In summary, strengthening the inoculation of Saccharomyces cerevisiae to ferment Pu-erh tea can improve the aroma of Pu-erh ripe tea, increase its sweetness and mellow taste, thereby improving the flavor of Pu-erh tea.

[0006] At present, there is no research on strengthening the fermentation of Pu-erh tea with yeast strains to increase its soluble sugar content. The inventor has conducted research on strengthening the fermentation of Pu-erh tea with Saccharomyces cerevisiae PETY001 and developed a fermentation method and its use for increasing the soluble sugar content of Pu-erh ripe tea. Summary of the Invention

[0007] In order to overcome the defects of the prior art and avoid the instability of the quality and safety risks caused by uncontrollable fermentation microorganisms in Pu-erh ripe tea, the present invention provides a fermentation method and its use for increasing the soluble sugar content of Pu-erh ripe tea.

[0008] Specifically, the present invention is realized through the following technical solutions:

[0009] In the first aspect, the present invention provides a fermentation method for increasing the soluble sugar content of Pu-erh ripe tea. The fermentation method includes the following steps: during the third and fourth turning piles in the Pu-erh tea fermentation process, continuously inoculate the fermentation agent of Saccharomyces cerevisiae PETY001 for strengthened fermentation to obtain a Pu-erh ripe tea with an increased soluble sugar content. The Saccharomyces cerevisiae PETY001 is classified and named as Saccharomyces cerevisiae, with the preservation number of CGMCC No. 26063, the preservation time of November 07, 2022, and the preservation location of the China General Microbiological Culture Collection Center (CGMCC). The address of the preservation unit is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] As an alternative, in the above fermentation method, the fermentation method comprises the following steps:

[0011] (1) Using sun-dried green tea of Yunnan big-leaf tea tree as raw material, weigh 30 kg of sun-dried green tea, add 9 L of distilled water according to 30% of the raw material quality in terms of moisture content, stir and mix evenly, and then put it into a 304 stainless steel fermentation frame for fermentation;

[0012] (2) Insert intelligent temperature recorders at different parts of the tea pile to monitor and record the temperature. When the temperature of the tea pile is above 55 °C, untie the tea pile to cool down, mix the tea leaves, sprinkle water during the mixing process to keep the moisture content of the tea leaves at about 30%, and turn over the mixed tea leaves and stack them up together;

[0013] (3) Control the temperature and the moisture content of the tea leaves. Every 5 - 7 days, perform the operations of untie the pile, sprinkle water, mix evenly, and re-stack;

[0014] (4) When turning the pile for the third time during fermentation, add 60 mL of Saccharomyces cerevisiae PETY001 bacterial suspension, mix evenly and continue fermentation for 7 days; continue to inoculate with the fermenting agent. When turning the pile for the fourth time during fermentation, add 60 mL of Saccharomyces cerevisiae PETY001 bacterial suspension, mix evenly and continue fermentation for 7 days, then stop fermentation, spread the tea leaves to a thickness of 5 - 6 cm, and naturally dry until the moisture content is lower than 12%.

[0015] As an alternative, in the above fermentation method, the preparation method of the Saccharomyces cerevisiae PETY001 bacterial suspension comprises the following steps:

[0016] Inoculate Saccharomyces cerevisiae PETY001, which is isolated from the fermentation of Pu-erh ripe tea and has been detected not to produce mycotoxins, into a wort agar medium, culture it at 28 °C for 72 h, elute the strain with sterile physiological saline, after counting, adjust the strain concentration to 1×10 7 CFU / mL with sterile physiological saline. Subsequently, inoculate 1 mL of the bacterial suspension into 100 mL of tea medium, culture it with shaking at 28 °C and 120 r / min for 2 d. After counting the bacteria, adjust the bacterial concentration to 1×10 7 CFU / mL with sterile physiological saline to obtain the Saccharomyces cerevisiae PETY001 bacterial suspension.

[0017] As an alternative, in the above fermentation method, the preparation method of the tea medium comprises the following steps:

[0018] Take 50 g of sun-dried green tea, add 500 mL of distilled water, extract it with boiling water for 30 min, then filter it under reduced pressure. Continue to add 500 mL of distilled water, extract it with boiling water, and then filter it under reduced pressure and make up the volume to 1000 mL, dispense it, and sterilize it at 121 °C for 20 min.

[0019] As an alternative, in the above fermentation method, the third turning of the pile is carried out about 20 days after the start of fermentation, and the fourth turning of the pile is carried out about 27 days after the start of fermentation.

[0020] As an alternative, in the above fermentation method, the fermentation method further comprises the following steps:

[0021] (5) After stopping fermentation, when the tea pile is untied, samples are taken from the upper, middle and lower layers respectively and mixed evenly. The separation culture, plate colony counting method and ITS amplicon sequencing method are used to detect the mycotoxins, Saccharomyces cerevisiae colonies and soluble sugar content of the fermented tea samples.

[0022] In the second aspect, the present invention provides Pu-erh ripe tea with increased soluble sugar content prepared by using the fermentation method described in the first aspect above. The Pu-erh ripe tea does not contain mycotoxins, the Saccharomyces cerevisiae colonies and the soluble sugar content are increased, and the sensory quality shows that the aroma is a special mushroom aroma, the taste shows a heavy texture, smoothness, sweetness and aftertaste are increased, while the bitterness, astringency and sourness are significantly reduced.

[0023] In the third aspect, the present invention provides a Saccharomyces cerevisiae PETY001, which is used to prepare the Pu-erh ripe tea with increased soluble sugar content described in the second aspect above. The taxonomic name of the Saccharomyces cerevisiae PETY001 is Saccharomyces cerevisiae, the preservation number is CGMCC No. 26063, the preservation time is November 7, 2022, and the preservation location is the China General Microbiological Culture Collection Center (CGMCC), and the address of the preservation unit is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

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

[0025] (1) During the pile-fermentation process of the present invention, non-toxin-producing Saccharomyces cerevisiae is inoculated for fermentation, and then the microorganisms are controlled to process Pu-erh ripe tea. The present invention uses the methods of separation culture, plate colony counting method, ITS amplicon sequencing method and mycotoxin detection to detect the Pu-erh ripe tea prepared by the present invention, and to detect the microbial community composition of the fermented tea samples. It is found that the inoculated bacteria have become the dominant bacteria and participated in the fermentation of Pu-erh ripe tea.

[0026] (2) The Pu-erh ripe tea prepared by the method of the present invention does not detect mycotoxins. The Saccharomyces cerevisiae colonies in the Pu-erh tea strengthened by inoculating Saccharomyces cerevisiae are significantly increased compared with the control group, rich in Saccharomyces cerevisiae, and the soluble sugar content is increased.

[0027] (3) The sensory quality of the Pu-erh ripe tea prepared by the method of the present invention shows that the aroma presents a special mushroom aroma, and the taste shows a thick texture, smoothness, sweetness and aftertaste, which are increased compared with the control tea, while the bitterness, astringency and sourness are significantly reduced compared with the control tea. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Photos of plates with partial fungal colony counts.

[0029] Figure 2 : Sensory characteristics of the fermented sample's dry tea, tea soup and tea leaves. Among them, A. Sensory characteristics of the fermented sample's dry tea, tea soup and tea leaves; B. Color parameters of the fermented sample's tea soup; C. Taste characteristics of the fermented sample's tea soup.

[0030] Figure 3 : 36 tea characteristic components in different tea samples. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the scope of the present invention.

[0032] For those techniques or conditions not specified in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For those reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through regular channels.

[0033] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

[0034] Example 1: A method for fermenting Pu-erh ripe tea rich in Saccharomyces cerevisiae, the specific implementation method is as follows:

[0035] (1) Raw material preparation: Prepare 30 kg of third-grade raw material of sun-dried green tea of large-leaf variety, with a water content of 7-9%.

[0036] (2) Preparation of Saccharomyces cerevisiae PETY001 bacterial suspension: The Saccharomyces cerevisiae PETY001 isolated and purified from the fermentation of Pu-erh ripe tea was inoculated into a malt extract agar medium and cultured at 28 °C for 72 h. The strain was eluted with sterile normal saline. After counting, the strain concentration was adjusted to 1×10 7CFU / mL. For the preparation of the tea medium, 50 g of sun-dried tea is added with 500 mL of distilled water, extracted with boiling water for 30 min, then filtered under reduced pressure. Another 500 mL of distilled water is added, extracted with boiling water, and then filtered under reduced pressure and made up to 1000 mL, dispensed, and sterilized at 121 °C for 20 min. Subsequently, 1 mL of the bacterial suspension is inoculated into 100 mL of the tea medium, and cultured with shaking at 28 °C and 120 r / min for 2 d. After counting the bacteria, the bacterial concentration is adjusted to 1×10 7 CFU / mL to obtain the Saccharomyces cerevisiae PETY001 bacterial suspension.

[0037] (3) Moisture content adjustment: According to 30% of the raw material quality for the moisture content, 9 L of distilled water is sprayed and added to 30 kg of sun-dried tea, stirred and mixed evenly, and then put into a 304 stainless steel fermentation frame for fermentation; intelligent temperature recorders are inserted at different parts of the tea pile to monitor and record the temperature, control the temperature and the moisture content of the tea. Every 5 - 7 days, operations such as piling removal, watering, mixing, and re-piling are carried out; when turning the pile for the third time during fermentation (about 20 days), 60 mL of the Saccharomyces cerevisiae PETY001 bacterial suspension is added, mixed evenly and continue to ferment for 7 days; continue to inoculate with the fermentation agent, when turning the pile for the fourth time during fermentation (about 27 days), 60 mL of the Saccharomyces cerevisiae PETY001 bacterial suspension is added, mixed evenly and continue to ferment for 7 days. Pile fermentation: The pile height is 100 cm - 120 cm, turn the pile once every 5 - 7 days. After turning the pile twice and within 48 hours after turning the pile, the temperature needs to be controlled at 60 °C, and the temperature of 60 °C cannot exceed 72 hours. If the temperature exceeds 60 °C for more than 72 h, the pile can be turned.

[0038] (4) Natural drying: After spreading the fermented ripe tea wool material for 5 - 6 m, it is naturally dried until the moisture content is lower than 12%.

[0039] (5) Dry spreading.

[0040] (6) Packaging, finished product.

[0041] Example 2: Colony counting results of Saccharomyces cerevisiae

[0042] The fungal colonies of the fermentation samples were counted using the wort medium to study the growth of fungi during the fermentation process. The total number of fungal colonies of each sample is shown in Table 1, and some counting plates are shown in Figure 1 . It can be seen from Table 1 that the number of fungal colonies in the blank sample is the least, among which CK3 is 4.9×10 4 CFU / g, and CK5 is 4.6×10 4 CFU / g. After artificially inoculating yeast, the number of J3 yeast is 7.8×105CFU / g, and J5 is 6.7×105CFU / g. It can be seen that there are a large number of yeasts in the tea samples fermented with the inoculated yeast after drying, indicating that yeasts can survive during the pile fermentation process of Pu-erh tea and act on the fermentation process.

[0043] Table 1 Yeast colony counts of each tea sample

[0044]

[0045] Example 3: Mycotoxin detection results

[0046] The results of mycotoxin detection in tea samples fermented with yeast are shown in Table 2.

[0047] Table 2 Mycotoxin test results

[0048]

[0049] Example 4: Tea Evaluation Results

[0050] Sensory evaluation of fermented tea samples was conducted. The results of sensory evaluation of tea are shown in Figure 2 A. From the picture, we can see that except for the CK tea sample, which is gray-brown, has stems, and has loose strips, the other tea samples are all dark brown, have stems, and have loose strips. The tea soup color of CK, F2, F3, and F4 is bright reddish brown; the soup color of F2 is bright dark brown. The leaf bottoms are all dark brown, with red stems and red leaves. Figure 2 B The results show that the higher L values of CK (63.35±0.02) and F2 (463.72±1.18) indicate higher brightness; the higher a value of F1 (46.25±0.03) indicates that its soup color is redder and darker; the higher b value of CK (91.56±0.2) indicates that its yellowness is higher.

[0051] The bitterness, astringency, sourness, thickness, sweetness and aroma of the fermented tea samples were scored by Figure 2 C The results showed that the aroma (7.50±0.84), sweetness (6.67±0.82) and thickness (7.00±0.89) of F4 tea samples were higher than those of CK and other inoculated tea samples (P<0.05), indicating that the quality of the tea samples had changed, resulting in a more obvious aroma, higher sweetness and more mellow taste. The sourness score of F4 tea samples (1.17±0.41) was lower than that of CK tea samples, indicating that the composition changes caused by F4 inoculation weakened the sourness of the tea leaves. The aroma (5.33±1.03, 5.83±1.47) and sweetness (5.83±0.41, 5.67±0.52) scores of F1 and F2 tea samples were lower than those of CK tea samples, indicating that the aroma and sweetness of F1 and F2 tea samples were not as good as those of CK tea samples, and the effect of yeast inoculation on the taste of these two samples was not good. In general, the fourth fermentation is enhanced by adding yeast, and the tea tastes best.

[0052] Example 5: Tea ingredient determination results

[0053] To further optimize the inoculation time, the chemical components of the samples, such as water content, water extract, soluble sugar, tea polyphenols, free amino acids, and catechins, were determined (Table 3). After fermentation, the soluble sugar content decreased, and the soluble sugar content of the fermented tea samples was 4.08±0.22%-6.42±0.20%. The soluble sugar content of F1 and F2 decreased significantly compared with CK (P<0.05), suggesting that yeast might consume soluble sugar as a carbon source. The soluble sugar content of F4 increased significantly (P<0.05), suggesting that inoculating yeast at the fourth turning would promote the production of carbohydrates. The tea polyphenol content of the tea samples was 46.20±6.23mg / g-77.40±10.30mg / g. Compared with F2-CK, the tea polyphenol content of F1, F2, and F3 decreased significantly after fermentation with yeast. Among the samples inoculated with yeast, compared with CK, the theabrownin content increased, and the theabrownin content of F1 and F4 increased significantly (P<0.05).

[0054] Table 3 Chemical Component Contents of Tea Samples

[0055]

[0056] Note: There was no significant difference between the groups with the same superscript letters in the experimental results in the table, and there was a significant difference between the groups with different superscript letters, and P<0.05. When comparing each group, if there are both the same and different letters in the superscript, the same letters shall prevail, indicating that there is no significant difference between the groups.

[0057] The contents of catechins and amino acids in tea samples were determined by high performance liquid chromatography ( Figure 3 ). The catechin content of the fermented tea samples was 7.48±0.63mg / g-12.05±1.03mg / g. Compared with CK, the catechin content of F4 increased significantly (P<0.05). Among them, the contents of GC, C, EC, and CG in F4 were significantly higher than those in CK (P<0.05). The flavone and flavonoid glycoside content of the tea samples was 0.02±0.02mg / g-0.10±0.02mg / g. Compared with CK, the contents of taxifolin, myricetin, luteolin, and kaempferol in F4 increased significantly (P<0.05). The phenolic acid content of the tea samples was 0.31±0.12mg / g-1.36±0.17mg / g. Compared with CK, the ellagic acid content of F4 increased significantly (P<0.05). The alkaloid content of the tea samples was 5.53±0.36mg / g-8.42±0.15mg / g. Compared with CK, the caffeine and theophylline contents of F4 increased significantly (P<0.05). The theanine content of the tea samples was 0.34±0.09mg / g-1.02±0.08mg / g. Compared with CK, the theanine and aspartic acid contents of F4 decreased significantly (P<0.05).

[0058] Example 8: Determination Results of Tea Components in Scale-up Experiments

[0059] After determining the inoculum amount, scale-up experiments were carried out. It was found through measurement (see Table 4) that the soluble sugar content of the tea samples inoculated with yeast PETY001 was higher than that of other tea samples (7.38 ± 0.55%).

[0060] Table 4 Chemical Component Contents of Tea Samples

[0061]

[0062] Note: There is no significant difference between the groups with the same superscript letters in the experimental results in the table, and there is a significant difference between the groups with different superscript letters, and P < 0.05.

[0063] Example 9: Comparison of Soluble Sugars in Tea Samples Inoculated with Other Bacteria or Uninoculated Tea Samples Reported in the Literature

[0064] (1) Compared with the tea samples inoculated with Aspergillus niger and their control samples reported, the soluble sugar content of the tea samples inoculated with yeast PETY001 (7.38 ± 0.55%) was higher than that of other tea samples (see Liu Kunyi, Wang Liyan, et al. Research on Fermentation of Pu-erh Tea by Inoculating Aspergillus niger [J]. Journal of Light Industry, 2022, 37(04): 1-9.).

[0065] (2) Compared with the tea samples inoculated with Bacillus licheniformis and their control samples reported, the soluble sugar content of the tea samples inoculated with yeast PETY001 (7.38 ± 0.55%) was higher than that of other tea samples (see Liu Kunyi, Wang Liyan, et al. Quality and Microbial Community Analysis of Pu-erh Tea Fermented by Inoculating Bacillus licheniformis [J]. Journal of Food Science and Technology, 2022, 40(02): 108-118).

[0066] (3) Recent review articles reported that some Aspergillus strains can increase the content of tea polysaccharides in Pu-erh tea. In this study, it was found that compared with the control (5.05 ± 0.73%), the soluble sugar content increased when inoculated with Aspergillus luchuensis (6.43 ± 0.71%), and the soluble sugar content of the tea samples inoculated with Saccharomyces cerevisiae (7.38 ± 0.55%) increased significantly compared with that inoculated with Aspergillus luchuensis (p < 0.05) (see Hao Binxiu, Li Song, Tian Haixia, Ma Yue, Liu Haixin, Wang Chunling. Research Progress on Fermentation Microorganisms of Ripened Pu-erh Tea [J]. Food Research and Development, 2018, 39(08): 203-206).

[0067] (4) Compared with previous patent literatures, in this study, the soluble sugar content of tea samples was mainly increased by inoculating Saccharomyces cerevisiae to enhance fermentation, and the improvement effect was more significant than that of previous patent literatures (the tea polysaccharide content increased by about 5.3%). The soluble sugar content in this experiment increased by 46.14% compared with the control group (see Wu Xiaobin, Sun Zhaoyue, Li Jing, Wu Jie, Yu Yi. Application of a Saccharomyces cerevisiae and Lactobacillus in the fermentation of Pu-erh tea [P]. Shanghai: CN113749164A, December 7, 2021).

[0068] (5) The same Pu-erh ripe tea produced by Anning Haiwan Tea Industry Co., Ltd. in 2012, 2013, and from 2019 to 2021 was collected, and its soluble sugar content was detected. It was found by comparison (Table 5) that the soluble sugar content of tea samples increased significantly by inoculating Saccharomyces cerevisiae to enhance fermentation was significantly higher than that of commercially available tea samples.

[0069] Table 5 Chemical component content of tea samples

[0070]

[0071] Note: There is no significant difference between the groups with the same superscript letters in the experimental results in the table, and there is a significant difference between the groups with different superscript letters, and P < 0.05.

[0072] Example 10: Screening of yeast strains

[0073] The inventors screened 10 strains of yeasts in the laboratory and carried out fermentation experiments with yeasts respectively. It was detected that the soluble sugar content of tea fermented by strain No. 4 was significantly higher than that of tea samples fermented by other yeasts. Strain No. 4 is the Saccharomyces cerevisiae PETY001 used in this patent. The results are shown in Table 6.

[0074] Table 6 Chemical component content of tea samples

[0075]

[0076] Note: There is no significant difference between the groups with the same superscript letters in the experimental results in the table, and there is a significant difference between the groups with different superscript letters, and P < 0.05. When comparing between groups, if there are both the same and different letters in the superscript, take the same letters as the standard, that is, it means there is no significant difference between the groups.

[0077] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and its equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A fermentation method for increasing the soluble sugar content of ripe Pu-erh tea, characterized in that: The fermentation method includes the following steps: during the third and fourth turning operations in the Pu-erh tea fermentation process, Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) PETY001 fermenter is continuously inoculated for enhanced fermentation to obtain a ripe Pu-erh tea with increased soluble sugar content. The Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) PETY001 is taxonomically named Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), with a preservation number of CGMCC No. 26063, a preservation date of November 7, 2022, and a preservation location at the China General Microbiological Culture Collection Center (CGMCC). The address of the preservation unit is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The third turning operation is carried out 20 days after the start of fermentation, and the fourth turning operation is carried out 27 days after the start of fermentation.

2. The fermentation method according to claim 1, characterized in that: The fermentation method comprises the following steps: (1) Using the sun-dried green tea of Yunnan big-leaf tea tree as raw material, weighing 30 kg of sun-dried green tea, adding 9 L of distilled water according to the moisture content of 30% of the raw material quality, stirring and mixing evenly, and then putting it into a 304 stainless steel fermentation frame for fermentation; (2) Inserting intelligent temperature recorders at different parts of the tea pile to monitor and record the temperature. When the temperature of the tea pile is above 55 °C, untie the tea pile to cool down, mix the tea leaves, sprinkle water during the mixing process to keep the moisture content of the tea leaves at about 30%, and turn over the mixed tea leaves and stack them up together; (3) Controlling the temperature and the moisture content of the tea leaves, and performing the operations of untieing the pile, sprinkling water, mixing evenly, and re-stacking every 5 - 7 days; (4) When turning the pile for the third time during fermentation, adding 60 mL of Saccharomyces cerevisiae PETY001 bacterial suspension, mixing evenly and continuing fermentation for 7 days; continuing to inoculate the fermenting agent for fermentation. When turning the pile for the fourth time during fermentation, adding 60 mL of Saccharomyces cerevisiae PETY001 bacterial suspension, mixing evenly and continuing fermentation for 7 days, then stopping fermentation, spreading out the tea leaves to a thickness of 5 - 6 cm, and naturally drying until the moisture content is lower than 12%.

3. The fermentation method according to claim 2, wherein: The preparation method of the Saccharomyces cerevisiae PETY001 bacterial suspension comprises the following steps: The Saccharomyces cerevisiae PETY001, which is isolated from the fermentation of Pu-erh ripe tea and has been detected not to produce mycotoxins, is inoculated into the wort agar medium and cultured at 28 °C for 72 h. The strain is eluted with sterile normal saline. After counting, the strain concentration is adjusted to 1×10 7 CFU / mL with sterile normal saline. Subsequently, 1 mL of the bacterial suspension is inoculated into 100 mL of the tea medium and cultured with shaking at 28 °C and 120 r / min for 2 d. After counting the bacteria, the cell concentration is adjusted to 1×10 7 CFU / mL with sterile normal saline to obtain the Saccharomyces cerevisiae PETY001 bacterial suspension.

4. The fermentation method according to claim 3, wherein: The preparation method of the tea medium comprises the following steps: 50 g of sun-dried green tea, adding 500 mL of distilled water, extracting with boiling water for 30 min, then filtering under reduced pressure, continuing to add 500 mL of distilled water, extracting with boiling water, filtering under reduced pressure and making up the volume to 1000 mL, dispensing, and sterilizing at 121 °C for 20 min.

5. The fermentation method according to claim 2, characterized in that: The fermentation method further comprises the following steps: (5) After stopping fermentation, taking samples from the upper, middle and lower layers respectively when untying the tea pile, mixing evenly, and using the methods of isolation culture, plate colony counting and ITS amplicon sequencing to detect the mycotoxin, Saccharomyces cerevisiae colony and soluble sugar content of the fermented tea sample.

6. The Pu-erh ripe tea with increased soluble sugar content prepared by the fermentation method according to any one of claims 1 to 5, characterized in that: The Pu-erh ripe tea does not contain mycotoxin, the Saccharomyces cerevisiae colony and soluble sugar content increase, and the sensory quality shows that the aroma presents a special mushroom aroma, the taste shows a stronger sense of heaviness, smoothness, sweetness and aftertaste, while the bitterness, astringency and sourness are significantly reduced.

Images