Emericella coronata tea beverage and method for preparing the same

Abstract

The application discloses a Eurotium cristatum tea beverage and a preparation method thereof, and belongs to the fields of food biotechnology and functional beverages. The method innovatively uses tea leaf extraction liquid as the only nutrient medium, accesses Eurotium cristatum to perform full liquid submerged fermentation, and obtains a fully fermented tea beverage through heat treatment and solid-liquid separation. The process discards the traditional solid-state fermentation mode, changes the fermentation system into a homogeneous system, realizes accurate control of fermentation parameters, realizes in-situ efficient enrichment and complete water solubility of functional components, and solves the problems of low dissolution rate of active components, poor product stability, large batch difference and long process in the prior art. The process has high standardization, low production cost and green efficiency, is suitable for various teas, provides a brand-new technical scheme for functionalization and beverage application of Eurotium cristatum, and has broad industrialization prospects.

Description

Technical Field

[0001] This invention relates to the fields of food biotechnology and functional beverages, and particularly to a *Aspergillus cristatus* tea beverage and its preparation method. Background Technology

[0002] *Eurotium cristatum* is a characteristic probiotic fungus in traditional fermented teas (such as Fu brick tea and Qianliang tea). Its mycelium and ascospores form tiny golden-yellow cleistothecia on the surface of the tea leaves, commonly known as "golden flowers," and are the core microorganisms that determine the unique flavor, color, and functionality of traditional fermented teas. Modern pharmacological studies have confirmed that *Eurotium cristatum* can produce a variety of active metabolites during its growth and metabolism, including fungal polysaccharides, polyphenols, cellulases, pectinases, and polyphenol oxidases. These metabolites not only improve the taste and flavor of tea but also have multiple probiotic functions, such as regulating the balance of human intestinal flora, scavenging free radicals, antioxidation, and enhancing the body's immunity. Therefore, *Eurotium cristatum*-related products have broad application prospects in the field of functional foods and beverages.

[0003] Currently, the application of *Aurorus cristatus* in existing technologies mainly focuses on solid-state fermentation of tea leaves. The typical process route is: "tea raw material pretreatment → *Aurorus cristatus* inoculation → solid-state fermentation → fermented tea drying → pulverization → extraction → filtration → blending → finished product." While this traditional solid-state fermentation method can achieve the growth and reproduction of *Aurorus cristatus* on tea leaves, it suffers from numerous insurmountable technical defects due to the characteristics of the solid substrate, severely hindering the industrialization and quality improvement of *Aurorus cristatus*-related products.

[0004] Specifically, the shortcomings of traditional solid-state fermentation are mainly reflected in the following aspects: First, process parameters are difficult to control precisely. During solid-state fermentation, key parameters such as temperature, humidity, and aeration vary significantly in different regions, leading to uneven fermentation of *Aspergillus cristatus*, large fluctuations in strain growth status and metabolite synthesis, and consequently, significant differences in product quality between batches, making standardized production difficult. Second, the dissolution rate of active ingredients is low and easily lost. After solid-state fermentation, most of the metabolites of *Aspergillus cristatus* and active ingredients in tea (such as tea polyphenols and tea polysaccharides) are encapsulated in the tea fiber matrix, requiring complex subsequent processing such as extraction, crushing, and filtration to dissolve. However, the extraction process involves high temperature and prolonged stirring. Stirring and other operations can lead to a significant loss of heat-sensitive active ingredients (such as free flavonoid aglycones and volatile flavor compounds), reducing the product's functionality and flavor quality. Third, the final product has poor stability. Tea extracts produced by traditional processes contain a large number of undegraded macromolecules (such as cellulose, pectin, and insoluble polysaccharide complexes). These substances are prone to aggregation and flocculation during storage, leading to precipitation and stratification, which seriously affects the product's appearance and shelf life. Fourth, production efficiency is low and energy consumption is high. Solid-state fermentation typically takes 15-30 days, and subsequent drying, crushing, and extraction processes require a large amount of energy and manpower, resulting in high production costs that do not meet the requirements of green manufacturing and efficient industrial production.

[0005] Furthermore, *Aspergillus cristatus* produces certain antibacterial substances during its growth, which can inhibit the growth of some harmful bacteria. This characteristic has not been fully utilized in traditional solid-state fermentation. In existing liquid fermentation technologies, due to the complex composition of the substrate or the addition of exogenous nutrients, contamination by other microorganisms is easily caused, affecting the stability of the fermentation process and product quality. At the same time, the extracts of different tea types (such as green tea and oolong tea) contain microbial inhibitory components such as ester-type catechins, which can affect the growth and metabolism of *Aspergillus cristatus*. Current technologies have not proposed effective solutions to this problem, thus limiting the applicability of liquid fermentation technology.

[0006] Therefore, this invention proposes a liquid fermentation technology that can avoid the defects of traditional solid-state fermentation and achieve efficient, stable and standardized production of functional components of *Aspergillus cristatus*. It does not require the addition of exogenous nutrients, but directly uses tea extract as the sole nutrient matrix to achieve in-situ biotransformation and complete water solubility of functional components. At the same time, it solves the problems of inhibition and contamination of different tea matrices, and directly transforms them into a ready-to-drink beverage base with uniform quality and excellent stability. Summary of the Invention

[0007] This invention aims to overcome the aforementioned deficiencies of existing technologies and provide a novel liquid deep fermentation technology for *Aurotriarcha cristatus*. This technology uses pure tea extract as the sole organic nutrient source, without the need for any exogenous nutrients. By precisely controlling the liquid deep fermentation parameters, it achieves in-situ biotransformation and efficient enrichment of the active ingredients in tea by *Aurotriarcha cristatus*, producing a stable and homogeneous fully fermented tea beverage that can be directly used as the core ingredient for ready-to-drink functional beverages. Simultaneously, it solves problems such as uneven fermentation, loss of active ingredients, poor product stability, and lengthy processes in existing technologies, providing a novel technical solution for the industrial and standardized application of *Aurotriarcha cristatus*.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A *Eurotium cristatum* tea beverage includes the following steps: S1. Preparation of tea-based culture medium: using tea extract as the sole organic nutrient source, adjusting its total soluble sugar concentration to 0.5%-5.0% and pH to 4.0-6.5; S2. Inoculation and Fermentation: Inoculate a pure culture of *Aspergillus cristatus* into the tea-based culture medium at an inoculation concentration of [missing information]. CFU / mL; S3. Liquid submerged fermentation: Ferment for 3-10 days at 25-32℃, aeration rate of 0.5-1.5 vvm, and stirring conditions; S4. Termination and Separation: After fermentation is completed, the fermentation is terminated by heat treatment and sterilized, and then solid-liquid separation is performed to obtain the clarified fully fermented tea beverage.

[0009] Preferably, the tea leaves are selected from at least one of black tea, oolong tea, red tea, and green tea. For tea extracts containing strong microbial inhibitory components, preheating at 60-80°C for 10-30 minutes before inoculation can reduce the activity of the inhibitors and improve cell growth.

[0010] Secondly, this invention provides a fully fermented tea beverage made from *Aspergillus cristatus* prepared by the above method. The content of water-soluble polysaccharides derived from *Aspergillus cristatus* metabolism and tea component transformation in this tea beverage is increased by more than 50% compared to the same concentration of tea extract before fermentation, and / or the content of free flavonoid aglycones is increased by more than 30%. The tea beverage exhibits excellent physical stability, showing no visible precipitation or only slight, recoverable homogeneous flocculation after being stored at 4°C for 30 days.

[0011] Thirdly, the present invention provides a ready-to-drink functional beverage, characterized in that it contains not less than 10% (v / v) of the aforementioned *Aspergillus cristatus* fully fermented tea beverage. In a preferred embodiment, the beverage comprises, by weight percentage: 30%-70% of the fully fermented tea beverage, 20%-65% water, 0.1%-5.0% flavor modifier, and optionally 0-0.5% food additives.

[0012] The flavor modifiers include, but are not limited to, one or more of the following: sweeteners (such as white sugar, fructose syrup, erythritol, steviol glycosides), acidulants (such as citric acid, malic acid, lactic acid), and edible flavorings (such as tea flavorings, fruit flavorings). The food additives include, but are not limited to, one or more of the following: antioxidants (such as sodium D-isoascorbate), stabilizers (such as sodium carboxymethyl cellulose), and preservatives (such as potassium sorbate). Those skilled in the art can conventionally select and compound these additives within the above dosage range according to the flavor, taste, and shelf-life requirements of the target product.

[0013] Fourthly, the present invention provides the application of the above-mentioned fully fermented tea beverage of *Aspergillus cristatus* in the preparation of functional foods or beverages for regulating intestinal flora and / or providing antioxidant activity.

[0014] The beneficial effects of this invention are as follows: 1. Fundamental Innovation in Technological Approach: This technology is the first to propose and implement a fully liquid-based deep fermentation process for *Aspergillus cristatus* using pure tea extract as the sole nutrient substrate, completely abandoning the traditional "solid-state fermentation + extraction" model. This approach transforms the fermentation system from a heterogeneous "solid-gas-liquid" system to a homogeneous "liquid-gas" system, enabling precise online monitoring and control of key process parameters such as temperature, pH, dissolved oxygen, and cell concentration. This ensures the stability and reproducibility of the fermentation process, minimizing batch-to-batch product quality variations and truly achieving standardized production.

[0015] 2. Efficient in-situ enrichment and transformation of active ingredients: *Aspergillus cristatus* grows and metabolizes directly in liquid tea infusion. Its secreted extracellular enzymes (such as cellulase, pectinase, polyphenol oxidase, and β-glucosidase) can perform in-situ, efficient enzymatic hydrolysis and biotransformation of macromolecular substrates in tea infusion. For example, it degrades insoluble polysaccharide complexes, cellulose, and pectin in tea into water-soluble polysaccharides, improving their bioavailability; it also converts bound polyphenols and flavonoids with low bioavailability in tea into free flavonoid aglycones with higher bioavailability, achieving targeted biofortification of functional components and significantly enhancing the functional activity of the product.

[0016] 3. High standardization and excellent stability of the product: The liquid deep fermentation conditions are uniform, and the *Aspergillus cristatus* grows and is evenly distributed in the tea soup. The fermentation process is controllable, ensuring a high degree of consistency in the active ingredient spectrum, content, and flavor of the final product. At the same time, macromolecules are effectively degraded or transformed during the fermentation process, and the resulting fully fermented tea soup forms a stable colloidal system that has been fully utilized by microorganisms. This fundamentally solves the precipitation and stratification problems caused by the aggregation of macromolecular polymers in traditional tea extracts, and the product stability is significantly better than that of existing technology products.

[0017] 4. Simplified Process and Green Production: This invention achieves "one-step fermentation, direct soup production," eliminating multiple energy-intensive steps such as drying, crushing, extraction, and concentration after traditional solid-state fermentation. The process flow is shortened by more than 60%, and the production cycle is shortened to 3-10 days, greatly simplifying the production process and reducing production costs and energy consumption. At the same time, no harmful waste is generated during the fermentation process, and the extract can be fully utilized, which is in line with the development concept of green manufacturing and high efficiency and energy saving in the modern food industry.

[0018] 5. Wide range of applications and broad market prospects: The method of this invention is applicable to various types of tea such as black tea, oolong tea, red tea, and green tea, and can prepare fully fermented tea beverages with different flavors. The resulting fully fermented tea beverages can be directly used as core base materials to prepare various ready-to-drink functional beverages, and can also be used to prepare other functional foods, meeting consumers' demand for healthy, convenient, and high-quality foods, and has broad market prospects and industrialization value. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a flowchart illustrating the preparation process of the *Aspergillus cristatus* fully fermented tea beverage of the present invention. Figure 2 This is a flowchart illustrating the preparation process of the tea-based culture medium of the present invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0022] The *Aurotriarcha* strain used in the examples can be found at CGMCC No. 8730, which is deposited by the China General Microbiological Culture Collection Center.

[0023] All tea leaves used in the examples were commercially available high-quality tea leaves, all other reagents were food-grade or analytical grade, and all instruments and equipment were conventional equipment used in the food industry or conventional laboratory instruments.

[0024] The detection methods involved in the embodiments are as follows: 1. Determination of water-soluble polysaccharide content: The phenol-sulfuric acid method was used. Take 1 mL of sample, add 1 mL of phenol solution (5%), shake well, add 5 mL of concentrated sulfuric acid, heat in a boiling water bath for 15 minutes, cool to room temperature, and measure the absorbance at a wavelength of 490 nm. Use glucose as a standard to plot a standard curve and calculate the content of water-soluble polysaccharides in the sample.

[0025] 2. Determination of free flavonoid aglycone content: High performance liquid chromatography (HPLC) was used. The chromatographic conditions were as follows: C18 column (4.6 mm × 250 mm, 5 μm), mobile phase: methanol-0.1% formic acid aqueous solution (volume ratio 40:60), detection wavelength: 254 nm, column temperature: 30 ℃, flow rate: 1.0 mL / min, injection volume: 10 μL. A standard curve was plotted using quercetin as a standard, and the content of free flavonoid aglycones in the sample (calculated as quercetin equivalent) was calculated.

[0026] 3. Stability test: The samples were placed in refrigerated conditions at 4℃ and in room temperature conditions at 25℃ respectively for static storage. The clarity of the samples and whether there was precipitation or stratification were observed regularly. The stability period of the samples was recorded.

[0027] 4. Microbiological index testing: In accordance with the requirements of GB 7101-2021 "National Food Safety Standard for Beverages", the total bacterial count, coliform bacteria, and pathogenic bacteria (Salmonella, Staphylococcus aureus, Shigella) of the samples were tested.

[0028] Example 1: Preparation of a fully fermented tea beverage based on black tea Step 1: Preparation of tea-based culture medium. Take 100g of Anhua black tea, grind it to 40 mesh, add 1500mL of pure water, with a material-to-water ratio of 1:15 (g:mL), and extract at 95℃ for 15 minutes, stirring every 5 minutes during the extraction process. After extraction, filter with gauze to remove most of the tea residue, then centrifuge (4000 rpm, 8 minutes), and collect the supernatant to obtain the tea extract. The soluble total sugar content was determined to be 2.5% using the phenol-sulfuric acid method, and the pH was determined to be 5.5 using a precision pH meter. No adjustment is required, and it can be directly used as the fermentation culture medium.

[0029] Step 2: Inoculation and Fermentation. *Aspergillus cristatus* CGMCC No. 8730 was inoculated onto PDA slant medium and cultured at 29°C for 8 days. The slant was washed with sterile physiological saline, and the spore suspension was collected. The mycelium was removed by filtration to obtain a pure culture of *Aspergillus cristatus*. Under sterile conditions, this pure culture was inoculated into the aforementioned tea-based culture medium, and the initial bacterial concentration was adjusted to [value missing]. CFU / mL, shake well.

[0030] Step 3: Liquid deep fermentation. The inoculated tea-based culture medium was placed in a 5L fermentation tank, and the fermentation temperature was set to 30℃, the aeration rate to 1.0 vvm, and the stirring speed to 150 rpm for liquid deep fermentation for 6 days. During the fermentation process, the pH and dissolved oxygen concentration were monitored in real time. The pH was maintained between 5.0 and 5.5, and the dissolved oxygen concentration was maintained between 30% and 50%. No additional adjustments were made during the period.

[0031] Step 4: Termination and Separation. After fermentation, the fermentation liquid is pasteurized in a 90℃ water bath for 15 minutes to terminate fermentation and sterilize. After cooling to room temperature, 1.0% (by mass) of diatomaceous earth is added, stirred evenly, and then filtered to remove bacterial residues and macromolecular impurities. The mixture is then filtered through a 0.2μm microporous membrane to obtain a clear, reddish-brown fully fermented black tea liquor A made from *Aspergillus cristatus*.

[0032] Test results: The water-soluble polysaccharide content of tea infusion A was 1.52 g / L, compared to 0.84 g / L in the tea extract of the same concentration before fermentation, representing an increase of approximately 82%; the free flavonoid aglycone content was 45.2 mg / L, compared to 31.2 mg / L before fermentation, representing an increase of approximately 45%; the microbiological indicators met the requirements of GB 7101-2021; after refrigeration at 4℃ for 30 days, it remained clear and transparent with no sediment at the bottom of the bottle; after storage at room temperature of 25℃ for 6 months, there was no stratification or sedimentation, demonstrating excellent stability.

[0033] Comparative Example 1: Traditional Fu Brick Tea Extract Take 100g of commercially available Fu brick tea, grind it to 40 mesh, and prepare an extract using the same extraction conditions as in Example 1 (material-to-water ratio 1:15, extraction at 95℃ for 15 minutes, gauze filtration + centrifugation) to obtain a traditional Fu brick tea extract.

[0034] Test results: The water-soluble polysaccharide content of the extract was 0.88 g / L, and the free flavonoid aglycone content was 32.5 mg / L, both significantly lower than that of the fully fermented black tea soup A in Example 1; after refrigeration at 4℃ for 7 days, a large amount of flocculent precipitate appeared at the bottom of the bottle; after storage at room temperature of 25℃ for 1 month, obvious stratification occurred, and the stability was significantly inferior to that of the product of this invention.

[0035] Example 2: Preparation of Oolong Tea-Based Fully Fermented Tea Beverage (Example for Addressing Inhibitory Matrix) Step 1: Preparation of tea-based culture medium. Take 100g of Minnan Tieguanyin Oolong tea, grind it to 40 mesh, add 2000mL of pure water, with a material-to-water ratio of 1:20 (g:mL), extract at 90℃ for 10 minutes, filter quickly to obtain tea extract; place the extract in a 70℃ water bath for 20 minutes to reduce the antibacterial activity of ester-type catechins; after cooling to 28℃, determine its total soluble sugar content to be approximately 1.8%, pH 5.2, no adjustment required, and use it as a fermentation culture medium.

[0036] Step 2: Inoculation and Fermentation. Prepare a pure culture of *Aspergillus cristatus* (method as in Example 1), inoculate it into the above-mentioned tea-based culture medium, and adjust the initial bacterial concentration after inoculation to... CFU / mL, shake well.

[0037] Step 3: Liquid submerged fermentation. The inoculated culture medium was placed in a 5L fermenter, and the fermentation temperature was set to 28℃, the aeration rate to 0.8 vvm, and the stirring speed to 120 rpm for liquid submerged fermentation for 5 days. During the fermentation process, the pH and dissolved oxygen concentration were monitored in real time, and the pH was maintained between 5.0 and 5.2, and the dissolved oxygen concentration was maintained between 25% and 45%.

[0038] Step 4: Termination and Separation. After fermentation, the fermentation liquid is sterilized at 85℃ for 10 minutes, cooled to room temperature, and 0.8% (by mass) of diatomaceous earth is added. After filtration, it is filtered again through a 0.45μm microporous membrane to obtain a clear amber-colored fully fermented oolong tea liquor B.

[0039] Test results: The content of microbial water-soluble polysaccharides in tea soup B was 1.15 g / L, compared with 0.70 g / L in the extract of the same concentration before fermentation, an increase of about 64%; the content of free flavonoid aglycones increased by about 35% compared with before fermentation; the microbial indicators met the requirements of GB 7101-2021; no precipitation was observed after 30 days of refrigerated storage at 4℃, and good stability was observed after 6 months of storage at room temperature at 25℃; the flavor changed from the typical light and slightly astringent aroma of oolong tea to a mellow aroma of ripe fruit and a soft aroma of bacteria, with a significant improvement in taste.

[0040] Example 3: Preparation of Green Tea-Based Fully Fermented Tea Beverage Step 1: Preparation of tea-based culture medium. Take 100g of West Lake Longjing green tea, grind it to 40 mesh, add 1800mL of pure water, with a material-to-water ratio of 1:18 (g:mL), extract at 88℃ for 8 minutes, filter to obtain tea extract; place the extract in a 75℃ water bath for 15 minutes for heat treatment, cool to 28℃, and determine its total soluble sugar content to be 1.2% and pH to be 4.8. Adjust the pH to 5.0 with citric acid, and use it as fermentation culture medium.

[0041] Step 2: Inoculation and Fermentation. Prepare a pure culture of *Aspergillus cristatus* (method as in Example 1), inoculate it into tea-based culture medium, and adjust the initial bacterial concentration to... CFU / mL, shake well.

[0042] Step 3: Liquid submerged fermentation. The inoculated culture medium was placed in a 5L fermenter, fermented at 29℃, with an aeration rate of 1.1 vvm and a stirring speed of 160 rpm for 7 days. During fermentation, the pH was maintained between 4.8 and 5.2, and the dissolved oxygen concentration was maintained between 35% and 55%.

[0043] Step 4: Termination and Separation. The fermentation broth was sterilized at 90℃ for 12 minutes, cooled, and then filtered with 1.2% (by mass) diatomaceous earth. The broth was then filtered through a 0.2μm microporous membrane to obtain a pale yellow, clear, fully fermented green tea infusion C.

[0044] Test results: The water-soluble polysaccharide content of tea soup C was 0.98 g / L, compared with 0.58 g / L of the extract of the same concentration before fermentation, an increase of about 69%; the content of free flavonoid aglycones increased by about 42% compared with before fermentation; no precipitation was observed after 30 days of refrigerated storage at 4℃, and good stability was observed after 6 months of storage at room temperature at 25℃. It has a fresh green tea aroma and a light fungal aroma, and a mellow taste without bitterness.

[0045] Example 4: Preparation of ready-to-drink functional beverage (sugar-free) Formula (by weight percentage): 50% of the fully fermented black tea soup A prepared in Example 1, 47.45% of purified water, 2.0% of erythritol, 0.05% of steviol glycosides, 0.3% of citric acid, 0.05% of sodium D-isoascorbate, and 0.15% of black tea flavoring.

[0046] Preparation method: Add fully fermented black tea soup A and purified water to a mixing tank and stir evenly; then add erythritol, steviol glycosides, citric acid, sodium D-isoascorbate and black tea flavoring in sequence, and stir at 28℃ for 20 minutes to ensure that all components are completely dissolved and mixed; after mixing, use UHT ultra-high temperature instantaneous sterilization (137℃, 4 seconds), quickly cool to below 22℃, and aseptically fill to obtain sugar-free ready-to-drink functional beverage D.

[0047] Product characteristics: This beverage is a clear, reddish-brown liquid with a rich aroma of black tea and a mild mushroom fragrance. It has a mellow taste, a suitable sweetness (sugar-free), and no bitterness. The water-soluble polysaccharide content is 0.76 g / L, and the free flavonoid aglycone content is 22.6 mg / L. The microbiological indicators meet the requirements of GB 7101-2021. It exhibits excellent stability and shows no precipitation or stratification after 6 months of refrigerated storage at 4℃ or 12 months of room temperature storage at 25℃.

[0048] Example 5: Preparation of ready-to-drink functional beverage (fruit flavor) Formula (by weight percentage): 40% of the fully fermented oolong tea liquor B prepared in Example 2, 55.5% of purified water, 4.0% of white sugar, 0.2% of malic acid, 0.2% of lemon flavoring, 0.05% of xanthan gum, and 0.05% of potassium sorbate.

[0049] Preparation method: Add fully fermented oolong tea soup B and purified water to a mixing tank and stir evenly; then add white sugar, malic acid, lemon flavoring, xanthan gum and potassium sorbate, and stir at 26℃ for 25 minutes until all components are completely dissolved; pasteurize (88℃, 18 minutes), cool to below 25℃, and aseptically fill to obtain fruit-flavored ready-to-drink functional beverage E.

[0050] Product characteristics: This beverage is an amber-colored clear liquid with the aroma of oolong tea, lemon, and a mild mushroom aroma. It has a refreshing and sweet-sour taste with rich flavor layers. The water-soluble polysaccharide content is 0.46 g / L, and the free flavonoid aglycone content is 16.1 mg / L. The microbiological indicators meet the requirements of GB 7101-2021. It has good stability and can be stored at 4℃ for 6 months or at room temperature (25℃) for 12 months.

[0051] Experimental Example: In vitro functional verification To evaluate the bioactivity of the product of this invention, an in vitro simulated digestion-colonic fermentation model was used. The fully fermented tea infusions A and B prepared in Examples 1 and 2, and the traditional Fu brick tea extract from Comparative Example 1, were used as samples for the following experiments: 1. Simulated gastrointestinal digestive stability experiment Experimental method: Take 10 mL of each of the tea infusions A, B and Comparative Example 1, add simulated gastric juice (pH 2.0, containing pepsin), and digest at 37℃ with shaking for 2 hours; then add simulated intestinal juice (pH 7.5, containing trypsin and bile salts), and digest at 37℃ with shaking for 4 hours; after digestion, determine the retention rate of water-soluble polysaccharides and free flavonoid aglycones in the samples.

[0052] Experimental results: The digestibility and retention rates of water-soluble polysaccharides and free flavonoid aglycones in tea infusion A were 87% and 86%, respectively; the digestibility and retention rates of the two active ingredients in tea infusion B were 85% and 84%, respectively; while the digestibility and retention rates of the two active ingredients in the extract of Comparative Example 1 were only 71% and 70%. This indicates that the active ingredients in the fully fermented tea infusion of the present invention have stronger gastrointestinal digestibility and are more easily absorbed and utilized by the human body.

[0053] 2. Experiment assessing the potential of gut microbiota regulation Experimental methods: Tea infusions A and B, and extract of Comparative Example 1, after simulated gastrointestinal digestion, were mixed with fecal microbiota from healthy individuals and placed in an anaerobic incubator for anaerobic fermentation at 37°C for 24 hours. After fermentation, 16S rRNA sequencing technology was used to analyze the changes in the composition of fecal microbiota, focusing on detecting the relative abundance of Bifidobacterium, Lactobacillus (beneficial bacteria), and Clostridium (potentially harmful bacteria).

[0054] Experimental results: Compared with Comparative Example 1, tea infusion A increased the relative abundance of Bifidobacterium by an average of 15%, Lactobacillus by an average of 13%, and decreased the relative abundance of Clostridium by an average of 8%; tea infusion B increased the relative abundance of Bifidobacterium by an average of 12%, Lactobacillus by an average of 10%, and decreased the relative abundance of Clostridium by an average of 7%. This indicates that the fully fermented tea infusion of the present invention has a significant effect on regulating the balance of intestinal flora, effectively promoting the proliferation of beneficial bacteria and inhibiting the growth of potentially harmful bacteria.

[0055] 3. In vitro antioxidant activity experiment Experimental methods: The ORAC (Oxygen Radical Absorption Capacity) method was used, with Trolox (a water-soluble vitamin E analog) as the standard. The ORAC values ​​of tea infusions A and B and the extract of Comparative Example 1 were determined to evaluate their in vitro antioxidant activity.

[0056] Experimental results: The ORAC value of tea infusion A was 8500 μmol TE / L, the ORAC value of tea infusion B was 7800 μmol TE / L, while the ORAC value of the extract of Comparative Example 1 was only 6000 μmol TE / L. The ORAC values ​​of tea infusions A and B were increased by approximately 42% and 30% respectively compared with Comparative Example 1, indicating that the fully fermented tea infusion of the present invention has significantly enhanced in vitro antioxidant activity.

[0057] The equipment used in the method of this invention are all conventional equipment in the food industry, including tea extraction tanks, fermentation tanks, centrifuges, filtration equipment, sterilization equipment, blending tanks, aseptic filling machines, etc. No new special equipment is required, the equipment investment cost is low, and it is easy to upgrade existing production lines.

[0058] The fermentation process parameters of this invention are clear and controllable, the fermentation process is stable, the product quality difference between batches is small, and it is easy to achieve large-scale production. The production cycle is short (3-10 days), the production efficiency is high, and it has significant economic benefits.

[0059] The resulting fully fermented tea beverage made from *Aspergillus cristatus* has uniform and stable quality and can be directly supplied to beverage companies as a core raw material for developing functional ready-to-drink products with health claims such as "helping to regulate intestinal flora" and "antioxidant". It can also be used to prepare other functional foods to meet the needs of different consumer groups. At the same time, the method of this invention is applicable to various types of tea and can develop products with different flavors, further expanding the market coverage of the products and possessing broad market prospects and industrialization value.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a fully fermented tea beverage of *Aspergillus cristatus*, characterized in that, Includes the following steps: Using tea extract as a liquid culture medium, a pure culture of *Aspergillus cristatus* was inoculated and subjected to deep liquid fermentation under aeration and stirring conditions. After fermentation, the fully fermented tea beverage was obtained through heat treatment and solid-liquid separation.

2. The method according to claim 1, characterized in that, The tea extract is the only organic nutrient source in the fermentation system, and the tea used as raw material is selected from at least one of black tea, oolong tea, red tea, and green tea.

3. The method according to claim 1, characterized in that, The initial total soluble sugar concentration of the tea extract is 0.5%-5.0% (by mass / volume), and the pH is 4.0-6.

5.

4. The method according to claim 1, characterized in that, For tea extracts containing microbial inhibitory components, a preheating treatment is performed before inoculation. The preheating treatment temperature is 60-80℃ and the time is 10-30 minutes.

5. The method according to claim 1, characterized in that, The pure culture of *Aspergillus cristatus* is a suspension of *Aspergillus cristatus* spores. The conditions for the liquid submerged fermentation are: the initial bacterial concentration after inoculation is... CFU / mL, fermentation temperature 25-32℃, fermentation time 3-10 days, aeration rate 0.5-1.5 vvm, stirring speed 100-250 rpm.

6. A fully fermented tea beverage of *Aspergillus cristatus* prepared by the method according to any one of claims 1-5.

7. The fully fermented tea beverage of *Aspergillus cristatus* according to claim 6, characterized in that, Its water-soluble polysaccharide content is more than 50% higher than that of tea extract of the same concentration before fermentation, and / or its free flavonoid aglycone content is more than 30% higher than that of tea extract of the same concentration before fermentation.

8. A ready-to-drink functional beverage, characterized in that, The beverage comprises the fully fermented tea beverage of *Aspergillus cristatus* as described in any one of claims 6-7, wherein the volume proportion of the fully fermented tea beverage in the beverage is not less than 10%.

9. The ready-to-drink functional beverage according to claim 8, characterized in that, By weight percentage, it comprises: 30%-70% of the *Aspergillus cristatus* fully fermented tea beverage, 20%-65% water, 0.1%-5.0% flavor modifier, and 0-0.5% food additives.

10. The use of the fully fermented tea beverage of *Aspergillus cristatus* as described in claim 6 or 7 in the preparation of functional foods or beverages for regulating intestinal flora and / or providing antioxidant activity.

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