A special nitrogen source concentrate for promoting tea wine fermentation and a preparation method and application thereof

Abstract

The application discloses a special nitrogen source concentrated solution for promoting tea wine fermentation and application, and the concentrated solution is a liquid concentrate of L-theanine, wherein the mass concentration of L-theanine is 100-200 g / L, and the ratio of the total mass of tea polyphenols to the total mass of L-theanine is not higher than 0.1:1. The preparation method comprises the following steps: taking tea leaves or by-products of a high-theanine tea tree variety as raw materials, and performing low-temperature ethanol extraction, solid-liquid separation, membrane separation pre-concentration, ion exchange chromatography purification, ammonia water elution, reverse osmosis concentration and inert gas blowing and ammonia removal. When the concentrated solution is used for tea wine fermentation, it can be directly added to tea extraction liquid. The application can effectively solve the contradiction between the nitrogen source deficiency and the flavor retention in tea wine fermentation, significantly shortens the fermentation period, improves the ethanol yield, and improves the clarity of the tea wine under the premise of almost not introducing bitter substances such as tea polyphenols, and is suitable for the standardized and efficient production of high-quality tea wine.

Description

Technical Field

[0001] This invention belongs to the field of food fermentation and brewing technology, specifically relating to a special nitrogen source concentrate for promoting tea and wine fermentation, its preparation method, and its application. Background Technology

[0002] Tea wine is an innovative fermented beverage that combines the flavors of tea and wine, and it aligns with the trend of healthy consumption, showing significant market potential. However, its industrial development has long been constrained by a core technological bottleneck: the inherent yeast-assimilated nitrogen in tea extract is severely lacking, resulting in slow fermentation initiation, excessively long cycles, and low efficiency, which seriously affects production efficiency and product quality stability.

[0003] To overcome the aforementioned nitrogen source bottleneck, existing technologies mainly focus on supplementing exogenous nitrogen, but all have significant drawbacks. Supplementing with inorganic nitrogen sources, such as adding diammonium hydrogen phosphate or ammonium sulfate, provides ammonium nitrogen quickly, but its absorption by yeast is accompanied by proton release, easily causing a sharp drop in the pH of the fermentation broth. This creates acid stress on yeast cells, inhibiting fermentation activity and potentially promoting the production of undesirable flavor compounds such as hydrogen sulfide, thus disrupting the delicate flavor profile of the tea wine. Supplementing with general organic nitrogen sources, such as adding yeast extract, peptone, or common amino acids (such as glutamic acid), introduces complex amino acid profiles or non-tea-derived flavor compounds while providing nitrogen. This may mask or interfere with the unique fresh tea aroma and mellow taste of the tea wine, leading to a "distorted" flavor profile.

[0004] More fundamentally, the tea-wine fermentation system suffers from an inherent contradiction that cannot be resolved by adjusting traditional raw material ratios: the sufficient nitrogen source required for fermentation and the desired flavor balance in the tea infusion are mutually constraining. While tea leaves themselves contain nitrogen (primarily in the form of theanine), their concentration and extraction rate are far insufficient to support efficient fermentation. Calculations show that even using tea varieties with relatively high theanine content (e.g., 5%), under conventional tea extraction ratios (e.g., 0.6% tea-to-water ratio), the theanine concentration in the resulting tea extract is still far below the threshold required for vigorous yeast metabolism. Simply increasing the amount of tea leaves to improve the initial nitrogen concentration would significantly increase the concentrations of flavor compounds such as tea polyphenols and caffeine, inevitably leading to excessive bitterness and deterioration of the sensory quality. Therefore, within the framework of traditional processes, "simultaneously providing ideal flavor and sufficient nitrogen source for fermentation through tea leaves" is a technically intractable dilemma.

[0005] In summary, existing nitrogen source replenishment strategies either have negative impacts or are costly, and none of them fundamentally decouple or independently optimize the two key factors of "supply of flavor substances" and "guarantee of fermentation dynamics." Therefore, developing a specialized additive that can accurately and efficiently replenish fermentation nitrogen sources without introducing any interfering flavors and can synergize with any tea flavor matrix has become an urgent technological need to overcome the bottlenecks in the development of the tea and wine industry. Summary of the Invention

[0006] To resolve the contradiction between insufficient nitrogen source and flavor preservation in tea and wine fermentation, the present invention aims to provide a special nitrogen source concentrate for promoting tea and wine fermentation, its preparation method, and its application.

[0007] The present invention is specifically implemented using the following technical solutions: The first aspect of this invention is to provide a special nitrogen source concentrate for promoting tea and wine fermentation. This concentrate is a liquid enrichment of L-theanine, characterized by a combination of high concentration and high purity: the mass concentration of L-theanine is as high as 100–200 g / L, while the ratio of the total mass of tea polyphenols to the total mass of L-theanine is strictly controlled to be no higher than 0.1:1.

[0008] Preferably, the concentrate is derived from tea leaves of high-theanine tea varieties and / or their processing by-products. This product design brings significant benefits: First, the high concentration of theanine enables it to act as a highly efficient fermentation promoter, satisfying the yeast's vigorous demand for assimilable nitrogen with minimal addition; second, the extremely low proportion of tea polyphenols ensures that it introduces almost no additional bitter substances into the fermentation system when supplementing nitrogen, thus perfectly resolving the contradiction between "nitrogen supplementation" and "bitterness enhancement" in traditional supplementation methods, achieving decoupling of fermentation dynamics and flavor purity.

[0009] The second aspect of the present invention is to provide a method for preparing the above-mentioned special nitrogen source concentrate. The method includes the following steps: (1) low-temperature selective extraction: mixing high-theanine tea tree raw materials with 30% to 65% (v / v) ethanol aqueous solution and extracting under mild conditions of 15 to 40°C; (2) solid-liquid separation: filtering to obtain primary extract; (3) combined purification and enrichment: the primary extract is preliminarily concentrated and impurities removed by membrane separation technology, and then purified by ion exchange chromatography; (4) elution and post-treatment: after elution with food-grade ammonia water, the eluent is concentrated by membrane concentration technology, and residual ammonia is removed by inert gas purging at the same time. The beneficial effects of this method are as follows: it inhibits the excessive dissolution of tea polyphenols from the source through low-temperature ethanol extraction; the combination of membrane separation and ion exchange achieves highly selective purification and enrichment of theanine, which is the key to obtaining products with low tea polyphenol content; finally, through non-thermal membrane concentration combined with gas purging, it simultaneously achieves efficient concentration and complete deammoniation at low temperature, ensuring the activity and food safety of the final product, forming a green and efficient complete process chain.

[0010] A third aspect of the present invention is to provide the application of a special nitrogen source concentrate in the preparation of tea wine.

[0011] Furthermore, the method for preparing tea wine using a special nitrogen source concentrate includes: (1) preparing tea extract using tea leaves; (2) adding sucrose and the special nitrogen source concentrate to the tea extract to prepare a fermentation base, so that the final concentration of L-theanine is 0.2 to 1.0 g / L; and (3) inoculating with brewer's yeast for static fermentation.

[0012] This invention establishes a modular and precisely controllable new model for tea wine production. Producers can freely choose any tea leaves with suitable flavor to construct the basic flavor of the tea wine, and then precisely and efficiently meet the nitrogen source requirements for fermentation by independently adding the concentrated liquid of this invention. This fundamentally breaks the dual constraint of tea leaf quantity on flavor and fermentation efficiency in traditional processes, enabling a significant increase in the fermentation speed and ethanol yield of tea wine without excessively increasing the amount of tea leaves (avoiding bitterness). Therefore, this patent provides a reliable technical solution for the stable and efficient production of high-quality tea wine. Attached Figure Description

[0013] Figure 1 This is a graph showing the effect of different nitrogen sources on the change of alcohol content over time during the tea wine fermentation process in Example 2. Figure 2 This is a graph showing the effect of different nitrogen sources on pH value over time during tea wine fermentation in Example 2. Figure 3 This is a graph showing the effect of different nitrogen sources on the change of alcohol content over time during the tea wine fermentation process in Example 4. Figure 4 This is a comparison chart of the turbidity of tea wine samples from different nitrogen source groups after fermentation in Example 4. Detailed Implementation

[0014] The present invention will be further described below with reference to specific embodiments in order to better understand the technical solution.

[0015] Example 1: Preparation of Special Nitrogen Source Concentrate A Raw material pretreatment: Take 150 g of the finished dried tea of ​​the commercially available high-theanine tea tree variety "Golden Bud", grind it with a high-speed grinder, pass it through a 40-mesh sieve, and set it aside.

[0016] Low-temperature selective extraction: Mix the above tea powder with 3 L of 45% (v / v) food-grade ethanol aqueous solution and place it in a 5 L glass beaker. Place the beaker in a circulating water bath with a temperature control accuracy of ±0.5℃, and maintain the extraction temperature at 25±1℃ for 150 minutes.

[0017] Solid-liquid separation: After extraction, the residue was separated by vacuum filtration using a Buchner funnel and medium-speed qualitative filter paper. The residue was then finely filtered through a 0.45 μm filter membrane to obtain approximately 2.85 L of clear primary extract.

[0018] Nanofiltration membrane pre-concentration: The primary extract was pumped into a laboratory nanofiltration membrane separation device (membrane area 0.1 m², polyamide material, molecular weight cutoff 200 Da). Concentration was carried out under an operating pressure of 2.0 MPa and a temperature of 25°C until the feed volume was reduced to approximately 300 mL, yielding the pre-concentrated solution.

[0019] Ion exchange chromatography: The pH of the pre-concentrated solution was precisely adjusted to 3.5 using 0.1 mol / L dilute hydrochloric acid. It was then passed at a flow rate of 1.5 BV / h through a container filled with 50 mL of strongly acidic cation exchange resin (model 001×7, H...). + A glass chromatography column (1.6 cm × 30 cm) of type 1 was used. The column was washed with ultrapure water until the conductivity of the effluent was close to that of ultrapure water to remove unadsorbed impurities.

[0020] Elution: Elution was performed using a 0.5 mol / L food-grade ammonia solution. The eluent was collected in fractions by time or volume using an automated fraction collector. Each eluent sample was taken and analyzed offline using a ninhydrin post-column derivatization-amino acid analyzer. Fractions with high theanine content were combined to obtain approximately 120 mL of enriched eluent.

[0021] Reverse osmosis membrane concentration: The combined eluent was transferred to a small reverse osmosis membrane experimental machine (operating pressure 3.0 MPa, temperature 30℃) and concentrated to a final volume of approximately 40 mL.

[0022] Nitrogen purging for ammonia removal: The concentrate was transferred to a 100 mL glass bottle equipped with a sintered sand core bubbler, and the bottle was placed in a 30℃ water bath. High-purity nitrogen gas was introduced into the liquid at a constant flow rate of 0.2 L / min for 2 hours. A dedicated nitrogen source concentrate A was obtained. The concentration of L-theanine was determined to be 158 g / L using an amino acid analyzer. The total amount of tea polyphenols was determined to be 12.6 g / L using the Folin-Ciocalteu method, and the mass ratio of tea polyphenols to theanine was calculated to be 0.080:1. Ammonia residue was determined to be 3.8 mg / kg according to the national food safety standard method. This sample is designated as concentrate A.

[0023] Example 2: Application of Concentrate A in the Fermentation of Fuzhuan Tea Wine Preparation of fermentation substrate: In a 5 L fermenter, add 600 g of sucrose and 12.66 mL of the special nitrogen source concentrate A prepared in Example 1, labeled as concentrate group A (final theanine concentration 0.5 g / L); a control group was prepared by adding only 600 g of sucrose; and a control group was prepared by adding 600 g of sucrose and 2 g of ammonium sulfate (inorganic nitrogen source) (final ammonium sulfate concentration 0.5 g / L). Add approximately 3.5 L of purified water and stir until the sucrose is completely dissolved. Adjust the volume to 4 L with purified water. Seal the fermenter and sterilize at 121°C for 15 minutes. Weigh 24.0 g of pulverized Fu brick tea powder and add it to the sterilized sucrose-nitrogen source mixture. Incubate at 90°C for 20 minutes. After extraction, filter through sterilized multi-layered gauze and a filter screen to thoroughly remove tea residue. Obtain the fermentation substrate, cool to room temperature, and set aside.

[0024] Inoculation and fermentation: The *Saccharomyces cerevisiae* T3 strain with preservation number CCTCC NO: M2017624 was inoculated into PDA medium and activated at 30℃ for 24 h to prepare a seed culture. The seed culture was then inoculated into the above fermentation substrate at an inoculation rate of 2% (v / v). The mixture was then placed in a constant temperature incubator at 28℃ for static fermentation.

[0025] Samples were taken every two days during fermentation, and alcohol content was monitored using a biosensor. Primary fermentation was considered complete when the alcohol content changed by less than 0.5% ABV for two consecutive days. Figure 1 As shown, the primary fermentation period for concentrate A was 10 days, with a final alcohol content of 11.6% ABV. Compared to the control group without any nitrogen source (primary fermentation period of 16 days, alcohol content of approximately 10.3% ABV), the fermentation period for concentrate A was shortened by 37.5%, and the ethanol yield increased by 12.6%. Although the primary fermentation period for the ammonium sulfate group was the same as that for concentrate A (10 days), the final alcohol content was 7.0% ABV. The pH of the fermentation was monitored. Figure 2It was found that the pH of the ammonium sulfate group dropped rapidly after fermentation, reaching as low as 2.48 on the 4th day of fermentation. This severely affected the growth and metabolism of the brewing yeast, resulting in a significantly lower alcohol content than the concentrated liquid A group and the control group.

[0026] Based on the above comparisons, the dedicated nitrogen source concentrate A can not only significantly reduce the fermentation cycle of tea wine, but also increase its alcohol content, making it an ideal nitrogen source.

[0027] Example 3: Preparation of Special Nitrogen Source Concentrate B Raw material pretreatment: Take 200 g of tea stem by-products after refining "Baiye No. 1" white tea, crush them with a high-speed pulverizer, pass them through a 40-mesh sieve, and set aside.

[0028] Low-temperature selective extraction: The above-mentioned tea stem powder was mixed with 4 L of 50% (v / v) food-grade ethanol aqueous solution (solid-liquid ratio 1:20) and placed in a 5 L glass bottle. The bottle was placed in a constant temperature water bath, and the extraction temperature was maintained at 35±1℃ for 120 minutes.

[0029] Solid-liquid separation and membrane pretreatment: After filtration through filter cloth and a 0.45 μm filter membrane, approximately 2.8 L of clear extract was obtained. This extract was then concentrated to approximately 300 mL using a nanofiltration membrane system with a molecular weight cutoff of 300 Da at 2.5 MPa and 25 °C.

[0030] Ion exchange purification: Adjust the pH of the concentrate to 4.0, load the sample into a chromatography column containing 50 mL of strong acid cation exchange resin (type 001×7), and wash the column with ultrapure water.

[0031] Elution and post-treatment: The solution was eluted with 0.4 mol / L ammonia water, collecting approximately 60 mL of theanine main fraction. This fraction was concentrated to approximately 20 mL using a reverse osmosis membrane (3.0 MPa, 30℃), and then purged with nitrogen gas at a flow rate of 0.3 L / min for 2 hours at 30℃. The final product was a dedicated nitrogen source concentrate B. Analysis showed that the L-theanine concentration was 118 g / L, the tea polyphenol concentration was 9.5 g / L, the mass ratio of L-theanine to polyphenols was 0.081:1, and the residual ammonia was 4.1 mg / kg. This sample was designated as concentrate B.

[0032] Example 4: Application of Concentrate B in the Fermentation of Tieguanyin Tea Wine Preparation of fermentation substrate: In a 5 L fermenter, add 800 g of sucrose and 23.7 mL of the special nitrogen source concentrate B prepared in Example 3, labeled as concentrate group B (final theanine concentration 0.7 g / L); a control group was prepared by adding only 800 g of sucrose; and a glutamic acid group was prepared by adding 800 g of sucrose and 2.8 g of glutamic acid (a common organic nitrogen source) (final glutamic acid concentration 0.7 g / L). Add approximately 3.5 L of purified water and stir until the sucrose is completely dissolved. Adjust the volume to 4 L with purified water. Seal the fermenter and sterilize at 121°C for 15 minutes. Weigh 24.0 g of pulverized Tieguanyin tea powder and add it to the sterilized sucrose-nitrogen source mixture. Incubate at 90°C for 20 minutes. After extraction, filter the mixture using sterilized multi-layered gauze and a filter screen to thoroughly remove tea residue. Obtain the fermentation substrate, cool to room temperature, and set aside.

[0033] Inoculation and fermentation: The *Saccharomyces cerevisiae* T3 strain with preservation number CCTCC NO: M2017624 was inoculated into PDA medium and activated at 30℃ for 24 h to prepare a seed culture. The seed culture was then inoculated into the above fermentation substrate at an inoculation rate of 2% (v / v). The mixture was then placed in a constant temperature incubator at 28℃ for static fermentation.

[0034] Samples were taken every two days during fermentation, and alcohol content was monitored using a biosensor. Primary fermentation was considered complete when the alcohol content changed by less than 0.5% ABV for two consecutive days. Figure 3 As shown, the primary fermentation period for concentrate B was 10 days, with a final alcohol content of 13.7% ABV. Compared to the control group without any nitrogen source (primary fermentation period of 20 days, alcohol content of 12.9% ABV), the fermentation period for concentrate B was shortened by 50%, and the ethanol yield was increased by 6.2%. The primary fermentation period for the glutamic acid group was 10 days, with an alcohol content of 13.1% ABV, which is very close to that of concentrate B. Further comparison of turbidity ( Figure 4 The study found that, compared to the control group, the turbidity of concentrate B decreased by 55.1%, while the turbidity of the concentrate with added glutamic acid increased by 34.1%. This indicates that concentrate B can also improve the clarity of tea wine.

[0035] In summary, the dedicated nitrogen source concentrate B can not only significantly reduce the fermentation cycle of tea wine and increase its alcohol content, but also improve its clarity and enhance the sensory quality of the product, making it an ideal nitrogen source.

Claims

1. A special nitrogen source concentrate for promoting tea and wine fermentation, characterized in that, The special nitrogen source concentrate is a liquid enrichment of L-theanine, wherein the mass concentration of L-theanine is 100-200 g / L, and the ratio of the total mass of tea polyphenols to the total mass of L-theanine in the concentrate is not higher than 0.1:

1.

2. The special nitrogen source concentrate for promoting tea and wine fermentation as described in claim 1, characterized in that, The special nitrogen source concentrate is prepared from tea leaves of high-theanine tea varieties and / or their processing by-products.

3. A method for preparing a special nitrogen source concentrate as described in any one of claims 1-2, characterized in that, Includes the following steps: 1) Mix the raw material of high-theanine tea varieties with an aqueous ethanol solution of 30% to 65% (v / v) and extract it at 15 to 40°C to obtain a mixed solution; 2) Solid-liquid separation: The mixture obtained in step 1) is subjected to solid-liquid separation to obtain the primary extract; 3) Combined purification and enrichment: The primary extract is purified and enriched sequentially by membrane separation technology and ion exchange chromatography; 4) Elution and post-treatment: The resin after ion exchange chromatography is eluted with ammonia solution to collect the eluent rich in L-theanine; then the eluent is concentrated by membrane filtration and purged with inert gas to remove residual ammonia to obtain the special nitrogen source concentrate.

4. The method for preparing a special nitrogen source concentrate as described in claim 1, characterized in that, In step 1), the concentration of the ethanol-water solution is 45%–55% (v / v), the extraction temperature is 20–30°C, the extraction time is 60–180 minutes, and the solid-liquid ratio is 1:15–40.

5. The method for preparing a special nitrogen source concentrate as described in claim 1, characterized in that, In step 3), the membrane separation technology is nanofiltration, with a molecular weight cutoff of 150-300 Da; the ion exchange chromatography uses a strongly acidic cation exchange resin, and the pH of the primary extract or the pre-concentrated feed solution after membrane separation is adjusted to 3.5-4.0 before sample loading.

6. The method for preparing a special nitrogen source concentrate as described in claim 1, characterized in that, In step 4), the ammonia water is food-grade ammonia water with a concentration of 0.2–0.8 mol / L; the membrane concentration is reverse osmosis membrane concentration with an operating pressure of 1–5 MPa and a temperature of 15–40°C; the inert gas is nitrogen, and the purge gas flow rate is 0.5–2 L / min per liter of feed liquid.

7. The application of the special nitrogen source concentrate according to any one of claims 1-6 in the preparation of tea wine.

8. The application as described in claim 7, characterized in that, The method for preparing tea wine using a special nitrogen source concentrate includes the following steps: 1) Tea infusion preparation: Mix tea leaves with water, extract and filter to obtain tea extract; 2) Preparation of fermentation substrate: Sucrose and the special nitrogen source concentrate are added to the tea extract to prepare the fermentation substrate, so that the final concentration of sucrose in the fermentation substrate is 100-300 g / L and the final concentration of L-theanine is 0.2-1.0 g / L. 3) Fermentation: Add brewing yeast to the fermentation substrate and let it ferment statically at 20-30℃.

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