An enzyme-subcritical water combined adaptive extraction method for directional enrichment of astringent components in tea leaves

Abstract

The application discloses an enzyme-subcritical water composite adaptive extraction method for directional enrichment of a tea astringency component, and mainly relates to the field of tea deep processing. The method comprises the following steps: first, high-temperature short-time pre-extraction is carried out by using the high permeability and low dielectric constant characteristics of subcritical water, and flavor precursors are efficiently dissolved; then, the enzyme kinetic model is used to control the enzyme hydrolysis degree, the reaction is terminated in a "golden flavor release window", and the astringency and bitterness are accurately balanced; finally, the target component is directionally intercepted through a nanofiltration membrane. The method has the beneficial effects that, by the cooperation of physical fields and biological enzymes, the problems of low conversion rate of the astringency component and unbalanced flavor in traditional extraction are solved, and the preparation of high-quality functional tea base material is realized.

Description

Technical Field

[0001] This invention relates to the fields of natural product extraction and deep processing of tea, specifically an enzyme-subcritical water complex extraction method for the targeted enrichment of the aftertaste components of tea. Background Technology

[0002] The flavor of tea is mainly composed of sensory characteristics such as bitterness, astringency, umami, and sweetness. Among these, "aftertaste" is one of the core indicators for evaluating high-quality tea (such as premium oolong tea and ancient tree tea). Studies have shown that the aftertaste of tea mainly comes from some bonded glycosides such as flavonoid glycosides and triterpenoid saponins. These components usually exist in a bound or complex form, forming relatively stable complex structures with cell wall polysaccharides, lignin, etc., making them difficult to completely release through conventional water extraction methods. Therefore, industrial extraction processes are often used for extraction.

[0003] However, existing industrial extraction processes mainly suffer from the following problems and pain points: (1) Low extraction rate: Most current instant tea and functional tea extraction processes use hot water extraction at 80-90℃ or multi-stage countercurrent extraction. Since bonded glycosides are often tightly bound to structures such as cellulose and lignin, conventional hot water is highly polar and cannot effectively dissolve these medium-polarity organic macromolecules, resulting in a large amount of sweet aftertaste precursors remaining in the tea residue and being discarded, causing a waste of resources.

[0004] (2) The degree of hydrolysis of glycosides is difficult to control precisely: On the one hand, if glycosides are not properly treated, many bonded glycosides are tasteless or weakly tasted, and human taste buds cannot directly perceive them, making it difficult to form a noticeable aftertaste; on the other hand, if conventional enzymatic hydrolysis methods are used (such as high doses of cellulase and long reaction time), the glycoside structure is easily completely hydrolyzed into monosaccharides and aglycones. Although this can increase the straightforward sweetness, it will weaken or even eliminate the delayed release flavor of "bitter first, then sweet, and long aftertaste", making the flavor monotonous.

[0005] (3) Risk of organic solvent residue: When extracting flavonoid glycosides and other components under laboratory conditions, organic solvents such as methanol and ethanol are often used. Although these methods have strong dissolving power, they pose problems such as solvent residue, safety and environmental pressure in large-scale food production, which is inconsistent with the current development direction of "Clean Label" and green processing.

[0006] (4) Insufficient utilization of low-value tea resources: Summer and autumn tea, pruned leaves, coarse old leaves and other raw materials have large output and low price. Under conventional hot water extraction conditions, they taste rough and astringent and lack sweetness. They are usually treated as low-grade raw materials or even discarded. There is still a lack of a process that can efficiently "activate" and utilize the potential sweetness components. Summary of the Invention

[0007] The purpose of this invention is to provide an enzyme-subcritical water complex extraction method for the targeted enrichment of the aftertaste components of tea. This method can efficiently dissolve and enrich the bound glycosides and other aftertaste precursors in tea in an all-water system. It can precisely control the degree of partial hydrolysis of glycosides through "restricted enzymatic hydrolysis", retain their bound structure and delayed release characteristics, ensure the quality of tea soup, and make full use of low-value tea raw materials, thus "turning waste into treasure".

[0008] To achieve the above objectives, the present invention employs the following technical solution: An enzyme-subcritical water complex extraction method for targeted enrichment of the aftertaste components of tea includes the following steps: S1. Subcritical water pre-extraction and modification: Tea raw materials are mixed with deionized water and placed in a high-pressure reactor; the mixture is treated under subcritical conditions of 1.5-5.0 MPa and 120-160℃ for 5-15 minutes; the high permeability and low dielectric constant of subcritical water are used to break down the cell walls of tea leaves and dissolve macromolecular glycoside precursors, resulting in a subcritical extract rich in precursors. S2. The extract obtained in step S1 is rapidly cooled to 40℃~50℃, the pH value is adjusted, and a compound enzyme preparation is added for enzymatic hydrolysis. The enzymatic hydrolysis process is controlled by monitoring the enzyme-catalyzed reaction rate: the reaction progress is calculated according to the Michaelis-Menten equation, and the end point of enzymatic hydrolysis is set as the window period that maximizes the "sweet aftertaste / bitter flavor ratio". When the concentration of reducing sugar in the reaction system increases to 15%~20% of the theoretical total amount that can be hydrolyzed, the reaction is immediately terminated by heating to inactivate the enzyme. S3. Targeted enrichment and separation: Centrifuge the enzyme hydrolysate after enzyme inactivation in step S2 to remove the precipitate, and concentrate the supernatant through a nanofiltration membrane with a molecular weight cutoff of 500-1000 Da to retain and enrich the target sweet aftertaste component, and obtain a high sweet aftertaste tea concentrate. S4. Solid-liquid separation and clarification: The concentrated juice after step S3 is subjected to solid-liquid separation and then filtered through a membrane to obtain a tea base rich in bonded glycosides with a sweet aftertaste.

[0009] Furthermore, in step S2, the compound enzyme preparation is composed of tanninase, cellulase and β-glucosidase in a mass ratio of 2:1:1; the amount of enzyme added is 0.1% to 0.5% of the dry weight of the substrate.

[0010] Furthermore, in step S2, the "theoretical total amount that can be hydrolyzed" is a theoretical value calculated based on the initial molar concentration of glycosides in the raw materials; the 15% to 20% enzymatic hydrolysis range corresponds to the stage where glycosidic bonds are broken but the parent nucleus is not degraded, and is the optimal flavor balance point determined by response surface methodology. Within this range, the bitter macromolecular polyphenols have been partially degraded, while gallic acid, which produces bitterness, has not yet accumulated in large quantities.

[0011] Furthermore, in step S1, the subcritical water treatment maintains the water in a liquid state through a pressure control system; after the treatment is completed, a flash cooling method is used to reduce the temperature of the liquid to below 60°C within 30 seconds to prevent the degradation of heat-sensitive components.

[0012] Furthermore, in step S3, the nanofiltration membrane separation process is carried out at an operating pressure of 0.5 to 1.5 MPa, and inorganic salts and monosaccharide molecules are removed by cross-flow filtration.

[0013] Furthermore, in step S2, when the amount of reducing sugar or glucose released in the reaction solution reaches 15% to 20% of the theoretical total, the system is immediately heated to 90 to 95°C and kept at that temperature for 3 to 5 minutes to inactivate the enzyme preparation, thereby terminating the enzymatic hydrolysis reaction.

[0014] Furthermore, the filter membrane in step S4 is an inorganic ceramic membrane with a pore size of 30-80 nm.

[0015] Furthermore, the obtained tea base can be used to prepare instant tea powder, tea beverage concentrate, or as a flavor-enhancing base in other foods.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention combines ultra-high pressure microjet cell disruption with subcritical water extraction to release large quantities of previously difficult-to-dissolve bonded glycosides and other precursors of the sweet aftertaste. It can successfully extract high-value sweet aftertaste functional components from inexpensive coarse old leaves, summer and autumn teas, and pruned leaves, which helps reduce the raw material costs of high-end instant teas and tea beverages and improves the utilization rate of low-grade raw materials.

[0017] 2. This invention uses water as the sole extraction medium throughout the entire process, adjusting temperature and pressure to maintain a subcritical state, thereby achieving enhanced dissolution of the target components in a green manner. The entire process does not introduce any organic solvents, completely eliminating the risk of solvent residue and aligning with the development trends of "clean label" and green food processing.

[0018] 3. By setting a limited enzymatic hydrolysis endpoint of "15% to 20% of the theoretical total amount", this invention can moderately improve its solubility and perceptibility while preserving the glycosidic bond structure and latent aftertaste characteristics. The "burst time" and "duration" of the aftertaste can be controlled by fine-tuning the endpoint according to different product needs, so as to achieve standardized and reproducible flavor production.

[0019] 4. When the end point of enzymatic hydrolysis is controlled within the range of 15% to 20% of the theoretical total amount, this invention can achieve the best balance between the intensity and duration of the aftertaste, exhibiting a non-linear window effect, thereby significantly improving the overall sensory experience of the tea soup. Detailed Implementation

[0020] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined in this application.

[0021] This invention describes an enzyme-subcritical water complex extraction method for the targeted enrichment of the aftertaste components of tea, the main structure of which includes the following steps: S1. Raw material pretreatment: Select one or more of the following as raw materials: summer and autumn tea, coarse old leaves and pruned leaves. Dry the raw materials to a moisture content of 8% to 12% and grind them to 20 to 40 mesh to obtain tea powder.

[0022] After obtaining the tea powder, the tea powder is subjected to ultra-high pressure micro-jet treatment 1-2 times under a pressure of 250-350 MPa, preferably 300 MPa, to cause strong shearing and cavitation destruction of the cell wall structure, exposing intracellular bonded glycosides and other effective components, thereby significantly improving the subsequent extraction efficiency.

[0023] S2. Subcritical water extraction: The pretreated tea powder from step S1 is placed into a subcritical water extraction vessel, and pure deionized water is added as the sole extraction medium. The tea powder is dynamically extracted for 5 to 15 minutes at a temperature of 120–160°C and a pressure of 1.5–5.0 MPa, preferably at a temperature of 125–135°C and a pressure of 3.5–4.5 MPa. The extraction time is preferably 15 minutes, resulting in an extract containing flavonoid glycosides, triterpenoid saponins, and other components related to the sweet aftertaste.

[0024] Under the above conditions, water is in a subcritical state, its dielectric constant decreases significantly, its polarity weakens, and its solubility is closer to that of moderately polar solvents such as ethanol. This significantly improves the solubility of organic macromolecules such as flavonoid glycosides and triterpenoid saponins. Compared to room temperature water or hot water at 80–90°C, subcritical water significantly enhances the dissolution capacity of these bonded glycosides, achieving enhanced "physical hard extraction" of the precursors to the sweet aftertaste of tea.

[0025] S3. Restricted Enzymatic Hydrolysis: The extract obtained in step S2 is rapidly cooled to 45°C using a heat exchanger or other means, and the pH is adjusted to 5.5. A compound enzyme preparation is added to the extract, and the enzymatic hydrolysis reaction is carried out under constant temperature stirring at 45°C. During the reaction, firstly, under the same extract system and conditions, the "theoretical total amount that can be hydrolyzed" is calculated based on the initial molar concentration of glycosides in the raw materials. Then, in actual production, when the increase in the reducing sugar concentration in the reaction system reaches 15% to 20% of the theoretical total amount that can be hydrolyzed, the system is immediately heated to 90 to 95°C and kept at this temperature for 3 to 5 minutes to inactivate the enzyme preparation, thereby terminating the enzymatic hydrolysis reaction.

[0026] The compound enzyme preparation includes: cellulase with an activity of 50 U / g and pectinase with an activity of 200 U / g, with a mass ratio of cellulase to pectinase of 1:3. The total amount of the compound enzyme preparation added is 0.2% to 0.5% of the solid content in the extract. Cellulase mainly acts on the cellulose skeleton structure in the cell wall, while pectinase helps degrade pectin-like substances and reduce the viscosity of the system. The synergistic effect of the two can effectively disrupt the polysaccharide network of the cell wall, improve mass transfer conditions, and thus promote the directional release and dissolution of sweetness-related components such as bonded glycosides.

[0027] Through the controlled restriction of the degree of enzymatic hydrolysis, the glycoside structure is cleaved from complex, insoluble long chains or complexes into shorter, more soluble structures, while still retaining some bonded characteristics. This "partially hydrolyzed" state allows the glycosides to continue to be gradually hydrolyzed and released by saliva and the oral microenvironment in the mouth and throat after the tea soup enters the mouth, forming a delayed-release flavor characteristic of "bitter at first, then sweet, with a long-lasting aftertaste".

[0028] Practice shows that when the enzymatic hydrolysis endpoint is controlled at 15% to 20% of the theoretically hydrolyzable total amount, the large molecular polyphenols that produce bitterness in the tea soup have been partially degraded, while gallic acid, which produces bitterness, has not yet accumulated in large quantities.

[0029] S4. Solid-liquid separation and clarification: The reaction liquid after step S is subjected to solid-liquid separation. First, large particulate suspended matter and some insoluble matter are removed by centrifugation. Then, it is filtered through an inorganic ceramic membrane with a pore size of 30-80 nm, preferably 50 nm, to remove large molecular proteins, enzyme inactivation precipitates and other high molecular impurities, so as to obtain a clear and transparent tea soup base rich in bonded glycosides and sweet aftertaste substances.

[0030] The tea base material prepared above can be used to prepare instant tea powder, tea beverage concentrate, or as a flavor-enhancing base material in other foods.

[0031] Example: An enzyme-subcritical water complex extraction method for targeted enrichment of the aftertaste components of tea, comprising the following steps: S1. Raw material pretreatment: Take 1 kg of summer and autumn green tea, dry it to a moisture content of about 10%, and pulverize it to a particle size of 20-40 mesh to obtain tea powder. If necessary, perform two ultra-high pressure micro-jet treatments under 300 MPa conditions to destroy the cell wall structure and expose intracellular bonded glycosides and other effective components. S2. Subcritical water extraction: The tea powder obtained in step S1 is mixed with deionized water at a material-to-liquid ratio of 1:10 to 1:20 and placed in a subcritical water extraction vessel. Pure deionized water is used as the sole extraction medium. The temperature is rapidly raised to 130°C and the pressure is controlled at 4.0 MPa for 18 minutes to extract the tea powder and obtain an extract containing flavonoid glycosides, triterpenoid saponins and other components related to the aftertaste. S3. Restricted enzymatic hydrolysis: Cool the extract obtained in step S2 to 45°C and adjust the pH to 5.5. Add a compound enzyme preparation with a mass fraction of 0.3% of the solids in the extract. The compound enzyme preparation consists of cellulase and pectinase in a mass ratio of 1:3. Perform a constant temperature stirring enzymatic hydrolysis reaction at 45°C for about 35 minutes. During the process, take samples at regular intervals to measure the reducing sugar concentration. When the increase in reducing sugar concentration reaches 15% to 20% of the theoretical total hydrolyzable amount, immediately heat the system to 95°C and keep it at that temperature for 5 minutes to inactivate the enzyme preparation, thereby terminating the enzymatic hydrolysis reaction. The "theoretical total hydrolyzable amount" is a theoretical value calculated based on the initial molar concentration of glycosides in the raw material; S4. Solid-liquid separation and clarification: The reaction liquid after step S is centrifuged to remove large particulate matter, and then filtered through an inorganic ceramic membrane with a pore size of about 50 nm to obtain a tea base rich in bonded glycosides with a sweet aftertaste.

[0032] Effect test: HPLC analysis, using the summer and autumn green tea of ​​this embodiment as an example, showed that compared with the tea liquor obtained by traditional 90℃ hot water extraction, the content of representative flavonoid glycosides (such as myricetin-3-galactoside) in the tea liquor of this embodiment increased by approximately 280%. Blind evaluation by a 10-person sensory evaluation panel revealed that the bitterness and astringency of the tea liquor of this embodiment were lower than that of the control group. Approximately 3 seconds after swallowing, a more pronounced sweet aftertaste appeared in the back of the mouth, lasting for more than 45 seconds, while the sweet aftertaste of the control group tea liquor generally disappeared within 10-15 seconds.

[0033] In summary, this invention combines ultra-high pressure microjet cell disruption with subcritical water extraction to release a large amount of previously difficult-to-dissolve bonded glycosides and other precursors of the sweet aftertaste. This helps reduce the raw material costs of high-end instant tea and tea beverages, improves the utilization rate of low-grade raw materials, uses water as the sole extraction medium throughout the process, and introduces no organic solvents, eliminating any solvent residue risks. This aligns with the development trends of "clean label" and green food processing. Furthermore, this invention can moderately improve the solubility and perceptibility of the sweet aftertaste while preserving the bonded structure and latent sweet aftertaste characteristics. The "burst point" and "duration" of the sweet aftertaste can be controlled by fine-tuning the endpoint according to different product needs, achieving standardized and reproducible flavor production. It also achieves an optimal balance between the intensity and duration of the sweet aftertaste, exhibiting a non-linear window effect, thereby significantly improving the overall sensory experience of the tea infusion.

Claims

1. A method for targeted enrichment of the aftertaste components of tea by enzyme-subcritical water complex extraction, characterized in that: Includes the following steps: S1. Subcritical water pre-extraction and modification: Tea raw materials are mixed with deionized water and placed in a high-pressure reactor; the mixture is treated under subcritical conditions of 1.5-5.0 MPa and 120-160℃ for 5-15 minutes; the high permeability and low dielectric constant of subcritical water are used to break down the cell walls of tea leaves and dissolve macromolecular glycoside precursors, resulting in a subcritical extract rich in precursors. S2. The extract obtained in step S1 is rapidly cooled to 40℃~50℃, the pH value is adjusted, and a compound enzyme preparation is added for enzymatic hydrolysis. The enzymatic hydrolysis process is controlled by monitoring the enzyme-catalyzed reaction rate: the reaction progress is calculated according to the Michaelis-Menten equation, and the end point of enzymatic hydrolysis is set as the window period that maximizes the "sweet aftertaste / bitter flavor ratio". When the concentration of reducing sugar in the reaction system increases to 15%~20% of the theoretical total amount that can be hydrolyzed, the reaction is immediately terminated by heating to inactivate the enzyme. S3. Targeted enrichment and separation: Centrifuge the enzyme hydrolysate after enzyme inactivation in step S2 to remove the precipitate, and concentrate the supernatant through a nanofiltration membrane with a molecular weight cutoff of 500-1000 Da to retain and enrich the target sweet aftertaste component, and obtain a high sweet aftertaste tea concentrate. S4. Solid-liquid separation and clarification: The concentrated juice after step S3 is subjected to solid-liquid separation and then filtered through a membrane to obtain a tea base rich in bonded glycosides with a sweet aftertaste.

2. The enzyme-subcritical water complex extraction method for targeted enrichment of the aftertaste components of tea according to claim 1, characterized in that: In step S2, the compound enzyme preparation is composed of tanninase, cellulase and β-glucosidase in a mass ratio of 2:1:1; the amount of enzyme added is 0.1% to 0.5% of the dry weight of the substrate.

3. The enzyme-subcritical water complex extraction method for targeted enrichment of the aftertaste components of tea according to claim 1, characterized in that: In step S2, the "theoretical total amount of hydrolysable" is a theoretical value calculated based on the initial molar concentration of glycosides in the raw materials; the 15% to 20% enzymatic hydrolysis range corresponds to the stage where glycosidic bonds are broken but the parent nucleus is not degraded. It is the optimal flavor balance point determined by response surface methodology. Within this range, the bitter macromolecular polyphenols have been partially degraded, while gallic acid, which produces bitterness, has not yet accumulated in large quantities.

4. The enzyme-subcritical water complex extraction method for targeted enrichment of the aftertaste components of tea according to claim 1, characterized in that: In step S1, the subcritical water treatment maintains the water in a liquid state through a pressure control system; after the treatment is completed, a flash cooling method is used to reduce the temperature of the liquid to below 60°C within 30 seconds to prevent the degradation of heat-sensitive components.

5. The enzyme-subcritical water complex extraction method for targeted enrichment of the aftertaste components of tea according to claim 1, characterized in that: In step S3, the nanofiltration membrane separation process is carried out at an operating pressure of 0.5 to 1.5 MPa, and inorganic salts and monosaccharide molecules are removed by cross-flow filtration.

6. The enzyme-subcritical water complex extraction method for targeted enrichment of the aftertaste components of tea according to claim 1, characterized in that: In step S2, when the amount of reducing sugar or glucose released in the reaction solution reaches 15% to 20% of the theoretical total, the system is immediately heated to 90 to 95°C and kept at that temperature for 3 to 5 minutes to inactivate the enzyme preparation, thereby terminating the enzymatic hydrolysis reaction.

7. The enzyme-subcritical water complex extraction method for targeted enrichment of the aftertaste components of tea according to claim 1, characterized in that: The filter membrane used in step S4 is an inorganic ceramic membrane with a pore size of 30-80 nm.

8. The enzyme-subcritical water complex extraction method for targeted enrichment of the aftertaste components of tea according to any one of claims 1-7, characterized in that: The resulting tea base can be used to prepare instant tea powder, tea beverage concentrate, or as a flavor-enhancing base in other foods.