Preparation method for optimizing flavor of illicium verum wine through synergy of biotransformation and alcohol reduction

Abstract

The invention discloses a preparation method for optimizing the flavor of illicium verum wine through synergy of biotransformation and alcohol reduction, and belongs to the technical field of wine preparation. The method comprises the following steps: mixing illicium verum essential oil, a sugar donor, an immobilized enzyme preparation and a wine base, and catalyzing ethanol and the sugar donor to generate glucoside conversion reaction by using alpha-glucosidase under a control condition. The immobilized enzyme preparation is prepared by taking nano cellulose as a core carrier, coating the core carrier with hydrophilic polymer polyvinyl alcohol (PVA) to form a shielding layer, modifying chitosan, crosslinking and activating dialdehyde starch, and covalently coupling glycosidase. By utilizing the synergistic effect of enzymatic glucoside conversion and physical volatilization, the alpha-ethyl glucoside with a healthy function is generated while the alcoholic strength is reduced, and acetic acid accumulation caused by traditional oxidative alcohol reduction is avoided; meanwhile, the PVA hydrophilic shielding layer effectively blocks non-specific adsorption of the carrier to the hydrophobic star anise essential oil, so that the retention rate of flavor substances is increased, and the problems of boring flavor and rancidity of mouth feel are solved.

Description

Technical Field

[0001] This invention relates to the field of winemaking technology, and more specifically, to a method for optimizing the flavor of star anise wine through biotransformation and synergistic alcohol reduction. Background Technology

[0002] Anise liqueur is renowned in the market for its unique anethole aroma and slightly sweet taste. However, traditional anise liqueurs typically require a high alcohol content (>40% vol) to maintain the solubility and stability of hydrophobic anise oil, which contradicts the modern consumer trend towards lower-alcohol, healthier drinking. Therefore, developing a low-alcohol anise liqueur with a harmonious flavor has become a hot topic in the industry.

[0003] Current alcohol reduction technologies are mainly divided into physical methods and biological methods. Physical methods (such as vacuum distillation and reverse osmosis membranes) remove alcohol but are very likely to cause the simultaneous loss of volatile aroma components (especially trans-anetinoside in star anise oil), resulting in a bland flavor and a watery taste in the spirit.

[0004] Biological methods primarily rely on microbial fermentation or enzyme catalysis. However, existing enzymatic alcohol reduction technologies generally suffer from serious mechanistic flaws: current schemes mostly employ alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) systems, a reaction pathway that oxidizes ethanol to acetaldehyde, which is ultimately oxidized to acetic acid. This means that theoretically, every 1% vol reduction in alcohol content will produce approximately 10 g / L of acetic acid. If the alcohol reduction is significant, the final product will become a strongly pungent, acidic "anise vinegar," severely disrupting the sensory balance of the wine. Furthermore, commonly used enzyme carriers (such as unmodified cellulose and silica gel) often have porous structures, easily and non-specifically adsorbing hydrophobic aroma molecules, further deteriorating the flavor.

[0005] In view of this, the present invention proposes a method for preparing star anise wine based on transglycosylation biosynthesis. Summary of the Invention

[0006] The purpose of this invention is to utilize the transglycosylation activity of α-glucosidase to reduce the water activity of the system in the presence of a high concentration of sugar donor, catalyzing the transglycosylation reaction between ethanol and glucose to generate α-ethyl glucoside. α-EG is a non-volatile, alcohol-free natural component with a mellow bittersweet taste and has been proven to have hepatoprotective, antioxidant, and body-enhancing functions. This pathway achieves alcohol reduction while avoiding the formation of acetic acid, and utilizes a PVA-modified hydrophilic carrier to construct an "anti-adsorption barrier," thereby truly realizing synergistic flavor optimization through biotransformation.

[0007] To achieve the above objectives, this invention provides a method for preparing star anise liquor with optimized flavor through biotransformation and synergistic alcohol reduction, comprising the following steps: S1.1 Mix star anise essential oil, sugar donor, enzyme preparation and base wine, and react at a temperature of 30-45℃ and a pH of 4.5-5.5 for 24-72 hours. The enzyme preparation is prepared by covalently coupling α-glucosidase with nanocellulose that has been hydrophilically shielded by polyvinyl alcohol, modified by chitosan amylation and activated by dialdehyde starch crosslinking. S1.2 After the reaction is completed, cool to room temperature, filter to separate the solid-phase enzyme preparation and other insoluble substances, and obtain a wine that has reduced alcohol content and is rich in α-ethyl glucoside. S1.3. Finely adjust the sugar-acid ratio in the de-alcoholized wine, add potassium citrate or sodium bicarbonate solution to adjust the pH of the wine to a palatable range (3.8-4.2), and adjust the alcohol concentration as needed to balance and optimize the flavor, thus obtaining star anise wine.

[0008] The final wine was found to contain PVA and cross-linking agent residues that were below the detection limits set by national food safety standards.

[0009] Preferably, in step S1.1, the star anise essential oil is extracted using supercritical CO2 extraction. The amount of star anise essential oil added is 0.1%-0.5% of the total mass of the base liquor.

[0010] Star anise fragments are placed in the extraction tank of a supercritical CO2 extraction device. Supercritical CO2 is introduced at 40-60℃ and 30-40MPa for 2-4 hours for extraction. CO2 dissolves the volatile components in the essential oil under supercritical conditions. The extracted essential oil is separated from the CO2 gas through a depressurization process. The CO2 can be recovered and reused. The star anise essential oil is collected and stored under cold storage.

[0011] Preferably, in step S1.1, the enzyme preparation method is as follows: Food-grade polyvinyl alcohol is dissolved in deionized water and stirred at 400-500 rpm for 2 hours at 60-70℃ to obtain a polyvinyl alcohol solution with a mass concentration of 1%-5%. A nanocellulose aqueous dispersion with a concentration of 0.5-2.0 mg / mL was mixed with a polyvinyl alcohol solution and stirred continuously at 60-70℃ for 2 hours to coat the nanocellulose surface with polyvinyl alcohol. After the reaction was completed, the coated nanocellulose was washed three times with deionized water and then freeze-dried at -40℃ to -50℃ to obtain polyvinyl alcohol coated nanocellulose. Polyvinyl alcohol-coated cellulose nanoparticles were dispersed in an aqueous acetic acid solution with a pH of 4.0-5.0 to obtain a dispersion with a concentration of 0.5-2.0 mg / mL. A chitosan solution with a mass concentration of 1%-5% dissolved in an acidic medium was added, and the mixture was stirred at 25°C for 2 hours. The pH was then adjusted to neutral to allow the chitosan to precipitate. After the reaction, the mixture was washed and freeze-dried at -40°C to -50°C to obtain an aminated cellulose nanoparticle carrier. Aminated cellulose nanoparticles were dispersed in a phosphate buffer solution with a pH of 6.0-7.0, and food-grade dialdehyde starch solution was added to bring the final mass concentration to 1.0%-5.0%. The mixture was stirred and activated at 25°C for 1-2 hours. After the reaction was completed, the carrier was repeatedly washed by centrifugation with buffer solution to obtain the activated carrier. The activated carrier was redispersed in sodium acetate buffer at a mass ratio of 1:3 to 5.0-6.0. α-glucosidase solution was added, with an enzyme activity of 5000-10000 U / g carrier. The reaction was carried out at 4℃ and 100-200 rpm for 12-24 h. After the reaction was completed, glycine solution was added to a final concentration of 0.1-0.5 mol / L to block unreacted sites, and the reaction was continued for 1-2 h. Finally, the mixture was thoroughly washed with sterile water to obtain the enzyme preparation.

[0012] Preferably, the dry basis mass ratio of the nanocellulose to polyvinyl alcohol is 1:0.5-2.0.

[0013] Preferably, the amount of chitosan added is 1%-5% of the mass of nanocellulose.

[0014] Preferably, the dry basis mass ratio of the α-glucosidase to the aminated nanocellulose carrier is 0.1-0.3:1.

[0015] Preferably, the amount of enzyme preparation added is 0.1%-0.5% of the total mass of the base liquor; In S1.1, the sugar donor is anhydrous glucose or maltose, and the amount added is 10%-20% of the total mass of the base alcohol, in order to generate a high-concentration substrate environment to promote the transglycosylation reaction.

[0016] Preferably, in S1.1, the base wine is a mixed wine with wine as the base and adjusted to an alcohol volume fraction of 8%-18% using deionized water.

[0017] Preferably, in step S1.3, the alcohol concentration is adjusted by adding deionized water to adjust the alcohol volume fraction of the wine to 2%-8%.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: In the biotransformation-assisted alcohol reduction method for optimizing the flavor of star anise liquor of this invention, an innovative PVA hydrophilic shielded enzyme carrier system is constructed, and the transglycosylation of α-glucosidase is used to replace the traditional oxidative alcohol reduction pathway. First, the polyvinyl alcohol hydration membrane on the outer layer of modified nanocellulose can effectively block the non-specific binding of hydrophobic star anise essential oil to the carrier, significantly improving the retention rate of flavor substances. Second, the transglycosylation reaction is used to convert ethanol into α-ethyl glucoside (α-EG), which has liver-protective, antioxidant, and mellow taste properties. While achieving efficient alcohol reduction, the formation of acetic acid is avoided, eliminating the problem of liquor spoilage caused by traditional enzymatic alcohol reduction. In addition, the immobilized enzyme preparation has good stability in the high-alcohol environment of the liquor base and can be reused, ensuring the dual effects of batch consistency and flavor optimization. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Raw materials and reagents instructions 1. α-Glucosidase (EC3.2.1.20): Food grade, derived from Aspergillus niger, with transglycosylation activity ≥50,000 U / mL, purchased from Novozymes (China) Biotechnology Co., Ltd.

[0021] 2. Nanocellulose (CNF): 10-20nm in diameter, purchased from Guilin Qihong Technology Co., Ltd.

[0022] 3. Polyvinyl alcohol (PVA): Degree of polymerization 1700, degree of alcoholysis 99%, food grade.

[0023] 4. Dialdehyde starch (DAS): Oxidation degree 80%, food-grade crosslinking agent, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0024] 5. Base wine: Chardonnay dry white wine is used as the base, and the alcohol volume fraction is adjusted to 12% vol through distillation to serve as the simulated base wine.

[0025] Determination methods To scientifically evaluate the alcohol reduction effect and flavor optimization degree, the following methods were used for testing: 1. Decrease in alcohol content and product analysis (HPLC method): Alcohol content determination: The alcohol content (%vol) before and after the reaction was determined using an Anton Paar density meter.

[0026] Determination of α-EG and acetic acid content: High performance liquid chromatography (HPLC) was used. Chromatographic column: Aminex HPX-87H; mobile phase: 5 mL H2SO4; flow rate: 0.6 mL / min; detector: RI differential refractive index detector.

[0027] Indicator logic: α-EG represents beneficial conversion, and acetic acid represents the degree of rancidity.

[0028] 2. Retention rate of core flavor compounds (GC-MS method): The trans-anestin content was determined by headspace solid-phase microextraction (HS-SPME) combined with GC-MS. The internal standard was 3-octanol.

[0029] Retention rate (%) = (concentration after reaction / concentration before reaction) × 100%.

[0030] 3. Sensory comprehensive evaluation (quantitative descriptive analysis): A panel of 10 trained evaluators conducted blind tastings of the samples. The scoring dimensions included: "Aroma Typicality" (whether the star anise aroma was strong), "Acidity" (whether there was a pungent vinegar smell), "Full Body" (whether the wine was full-bodied), and "Overall Harmony." The maximum score was 10 points. Example 1:

[0031] S1.1 Enzyme Preparation: 1. PVA coating: Food-grade PVA was dissolved in deionized water to prepare a 3% solution. The nanocellulose (CNF) suspension and the PVA solution were mixed at a dry weight ratio of 1:1, stirred at 65°C for 2 hours, and then freeze-dried to obtain the PVA-CNF complex.

[0032] 2. Chitosan modification: The PVA-CNF complex was dispersed in a 1% acetic acid aqueous solution at pH 4.5. Chitosan (3% of the CNF mass) was added and dissolved. After stirring for 1 hour, 1M NaOH was slowly added dropwise to adjust the pH to 7.0, allowing chitosan to precipitate and form a film on the carrier surface. The film was then washed with water and freeze-dried.

[0033] 3. Activation and Coupling: The modified carrier was dispersed in pH 6.5 phosphate buffer, and 3% dialdehyde starch (DAS) solution was added. Cross-linking activation was performed at 25°C for 2 hours. After washing, α-glucosidase (enzyme-to-carrier ratio of 0.2:1) was added, and adsorption was carried out at 4°C with stirring for 18 hours. After the reaction, the mixture was washed with buffer containing 0.1 M maltose to obtain the immobilized enzyme preparation.

[0034] Construction of the S1.2 reaction system: Add star anise oil (0.3% w / w) to a 12% vol base spirit. Add anhydrous glucose as a sugar donor to bring the final concentration to 250 g / L. Adjust the pH to 5.0.

[0035] S1.3 Biotransformation: The above-mentioned immobilized enzyme preparation was added at a rate of 20 g / L, and the reaction was carried out under shaking conditions at 35°C and 120 rpm for 48 hours.

[0036] S1.4 Post-processing: The enzyme preparation was recovered by filtration. The filtrate was filtered through a 0.45 μm membrane, and the sugar-acid ratio was finely adjusted to obtain the final product. Example 2:

[0037] The only difference from Example 1 is that the concentration of glucose added in S1.2 is adjusted to 100g / L. Example 3:

[0038] The only difference from Example 1 is that the dry basis mass ratio of PVA to nanocellulose in S1.1 is adjusted to 1:0.2.

[0039] Comparative Example 1: The difference is that glucose is not added. The enzyme preparation is changed to an immobilized "alcohol dehydrogenase + acetaldehyde dehydrogenase" system, and the coenzyme NAD+ is added. The reaction conditions are the same: 35℃, 48h.

[0040] Comparative Example 2: The difference lies in the fact that the PVA coating step is omitted in the enzyme preparation process, and the nanocellulose is directly modified with chitosan and coupled with enzymes.

[0041] Comparative Example 3: Using free enzymes The difference lies in the fact that no carrier is used; instead, free α-glucosidase of equal activity is added directly to the wine base. After the reaction, the enzyme protein is removed through an ultrafiltration membrane.

[0042] Group Initial alcohol content (%vol) Final alcohol content (%vol) alcohol reduction range α-EG formation amount (g / L) Total acid content (g / L) Trans-anetinoside retention rate Sensory evaluation score (0-10 points) Example 1 12.0 9.8 18.3% 25.6 4.2 96.5% 9.2 Example 2 12.0 10.5 12.5% 11.2 4.3 96.0% 8.1 Example 3 12.0 9.9 17.5% 24.8 4.2 85.4% 7.8 Comparative Example 1 12.0 9.5 20.8% ND (Not Detected) 22.8 94.0% 3.5 Comparative Example 2 12.0 10.0 16.7% 23.5 4.3 68.2% 7.2 Comparative Example 3 12.0 10.2 15.0% 18.9 4.5 96.2% 7.5 Table 1. Comparison of performance data between each embodiment and the comparative example. 1. Rationality analysis of alcohol reduction mechanism and product distribution (Example 1 vs. Comparative Example 1): Example 1 (the present invention): The alcohol content decreased from 12.0% vol to 9.8% vol, a reduction of 18.3%. HPLC analysis showed the formation of 25.6 g / L of α-ethyl glucoside (α-EG).

[0043] Based on the stoichiometric ratio of the reaction (ethanol: α-EG ≈ 1:4.5), the production of 25.6 g / L of α-EG consumes approximately 5.7 g / L of ethanol (approximately 0.72% vol). This means that of the total 2.2% vol alcohol reduction, approximately 33% originates from specific enzymatic biotransformation, while the remaining approximately 67% comes from accompanying physical volatilization during the 35°C reaction process. This synergistic effect of "biotransformation + physical volatilization" achieves both a significant reduction in alcohol content and the conversion of ethanol into beneficial flavor compounds.

[0044] Comparative Example 1 (oxidation method): Although the alcohol content decreased to 9.5% vol, the total acid content surged from 4.2 g / L at the base to 22.8 g / L. This is because the oxidase system directly converted the consumed ethanol into acetic acid (theoretically, about 7.9 g / L of acid is produced per 1% vol of ethanol consumed, after deducting the volatile portion). The acidity of 22.8 g / L is close to that of vinegar, resulting in a sensory score of only 3.5 points, confirming the infeasibility of the oxidation method in reducing the alcohol content of flavored wines.

[0045] 2. The crucial role of the carrier's hydrophilic barrier in flavor preservation (Example 1 vs. Example 3 vs. Comparative Example 2): Example 1 (sufficient PVA): The trans-anetinoside retention rate was as high as 96.5%, indicating that the hydration layer constructed by PVA effectively blocked the contact between the hydrophobic essential oil and the cellulose carrier.

[0046] Comparative Example 2 (without PVA): The retention rate plummeted to 68.2%. Due to the loss of PVA shielding, the high specific surface area of ​​nanocellulose resulted in strong physical adsorption of hydrophobic anethole, leading to the loss of the core aroma.

[0047] Example 3 (small amount of PVA): The retention rate was 85.4%. This gradient change further confirms that the thickness / integrity of the shielding layer is positively correlated with the flavor retention rate, and the amount of PVA coating is a key parameter for flavor optimization.

[0048] 3. Effect of enzyme formulation on reaction efficiency (Example 1 vs. Comparative Example 3): Although the flavor retention rate (96.2%) of Comparative Example 3 (free enzyme) was comparable to that of Example 1, its α-EG production (18.9 g / L) was significantly lower than that of Example 1 (25.6 g / L). The main reason for this difference lies in the enzyme's stability: in alcohol-containing systems and during long-term (48 h) reactions, free enzymes are prone to conformational changes and inactivation; while the immobilized enzyme of this invention, due to the rigid support of the carrier, has a more stable spatial conformation, thus maintaining higher catalytic efficiency. In addition, a comparison of the data of Example 3 (low PVA, α-EG production 24.8 g / L) and Example 1 shows that the presence of the PVA layer and its thickness variation did not significantly hinder the substrate (ethanol / glucose) contact enzyme active site. This indicates that the carrier design of this invention successfully constructs an "anti-adsorption barrier" without sacrificing enzyme mass transfer efficiency, achieving a win-win situation of flavor retention and efficient conversion. Furthermore, the finished wine of Example 1 was clarified, while Comparative Example 3 required an additional ultrafiltration step to remove proteins, resulting in higher process costs.

[0049] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing star anise liquor with optimized flavor through biotransformation and synergistic alcohol reduction, characterized in that, Includes the following steps: S1. Mix star anise essential oil, sugar donor, immobilized enzyme preparation and base wine; S2. The reaction is carried out under controlled temperature and pH conditions, using the immobilized enzyme preparation to catalyze the transglycosylation reaction between ethanol in the base alcohol and sugar donor, producing... α -Ethyl glucoside; S3. After the reaction is complete, the immobilized enzyme preparation is separated, and the wine is blended to obtain the finished product; The immobilized enzyme preparation is prepared by covalently coupling a glycosidase with transglycosylation activity after the following steps: the preparation involves using nanocellulose as the core carrier, coating it with a hydrophilic polymer to form a shielding layer, modifying it with polysaccharide substances through amylation, activating it with a cross-linking agent, and then using nanocellulose as the core carrier.

2. The method for preparing star anise wine with optimized flavor through biotransformation and synergistic alcohol reduction according to claim 1, characterized in that, The preparation steps of the immobilized enzyme preparation include: The nanocellulose dispersion was mixed with a polyvinyl alcohol solution and dried to obtain polyvinyl alcohol-coated nanocellulose. The polyvinyl alcohol-coated nanocellulose was dispersed in an acidic medium, and the surface was modified with chitosan by surface amylation. The pH was adjusted to allow the chitosan to deposit and solidify. The aminated carrier was activated using dialdehyde starch as a crosslinking agent; The activated carrier was contacted with an α-glucosidase solution to carry out a coupling reaction.

3. The method for preparing star anise wine with optimized flavor through biotransformation and synergistic alcohol reduction according to claim 2, characterized in that, During the preparation of the immobilized enzyme preparation, the amounts of each component satisfy the following relationship: The dry basis mass ratio of the nanocellulose to polyvinyl alcohol is 1:0.2 to 1:2.0; The amount of chitosan added is 1% to 5% of the mass of nanocellulose; The mass concentration of the dialdehyde starch solution is 1.0% to 5.0%; The dry basis mass ratio of the α-glucosidase to the carrier is 0.1:1 to 0.3:

1.

4. The method for preparing star anise wine with optimized flavor through biotransformation and synergistic alcohol reduction according to claim 1, characterized in that, The glycosidase with transglycosylation activity is selected from Aspergillus niger, Aspergillus oryzae, or Saccharomyces cerevisiae. α - At least one of the glucosidases.

5. The method for preparing star anise wine with optimized flavor through biotransformation and synergistic alcohol reduction according to claim 1, characterized in that, The sugar donor is selected from at least one of anhydrous glucose, glucose monohydrate, maltose, or maltodextrin; and in step S1, the initial concentration of the sugar donor in the reaction system is 100 g / L to 400 g / L.

6. The method for preparing star anise wine with optimized flavor through biotransformation and synergistic alcohol reduction according to claim 1, characterized in that, The conditions for the enzymatic transglycosylation reaction described in step S2 are: temperature 30°C to 45°C, pH 4.5 to 5.5, and reaction time 24 to 72 hours.

7. The method for preparing star anise wine with optimized flavor through biotransformation and synergistic alcohol reduction according to claim 1, characterized in that, The star anise essential oil is obtained by supercritical CO2 extraction; in step S3, the amount of star anise essential oil added is 0.1% to 0.5% of the total mass of the base wine.

8. The method for preparing star anise wine with optimized flavor through biotransformation and synergistic alcohol reduction according to claim 1, characterized in that, In step S1, the amount of the immobilized enzyme preparation added is 0.1% to 0.5% of the total mass of the base wine.

9. The method for preparing star anise wine with optimized flavor through biotransformation and synergistic alcohol reduction according to claim 1, characterized in that, In step S2, the wine base is a wine base whose alcohol volume fraction has been adjusted to 8% vol to 18% vol by distilled water or deionized water.

10. The method for preparing star anise wine with optimized flavor through biotransformation and synergistic alcohol reduction according to claim 1, characterized in that, The preparation described in step S3 includes: adding citrate, tartrate or lactate buffer to the wine after separating the enzyme preparation to adjust the pH to 3.8 to 4.2, and adjusting the sugar-acid ratio.