A high-purity ginkgo wine and its preparation method

Abstract

This invention belongs to the field of alcoholic beverage processing technology, specifically relating to a high-purity ginkgo wine and its preparation method. The preparation method comprises the following steps: ginkgo slurry undergoes preliminary enzymatic hydrolysis with neutral protease and amylase, followed by secondary enzymatic hydrolysis with pectinase and sodium bisulfite; brewing yeast fermentation broth is added for pre-fermentation; glutamine transaminase, *Saccharomyces cerevisiae* fermentation broth, and *Candida valerate* fermentation broth are added for post-fermentation; and finally, distillation and filtration are performed to obtain the ginkgo wine. This invention achieves complete decomposition and fermentation of ginkgo kernels through a two-step enzymatic hydrolysis and two-step fermentation process. The preparation method has advantages such as simple process and low cost, which is beneficial for industrial production; the prepared ginkgo wine has advantages such as clarity, strong aroma, high alcohol content, and low acidity.

Description

Technical Field

[0001] This invention belongs to the field of alcoholic beverage processing technology, specifically relating to a high-purity ginkgo wine and its preparation method. Background Technology

[0002] ginkgo( Ginkgo biloba Ginkgo biloba (L.) belongs to the Ginkgoaceae family and is a deciduous tree, also known as "duck's foot tree" or "grandfather-grandson tree." In ancient times, it was called "white fruit." Ginkgo kernels are one of China's famous dried fruits, with a delicious, chewy, and smooth texture, and are rich in nutrients. Ginkgo nuts contain abundant flavonoids, terpenes, phenols, and amino acids, possessing extremely high medicinal and edible value. Among them, flavonoids can protect capillary permeability, dilate coronary arteries, and restore arterial elasticity, thereby lowering blood pressure and achieving a therapeutic effect on hypertension. Due to its unique and effective components, ginkgo kernels have been known as a "food and medicine of the same origin" in folk medicine throughout history. Li Shizhen's *Compendium of Materia Medica* records that "cooked ginkgo warms the lungs and benefits qi, relieves asthma, reduces urination, and stops leukorrhea; raw ginkgo reduces phlegm, clears toxins, and kills parasites." Regular consumption has a significant effect on improving brain function and delaying brain aging in the elderly. Ginkgo nuts can be used as a medicinal material, with good effects on treating Alzheimer's disease, clearing blood vessels, improving memory, and treating schizophrenia. Ginkgo fruit was listed in the directory of food and medicine with the same origin as medicinal herbs in 1992. According to the list of foods with therapeutic effects recorded in ancient herbal literature, ginkgo nuts can also be used as a cough suppressant and asthma reliever. The development of ginkgo products has broad application prospects and enormous economic value. Due to the significant nutritional value and health benefits of ginkgo fruit, the methods of consumption have diversified with the development and utilization of ginkgo products; ginkgo wine has become a popular beverage in recent years.

[0003] Currently, ginkgo wine is prepared using methods such as decoction and sedimentation, extraction and blending of active ingredients, and co-fermentation with ginkgo and glutinous rice or rice. However, ginkgo wine prepared using existing technologies still suffers from problems such as low content of active ginkgo ingredients, poor taste, low purity, and impure flavor due to the co-fermentation of multiple raw materials. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the first objective of the present invention is to provide a method for preparing high-purity ginkgo wine, which has the advantages of simple process and low raw material price.

[0005] The second objective of this invention is to provide a ginkgo wine prepared by the above method, wherein the ginkgo wine has low acidity, a medium-full taste, and high alcohol content.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing high-purity ginkgo wine, the method comprising the following steps:

[0008] (1) Remove the shells from the ginkgo nuts, steam them, crush them, and obtain a slurry;

[0009] (2) Add purified water to the slurry obtained in step (1), add neutral protease and amylase, perform enzymatic hydrolysis, and after inactivation of enzymes, obtain preliminary enzymatic hydrolysate;

[0010] (3) Add pectinase and sodium bisulfite to the preliminary enzymatic hydrolysate obtained in step (2) and enzymatically hydrolyze to obtain the final enzymatic hydrolysate;

[0011] (4) Pre-fermentation: Add brewing yeast fermentation liquid to the ginkgo hydrolysate obtained in step (3) for pre-fermentation. When the mass volume percentage concentration of residual reducing sugar drops to below 3%, stop the pre-fermentation to obtain pre-fermented mash.

[0012] (5) Post-fermentation: Glutamine transaminase, *Saccharomyces cerevisiae* fermentation broth, and *Candida vallis* ( Candida guard danger Add the fermentation liquid to the pre-fermentation mash obtained in step (4) for post-fermentation. When the mass volume percentage concentration of residual reducing sugar drops to below 1%, stop the post-fermentation to obtain the post-fermentation mash.

[0013] (6) The post-fermented mash obtained in step (5) is distilled and filtered to obtain ginkgo wine.

[0014] Furthermore, in step (1), the steaming time is 30-50 min.

[0015] Further, in step (2), the mass-to-volume ratio of the slurry and purified water is 1 g: (5-10) mL; the mass ratio of the slurry, neutral protease and amylase is (30-45): (1-2): (1-7).

[0016] Furthermore, in step (2), the enzyme activities of the neutral protease and amylase are both 20,000-30,000 U / g.

[0017] Further, in step (2), the enzymatic hydrolysis temperature is 45-50℃ and the time is 1-2 h; the enzyme inactivation temperature is 100℃ and the time is 1 min.

[0018] Furthermore, in step (3), the pectinase activity is 50,000 U / g.

[0019] Further, in step (3), the mass ratio of the preliminary enzymatic hydrolysate, pectinase and sodium bisulfite is 50:(1-5):(1-3).

[0020] Furthermore, in step (3), the enzymatic hydrolysis temperature is 50-55℃ and the time is 2-3 h.

[0021] Further, in step (4), the OD of the brewing yeast fermentation broth... 600 =1.8-2.2.

[0022] Further, in step (4), the weight ratio of the brewing yeast fermentation liquid and the enzymatic hydrolysate is (0.02-0.05):1.

[0023] Furthermore, in step (4), the temperature of the pre-fermentation is 20-35℃.

[0024] Further, in step (5), the OD of the *Saccharomyces cerevisiae* fermentation broth... 600 = (1.5-2); OD of the Candida valerate fermentation broth 600 = (1.2-1.5).

[0025] Further, in step (5), the mass ratio of the pre-fermentation mash, glutamine transaminase, *Saccharomyces cerevisiae* fermentation broth and *Candida valerate* fermentation broth is (20-25): (1-3): (0.2-0.5): (0.3-0.5).

[0026] Furthermore, in step (5), the temperature of the post-fermentation is 25-30°C.

[0027] Furthermore, in step (5), the Candida valerate was purchased from the China General Microbiological Culture Collection Center, with accession number CGMCC 2.5520.

[0028] A ginkgo wine prepared by the above method.

[0029] Beneficial Effects: This invention achieves full decomposition and fermentation of ginkgo kernels through a two-step enzymatic hydrolysis and two-step fermentation process. During enzymatic hydrolysis, neutral protease further degrades ginkgolic acid, resulting in a lower total acid content in the final ginkgo wine. During fermentation, based on the fermentation of *Saccharomyces cerevisiae*, fermentation broths of *Candida albicans* and *Candida valerate* are added. By controlling the concentration of the fermentation broth, high-quality and high-purity ginkgo wine is prepared. The ginkgo wine preparation method provided by this invention has the advantages of simple process and low cost, which is beneficial for industrial production; the prepared ginkgo wine has the advantages of clarity, strong aroma, high alcohol content, low acidity, and high flavonoid content. Detailed Implementation

[0030] The technical solution 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.

[0031] Example 1

[0032] (1) Select ginkgo fruits that are free from mold, spoilage, pests and diseases, high maturity, plumpness and freshness. Remove the shell, peel, remove the core, steam for 35 minutes, and then pulp and crush to obtain pulp.

[0033] (2) Add purified water to the slurry obtained in step (1), with a mass-volume ratio of slurry to purified water of 1 g: 5 mL. Add neutral protease and amylase, both with an enzyme activity of 30000 U / g, adjust the pH to 7, and the mass ratio of slurry to neutral protease and amylase is 45:1:1. Enzymatically hydrolyze at 50℃ for 1 h, and then heat to 100℃ to inactivate the enzyme for 1 min to obtain the preliminary enzymatic hydrolysate.

[0034] (3) Add pectinase and sodium bisulfite to the preliminary enzymatic hydrolysate obtained in step (2). The activity of pectinase is 50,000 U / g. The mass ratio of pectinase to sodium bisulfite in the preliminary enzymatic hydrolysate is 50:1:1. Adjust the pH to 3.5 and enzymatically hydrolyze at 50°C for 2 h to obtain the final enzymatic hydrolysate.

[0035] (4) Pre-fermentation: Add brewer's yeast fermentation broth to the ginkgo hydrolysate obtained in step (3), and the OD of the brewer's yeast fermentation broth... 600 =2.2, the weight ratio of brewing yeast to ginkgo hydrolysate is 0.02:1, mix evenly, and carry out pre-fermentation at 20°C. When the mass volume percentage concentration of residual reducing sugar drops to below 3%, stop the pre-fermentation to obtain pre-fermented mash.

[0036] (5) Post-fermentation: Glutamine transaminase, *Saccharomyces cerevisiae* fermentation broth, and *Candida valerate* fermentation broth were added to the pre-fermentation mash obtained in step (4). The OD of the *Saccharomyces cerevisiae* fermentation broth was... 600 =1.5, OD of Candida valerate fermentation broth 600 =1.5, the mass ratio of pre-fermentation mash, glutamine transaminase, *Candida albicans* fermentation broth, and *Candida valerate* fermentation broth is 20:1:0.2:0.5. Mix thoroughly and carry out post-fermentation at 25°C. Stop post-fermentation when the residual reducing sugar concentration drops below 1%, obtaining post-fermentation mash; *Candida valerate* ( Candida spp. The accession number was purchased from the China General Microbiological Culture Collection Center, and the accession number is CGMCC 2.5520;

[0037] (6) The obtained post-fermented mash is distilled, the distillate is collected and filtered through a 0.3 μm microporous membrane to obtain ginkgo wine; after the wine is produced from the still, the heads should be removed and the tails should be removed when the alcohol content reaches below 30% vol. The body and tails should be stored separately.

[0038] (7) Aging and storage: The collected wine is stored in a sealed stainless steel storage tank for aging for 90 days to facilitate the coordination of substances in the wine and the transformation of harmful substances.

[0039] (8) Blending and bottling: The aged ginkgo liquor is blended, filtered, and bottled to obtain the finished ginkgo liquor.

[0040] The fermentation broths of *Saccharomyces cerevisiae*, *Candida albicans*, and *Candida vallis* used were all obtained by culturing at 28℃ for 40 h. The culture medium used was: yeast medium (1L): 5.0 g peptone, 3.0 g malt extract, 3.0 g yeast extract, and 3.0 g glucose. (Add the above ingredients to distilled water, bring the volume to 1000 mL, heat to dissolve, adjust the pH to 6.2, dispense, and autoclave at 121℃ for 20 min. (Note: For solid culture medium, add 20 g / L agar powder to this medium).

[0041] Example 2

[0042] (1) Select ginkgo fruits that are free from mold, spoilage, pests and diseases, high maturity, plumpness and freshness. Remove the shell, peel, remove the core, steam for 35 minutes, and then pulp and crush to obtain pulp.

[0043] (2) Add purified water to the slurry obtained in step (1), with a mass-volume ratio of slurry to purified water of 1 g: 10 mL. Add neutral protease and amylase, both with an enzyme activity of 20000 U / g, adjust the pH to 7, and the mass ratio of slurry to neutral protease and amylase is 30: 2: 7. Enzymatically hydrolyze at 50℃ for 1 h, and then heat to 100℃ to inactivate the enzyme for 1 min to obtain the preliminary enzymatic hydrolysate.

[0044] (3) Add pectinase and sodium bisulfite to the preliminary enzymatic hydrolysate obtained in step (2). The activity of pectinase is 50,000 U / g. The mass ratio of pectinase to sodium bisulfite in the preliminary enzymatic hydrolysate is 50:5:3. Adjust the pH to 3.5 and enzymatically hydrolyze at 50°C for 2 h to obtain the final enzymatic hydrolysate.

[0045] (4) Pre-fermentation: Add brewer's yeast fermentation broth to the ginkgo hydrolysate obtained in step (3), and the OD of the brewer's yeast fermentation broth... 600=1.8, the weight ratio of brewing yeast fermentation liquid to ginkgo enzymatic hydrolysate is 0.05:1, mix evenly, carry out pre-fermentation at 20°C, and stop pre-fermentation when the mass volume percentage concentration of residual reducing sugar drops to below 3% to obtain pre-fermented mash;

[0046] (5) Post-fermentation: Glutamine transaminase, *Saccharomyces cerevisiae* fermentation broth, and *Candida valerate* fermentation broth were added to the pre-fermentation mash obtained in step (4). The OD of the *Saccharomyces cerevisiae* fermentation broth was... 600 =2, OD of Candida valerate fermentation broth 600 =1.2, the mass ratio of glutamine transaminase and *Candida albicans* is 25:3:0.5:0.3. Mix thoroughly and carry out secondary fermentation at 25°C. Stop secondary fermentation when the residual reducing sugar concentration drops below 1%, obtaining the secondary fermented mash; *Candida albicans* ( Candida spp. The accession number was purchased from the China General Microbiological Culture Collection Center, and the accession number is CGMCC 2.5520;

[0047] (6) The obtained post-fermented mash is distilled, the distillate is collected and filtered through a 0.3 μm microporous membrane to obtain ginkgo wine; after the wine is produced from the still, the heads should be removed and the tails should be removed when the alcohol content reaches below 30% vol. The body and tails should be stored separately.

[0048] (7) Aging and storage: The collected wine is stored in a sealed stainless steel storage tank for 180 days to facilitate the coordination of substances in the wine and the transformation of harmful substances.

[0049] (8) Blending and bottling: The aged ginkgo liquor is blended, filtered, and bottled to obtain the finished ginkgo liquor.

[0050] The fermentation broths of *Saccharomyces cerevisiae*, *Candida albicans*, and *Candida vallis* used were all obtained by culturing at 28℃ for 40 h. The culture medium used was: yeast medium (1L): 5.0 g peptone, 3.0 g malt extract, 3.0 g yeast extract, and 3.0 g glucose. (Add the above ingredients to distilled water, bring the volume to 1000 mL, heat to dissolve, adjust the pH to 6.2, dispense, and autoclave at 121℃ for 20 min. (Note: For solid culture medium, add 20 g / L agar powder to this medium).

[0051] Comparative Example 1

[0052] (1) Select ginkgo fruits that are free from mold, spoilage, pests and diseases, high maturity, plumpness and freshness. Remove the shell, peel, remove the core, steam for 35 minutes, and then pulp and crush to obtain pulp.

[0053] (2) Add purified water to the slurry obtained in step (1), with a mass-volume ratio of slurry to purified water of 1 g: 10 mL. Add amylase with an enzyme activity of 30000 U / g, adjust the pH to 7, and the mass ratio of slurry to amylase of 45: 1. Enzymatically hydrolyze at 50℃ for 1 h, and then heat to 100℃ to inactivate the enzyme for 1 min to obtain the preliminary enzymatic hydrolysate.

[0054] (3) Add pectinase and sodium bisulfite to the preliminary enzymatic hydrolysate obtained in step (2). The activity of pectinase is 50,000 U / g. The mass ratio of pectinase to sodium bisulfite in the preliminary enzymatic hydrolysate is 50:1:1. Adjust the pH to 3.5 and enzymatically hydrolyze at 50°C for 2 h to obtain the final enzymatic hydrolysate.

[0055] (4) Pre-fermentation: Add brewer's yeast fermentation broth to the ginkgo hydrolysate obtained in step (3), and the OD of the brewer's yeast fermentation broth... 600 =2.2, the weight ratio of brewing yeast fermentation liquid to ginkgo enzymatic hydrolysate is 0.02:1. Mix evenly and carry out pre-fermentation at 20°C. When the mass volume percentage concentration of residual reducing sugar drops to below 3%, stop the pre-fermentation to obtain pre-fermented mash.

[0056] (5) Post-fermentation: Glutamine transaminase, *Saccharomyces cerevisiae* fermentation broth, and *Candida valerate* fermentation broth were added to the pre-fermentation mash obtained in step (4). The OD of the *Saccharomyces cerevisiae* fermentation broth was... 600 =1.5, OD of Candida valerate fermentation broth 600 =1.5 pre-fermentation mash, glutamine transaminase and *Candida albicans* in a mass ratio of 20:1:0.2:0.5, mixed evenly, and post-fermented at 25°C. Post-fermentation was stopped when the residual reducing sugar concentration dropped below 1%, yielding post-fermentation mash; *Candida albicans* ( Candida spp. The accession number was purchased from the China General Microbiological Culture Collection Center, and the accession number is CGMCC 2.5520;

[0057] (6) The obtained post-fermented mash is distilled, the distillate is collected and filtered through a 0.3 μm microporous membrane to obtain ginkgo wine; after the wine is produced from the still, the heads should be removed and the tails should be removed when the alcohol content reaches below 30% vol. The body and tails should be stored separately.

[0058] (7) Aging and storage: The collected wine is stored in a sealed stainless steel storage tank for aging for 90 days to facilitate the coordination of substances in the wine and the transformation of harmful substances.

[0059] (8) Blending and bottling: The aged ginkgo liquor is blended, filtered, and bottled to obtain the finished ginkgo liquor.

[0060] The fermentation broths of *Saccharomyces cerevisiae*, *Candida albicans*, and *Candida vallis* used were all obtained by culturing at 28℃ for 40 h. The culture medium used was: yeast medium (1L): 5.0 g peptone, 3.0 g malt extract, 3.0 g yeast extract, and 3.0 g glucose. (Add the above ingredients to distilled water, bring the volume to 1000 mL, heat to dissolve, adjust the pH to 6.2, dispense, and autoclave at 121℃ for 20 min. (Note: For solid culture medium, add 20 g / L agar powder to this medium).

[0061] Comparative Example 2

[0062] (1) Select ginkgo fruits that are free from mold, spoilage, pests and diseases, high maturity, plumpness and freshness. Remove the shell, peel, remove the core, steam for 35 minutes, and then pulp and crush to obtain pulp.

[0063] (2) Add purified water to the slurry obtained in step (1), with a mass-to-volume ratio of slurry to purified water of 1 g: 10 mL. Add neutral protease and amylase, both with an enzyme activity of 30000 U / g, adjust the pH to 7, and the mass ratio of slurry to neutral protease and amylase is 45: 1: 1. Enzymatically hydrolyze at 50℃ for 1 h, and then heat to 100℃ to inactivate the enzyme for 1 min to obtain the preliminary enzymatic hydrolysate.

[0064] (3) Add pectinase and sodium bisulfite to the preliminary enzymatic hydrolysate obtained in step (2). The activity of pectinase is 50,000 U / g. The mass ratio of pectinase to sodium bisulfite in the preliminary enzymatic hydrolysate is 50:1:1. Adjust the pH to 3.5 and enzymatically hydrolyze at 50°C for 2 h to obtain the final enzymatic hydrolysate.

[0065] (4) Pre-fermentation: Add brewer's yeast fermentation broth to the ginkgo hydrolysate obtained in step (3), and the OD of the brewer's yeast fermentation broth... 600 =2.2, the weight ratio of brewing yeast fermentation liquid to ginkgo enzymatic hydrolysate is 0.02:1. Mix evenly and carry out pre-fermentation at 20°C. When the mass volume percentage concentration of residual reducing sugar drops to below 3%, stop the pre-fermentation to obtain pre-fermented mash.

[0066] (5) Post-fermentation: Glutamine transaminase and *Saccharomyces cerevisiae* fermentation broth are added to the pre-fermentation mash obtained in step (4). The OD of the *Saccharomyces cerevisiae* fermentation broth is... 600 =1.5, the mass ratio of glutamine transaminase and yeast is 20:1:0.2. Mix them evenly and carry out post-fermentation at 25°C. When the mass volume percentage of residual reducing sugar drops to below 1%, stop post-fermentation to obtain post-fermented mash.

[0067] (6) The obtained post-fermented mash is distilled, the distillate is collected and filtered through a 0.3 μm microporous membrane to obtain ginkgo wine; after the wine is produced from the still, the heads should be removed and the tails should be removed when the alcohol content reaches below 30% vol. The body and tails should be stored separately.

[0068] (7) Aging and storage: The collected wine is stored in a sealed stainless steel storage tank for aging for 90 days to facilitate the coordination of substances in the wine and the transformation of harmful substances.

[0069] (8) Blending and bottling: The aged ginkgo liquor is blended, filtered, and bottled to obtain the finished ginkgo liquor.

[0070] The fermentation broths of both *Saccharomyces cerevisiae* and *Saccharomyces globosa* were obtained by culturing at 28℃ for 40 h. The culture medium used was: yeast medium (1L): 5.0g peptone, 3.0g malt extract, 3.0g yeast extract, and 3.0g glucose. (Add the above ingredients to distilled water, bring the volume to 1000mL, heat to dissolve, adjust the pH to 6.2, dispense, and autoclave at 121℃ for 20 min. (Note: For solid culture medium, add 20g / L agar powder to this medium).

[0071] Comparative Example 3

[0072] (1) Select ginkgo fruits that are free from mold, spoilage, pests and diseases, high maturity, plumpness and freshness. Remove the shell, peel, remove the core, steam for 35 minutes, and then pulp and crush to obtain pulp.

[0073] (2) Add purified water to the slurry obtained in step (1), with a mass-to-volume ratio of slurry to purified water of 1 g: 10 mL. Add neutral protease and amylase, both with an enzyme activity of 30000 U / g, adjust the pH to 7, and the mass ratio of slurry to neutral protease and amylase is 45: 1: 1. Enzymatically hydrolyze at 50℃ for 1 h, and then heat to 100℃ to inactivate the enzyme for 1 min to obtain the preliminary enzymatic hydrolysate.

[0074] (3) Add pectinase and sodium bisulfite to the preliminary enzymatic hydrolysate obtained in step (2). The activity of pectinase is 50,000 U / g. The mass ratio of pectinase to sodium bisulfite in the preliminary enzymatic hydrolysate is 50:1:1. Adjust the pH to 3.5 and enzymatically hydrolyze at 50°C for 2 h to obtain the final enzymatic hydrolysate.

[0075] (4) Pre-fermentation: Add brewer's yeast fermentation broth to the ginkgo hydrolysate obtained in step (3), and the OD of the brewer's yeast fermentation broth... 600 =2.2, the weight ratio of brewing yeast to ginkgo hydrolysate is 0.02:1, mix evenly, and carry out pre-fermentation at 20°C. When the mass volume percentage concentration of residual reducing sugar drops to below 3%, stop the pre-fermentation to obtain pre-fermented mash.

[0076] (5) Post-fermentation: Glutamine transaminase, *Saccharomyces cerevisiae* fermentation broth, and *Candida valerate* fermentation broth were added to the pre-fermentation mash obtained in step (4). The OD of the *Saccharomyces cerevisiae* fermentation broth was... 600 =1.5, OD of Candida valerate fermentation broth 600 =2, the mass ratio of pre-fermentation mash, glutamine transaminase, *Candida albicans* fermentation broth, and *Candida valerate* fermentation broth is 20:1:0.2:0.5. Mix thoroughly and carry out post-fermentation at 25°C. Stop post-fermentation when the residual reducing sugar concentration drops below 1%, obtaining post-fermentation mash; *Candida valerate* ( Candida spp. The accession number was purchased from the China General Microbiological Culture Collection Center, and the accession number is CGMCC 2.5520;

[0077] (6) The obtained post-fermented mash is distilled, the distillate is collected and filtered through a 0.3 μm microporous membrane to obtain ginkgo wine; after the wine is produced from the still, the heads should be removed and the tails should be removed when the alcohol content reaches below 30% vol. The body and tails should be stored separately.

[0078] (7) Aging and storage: The collected wine is stored in a sealed stainless steel storage tank for aging for 90 days to facilitate the coordination of substances in the wine and the transformation of harmful substances.

[0079] (8) Blending and bottling: The aged ginkgo liquor is blended, filtered, and bottled to obtain the finished ginkgo liquor.

[0080] The fermentation broths of *Saccharomyces cerevisiae*, *Candida albicans*, and *Candida vallis* used were all obtained by culturing at 28℃ for 40 h. The culture medium used was: yeast medium (1L): 5.0 g peptone, 3.0 g malt extract, 3.0 g yeast extract, and 3.0 g glucose. (Add the above ingredients to distilled water, bring the volume to 1000 mL, heat to dissolve, adjust the pH to 6.2, dispense, and autoclave at 121℃ for 20 min. (Note: For solid culture medium, add 20 g / L agar powder to this medium).

[0081] Comparative Example 4

[0082] (1) Select ginkgo fruits that are free from mold, spoilage, pests and diseases, high maturity, plumpness and freshness. Remove the shell, peel, remove the core, steam for 35 minutes, and then pulp and crush to obtain pulp.

[0083] (2) Add purified water to the slurry obtained in step (1), with a mass-to-volume ratio of slurry to purified water of 1 g: 10 mL. Add neutral protease and amylase, both with an enzyme activity of 30000 U / g, adjust the pH to 7, and the mass ratio of slurry to neutral protease and amylase is 45: 1: 1. Enzymatically hydrolyze at 50℃ for 1 h, and then heat to 100℃ to inactivate the enzyme for 1 min to obtain the preliminary enzymatic hydrolysate.

[0084] (3) Add pectinase and sodium bisulfite to the preliminary enzymatic hydrolysate obtained in step (2). The activity of pectinase is 50,000 U / g. The mass ratio of pectinase to sodium bisulfite in the preliminary enzymatic hydrolysate is 50:1:1. Adjust the pH to 3.5 and enzymatically hydrolyze at 50°C for 2 h to obtain the final enzymatic hydrolysate.

[0085] (4) Pre-fermentation: Add brewer's yeast fermentation broth to the ginkgo hydrolysate obtained in step (3), and the OD of the brewer's yeast fermentation broth... 600 =2.2, the weight ratio of brewing yeast to ginkgo hydrolysate is 0.02:1, mix evenly, and carry out pre-fermentation at 20°C. When the mass volume percentage concentration of residual reducing sugar drops to below 3%, stop the pre-fermentation to obtain pre-fermented mash.

[0086] (5) Post-fermentation: Glutamine transaminase, *Saccharomyces cerevisiae* fermentation broth, and *Candida valerate* fermentation broth were added to the pre-fermentation mash obtained in step (4). The OD of the *Saccharomyces cerevisiae* fermentation broth was... 600 =1.5, OD of Candida valerate fermentation broth 600 =1, the mass ratio of pre-fermentation mash, glutamine transaminase, *Candida albicans* fermentation broth, and *Candida valerate* fermentation broth is 20:1:0.2:0.5. Mix thoroughly and carry out post-fermentation at 25°C. Stop post-fermentation when the residual reducing sugar concentration drops below 1%, obtaining post-fermentation mash; *Candida valerate* ( Candida spp. The accession number was purchased from the China General Microbiological Culture Collection Center, and the accession number is CGMCC 2.5520;

[0087] (6) The obtained post-fermented mash is distilled, the distillate is collected and filtered through a 0.3 μm microporous membrane to obtain ginkgo wine; after the wine is produced from the still, the heads should be removed and the tails should be removed when the alcohol content reaches below 30% vol. The body and tails should be stored separately.

[0088] (7) Aging and storage: The collected wine is stored in a sealed stainless steel storage tank for aging for 90 days to facilitate the coordination of substances in the wine and the transformation of harmful substances.

[0089] (8) Blending and bottling: The aged ginkgo liquor is blended, filtered, and bottled to obtain the finished ginkgo liquor.

[0090] The fermentation broths of *Saccharomyces cerevisiae*, *Candida albicans*, and *Candida vallis* used were all obtained by culturing at 28℃ for 40 h. The culture medium used was: yeast medium (1L): 5.0 g peptone, 3.0 g malt extract, 3.0 g yeast extract, and 3.0 g glucose. (Add the above ingredients to distilled water, bring the volume to 1000 mL, heat to dissolve, adjust the pH to 6.2, dispense, and autoclave at 121℃ for 20 min. (Note: For solid culture medium, add 20 g / L agar powder to this medium).

[0091] Performance testing

[0092] 1. Alcohol content was determined according to GB / T15038-2006: distillation gravity method; total acidity was neutralized by sodium hydroxide titration; sensory evaluation: a 10-person evaluation panel conducted a comprehensive evaluation of the ginkgo wine based on color, flavor, and taste, and the average value was taken to determine the best solution. The scores corresponding to the degree of clarity were as follows: clear: 5 points; slightly cloudy: 4 points; cloudy: 3 points; somewhat cloudy: 2 points; the scores corresponding to the aroma were as follows: strong: 5 points; rich: 4 points; strong: 3 points; weak: 2 points; somewhat weak: 1 point. The above test and sensory evaluation results are shown in Table 1.

[0093] Table 1. Results of index testing for Ginkgo wine

[0094]

[0095] In addition, the taste evaluation results showed that the ginkgo wines prepared in Examples 1 and 2 had the most medium-bodied taste, while the ginkgo wines prepared in Comparative Examples 1-4 all showed a relatively thin body.

[0096] The content of flavonoids in the ginkgo wines prepared in Examples 1-2 and Comparative Examples 1-4 was determined by HPLC. The specific determination steps were as described in the literature (Xie Dong, Lu Minyi, Luo Yi. Determination of total flavonoid content in ginkgo leaf extract by HPLC [J]. Journal of Traditional Chinese Medicine, 2000, (03): 12-13.). The determination results are shown in Table 2. Table 2 Determination results of flavonoid content in ginkgo wines prepared in Examples 1-2 and Comparative Examples 1-4

[0097]

[0098] In the description of this specification, the references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0099] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A method for preparing high-purity ginkgo wine, characterized in that, The preparation method comprises the following steps: (1) Remove the shells from the ginkgo nuts, steam them, crush them, and obtain a slurry; (2) Add purified water to the slurry obtained in step (1), add neutral protease and amylase, perform enzymatic hydrolysis, and after inactivation of enzymes, obtain preliminary enzymatic hydrolysate; (3) Add pectinase and sodium bisulfite to the preliminary enzymatic hydrolysate obtained in step (2) and enzymatically hydrolyze to obtain the final enzymatic hydrolysate; (4) Pre-fermentation: Add brewing yeast fermentation liquid to the enzymatic hydrolysate obtained in step (3) for pre-fermentation. When the mass volume percentage concentration of residual reducing sugar drops below 3%, stop the pre-fermentation to obtain pre-fermented mash. (5) Post-fermentation: Glutamine transaminase, *Saccharomyces cerevisiae* fermentation broth, and *Candida valerate* fermentation broth are added to the pre-fermentation mash obtained in step (4) for post-fermentation. Post-fermentation is stopped when the residual reducing sugar concentration drops below 1%, yielding the post-fermentation mash. The OD of the *Candida valerate* fermentation broth is... 600 (1.2-1.5); (6) The post-fermented mash obtained in step (5) is distilled and filtered to obtain ginkgo wine.

2. The preparation method according to claim 1, characterized in that, In step (5), the Candida valerate was purchased from the China General Microbiological Culture Collection Center, with accession number CGMCC 2.5520.

3. The preparation method according to claim 1, characterized in that, In step (1), the steaming time is 30-50 minutes.

4. The preparation method according to claim 1, characterized in that, In step (2), the mass-to-volume ratio of the slurry and purified water is 1 g: (5-10) mL; The enzyme activities of the neutral protease and amylase are both 20,000-30,000 U / g; The mass ratio of neutral protease to amylase in the slurry is (30-45):(1-2):(1-7). The enzymatic hydrolysis is performed at a temperature of 45-50℃ for 1-2 hours.

5. The preparation method according to claim 1, characterized in that, In step (3), the pectinase activity is 50,000 U / g; The preliminary enzymatic hydrolysate has a pectinase and sodium bisulfite mass ratio of 50:(1-5):(1-3). The enzymatic hydrolysis is performed at a temperature of 50-55℃ for 2-3 hours.

6. The preparation method according to claim 1, characterized in that, In step (4), the OD of the brewing yeast fermentation broth 600 =1.8-2.

2.

7. The preparation method according to claim 1, characterized in that, In step (4), the weight ratio of the brewing yeast fermentation broth to the enzymatic hydrolysate is (0.02-0.05):1; The temperature for the pre-fermentation is 20-35℃.

8. The preparation method according to claim 1, characterized in that, In step (5), the OD of the *Saccharomyces cerevisiae* fermentation broth... 600 = (1.5-2); The temperature of the post-fermentation is 25-30℃.

9. The preparation method according to claim 1, characterized in that, In step (5), the mass ratio of the pre-fermentation mash, glutamine transaminase, *Saccharomyces cerevisiae* fermentation broth and *Candida vallis* fermentation broth is (20-25): (1-3): (0.2-0.5): (0.3-0.5).

10. Ginkgo wine prepared by the method according to any one of claims 1-9.

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