Brewing method of bergamot pear fruit wine
By using specific flavor inducers and multi-stage temperature-controlled fermentation technology, the problem of bland flavor in traditional pear wine has been solved, fermentation efficiency and flavor quality have been improved, and the unique flavor of pear wine has been preserved and its complexity enhanced.
Patent Information
- Application Number
- CN202511169119.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-20
- Publication Date
- 2025-10-17
AI Technical Summary
In the traditional pear wine brewing process, pear juice lacks ester synthesis precursors, resulting in a limited variety and low content of flavor compounds, which fails to meet consumers' demand for diverse flavors and health benefits in fruit wine.
By employing a specific combination of flavor inducers (L-proline, linoleic acid, manganese sulfate, and biotin) combined with multi-stage temperature control and dual-strain synergistic fermentation, the esterification pathway of *Calibic Mayer's yeast* is activated in a targeted manner. This is further enhanced by multi-enzyme hydrolysis treatment and precise adaptation to the metabolic needs of microorganisms, thereby improving fermentation efficiency and flavor complexity.
The content of key aroma esters and terpenes in pear wine is significantly increased, the fermentation rate and sugar conversion efficiency are improved, and pear wine with excellent flavor and stable quality is formed, which retains the unique flavor characteristics of pear.
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Figure CN120796016A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of fruit wine brewing, and particularly relates to a brewing method of fragrant pear fruit wine. BACKGROUND
[0002] With the increasing demand of consumers for healthy drinks, fruit wine is attracting more and more attention due to its rich nutritional ingredients and unique flavor. At present, fruit wine brewing technology is developing towards improving wine quality, retaining more nutritional ingredients and optimizing flavor characteristics.
[0003] Fragrant pear fruit wine is a low-alcohol beverage made from Korla fragrant pears through microbial fermentation. The traditional fragrant pear fruit wine brewing process mainly includes the steps of raw material pretreatment, single-strain constant-temperature fermentation, aging and clarification. However, fragrant pear juice naturally lacks ester synthesis precursors (such as medium-chain fatty acids) and key cofactors, resulting in a small number of flavor substances and low content in the fermentation product, and the fragrant pear fruit wine has a weak flavor. With the increasing demand of consumers for the flavor diversity and health attributes of fruit wine, how to improve fermentation efficiency and flavor quality through process optimization has become a key challenge in the industry. SUMMARY
[0004] To solve the above technical problems, the present application provides a brewing method of fragrant pear fruit wine. By adding a specific combination of inducers, combined with multi-stage temperature control and double-strain synergistic fermentation, the unique flavor characteristics of fragrant pear itself are effectively retained without adding flavoring spices, and the flavor complexity, fermentation efficiency and final quality of the fruit wine are simultaneously improved.
[0005] To achieve the above-mentioned application purposes, the present application adopts the following technical solutions: In a first aspect, a brewing method of fragrant pear fruit wine includes the following steps: S1: multi-enzyme hydrolysis treatment is performed on Korla fragrant pear fruit pulp to obtain enzyme-treated fruit pulp; the initial sugar content of the Korla fragrant pear fruit pulp is 18-24 °Bx, and the pH value is 3.0-3.8; S2: a flavor inducer combination is added to the enzyme-treated fruit pulp to obtain a fermentation substrate; the inducer combination includes L-proline, linoleic acid, manganese sulfate and biotin; the addition amount of L-proline is 10-40 mg / L; the addition amount of linoleic acid is 50-200 mg / L; the addition amount of manganese sulfate is 0.1-0.5 mg / L; and the addition amount of biotin is 0.05-0.2 mg / L; S3: multi-stage temperature control fermentation is performed on the fermentation substrate obtained in S2, and the multi-stage temperature control fermentation includes: First-stage fermentation: inoculate Saccharomyces cariibyckii (S. cariibyckii) and Saccharomyces bayanus (S. bayanus) in a ratio of 1:1, and ferment at 16-20℃ for 1-3 days, and then transfer to the second-stage fermentation; Meyerozyma caribbica Second-stage fermentation: inoculate S. cariibyckii and S. bayanus in a ratio of 1:1, and ferment at 20-24℃ for 1-3 days, and then transfer to the third-stage fermentation; S3: inoculating the S2 obtained fermentation material with Saccharomyces cerevisiae (S. cerevisiae) and continuing to ferment at 24-28℃ until the alcohol content reaches 8%-10% vol and the sugar content drops to ≤4 g / L, and then transferring to the third stage fermentation; Saccharomyces cerevisiae S3: inoculating the S2 obtained fermentation material with Saccharomyces cerevisiae (S. cerevisiae) and continuing to ferment at 24-28℃ until the alcohol content reaches 8%-10% vol and the sugar content drops to ≤4 g / L, and then transferring to the third stage fermentation; S3: inoculating the S2 obtained fermentation material with Saccharomyces cerevisiae (S. cerevisiae) and continuing to ferment at 24-28℃ until the alcohol content reaches 8%-10% vol and the sugar content drops to ≤4 g / L, and then transferring to the third stage fermentation; S4: collecting the wine liquid by squeezing and separating the S3 obtained fermentation material; and obtaining the pear wine by clarifying and aging the wine liquid.
[0006] Preferably, the enzymes used in the multi-enzyme hydrolysis treatment in step S1 are pectinase, cellulase and β-glucosidase.
[0007] More preferably, the specific operation of the multi-enzyme hydrolysis treatment in step S1 is: synchronously adding pectinase, cellulase and β-glucosidase to the Korla pear pulp, and standing for 0.5-2 hours at a temperature of 35-45℃; the addition amount of the pectinase is 0.01-0.03% (w / w) of the mass of the Korla pear pulp, the addition amount of the cellulase is 0.005-0.015% (w / w) of the mass of the Korla pear pulp, and the addition amount of the β-glucosidase is 0.003-0.008% (w / w) of the mass of the Korla pear pulp.
[0008] More preferably, after the multi-enzyme hydrolysis treatment, it further includes: adding sodium metabisulfite to the enzyme-hydrolyzed Korla pear pulp in an amount of 20-60 mg / L, and standing for 2-6 hours at 4-8℃.
[0009] Preferably, in step S2, the addition amount of L-proline is 30 mg / L; the addition amount of linoleic acid is 100 mg / L; the addition amount of manganese sulfate is 0.2 mg / L; and the addition amount of biotin is 0.15 mg / L.
[0010] Preferably, in step S3, the Kloeckera apiculata is Kloeckera apiculata CICC32557, and the inoculation amount is 5×10 5 -1×10 6 cells / mL; and the S. cerevisiae is S. cerevisiae EC1118, and the inoculation amount is 5×10 4 -1×10 5 cells / mL.
[0011] More preferably, the process parameters of the first stage fermentation are: fermentation temperature 16-20℃, stirring 1-2 times per day at a speed of 150 rpm, each stirring lasting for 5-10 minutes, and fermentation time 1-3 days.
[0012] More preferably, the process parameters of the second-stage fermentation are: fermentation temperature 24-28℃, fermentation time 4-6 days, stirring 2-3 times per day at a rotation speed of 200 rpm, and each stirring lasts for 5-15 minutes.
[0013] More preferably, the process parameters of the third-stage fermentation are: fermentation temperature 20-24℃, fermentation time 2-4 days, stirring once per day at a rotation speed of 100 rpm, and each stirring lasts for 10 minutes.
[0014] Preferably, before the step S1, the method further comprises a raw material pretreatment step: crushing the Korla fragrant pear to a particle size of 3-5 mm, and adding D-erythorbic acid in an amount of 0.03-0.09 g / kg of the mass of the pear pulp during the crushing process to obtain the Korla fragrant pear pulp.
[0015] Preferably, after sequentially adding chitosan and gelatin to the wine in S4, the wine is clarified at 10-15℃ for 48-72 h; the chitosan is added in an amount of 10-20 mg / L, and the gelatin is added in an amount of 20-40 mg / L; the clarified wine is subjected to membrane filtration and aging, the aging temperature is 5-15℃, the aging time is 2-6 months, and the wine is subjected to barrel turning treatment every 15-30 days during the aging process.
[0016] In a second aspect, the present application further provides a fragrant pear wine obtained by the above-mentioned brewing method.
[0017] The present application has the following beneficial effects: 1. By adding a specific flavor inducer combination (L-proline, linoleic acid, manganese sulfate, biotin), the esterification pathway of Meyerozyma caribbica is activated in a targeted manner, sufficient precursors and cofactors are provided, and the content of key aroma esters (such as ethyl hexanoate and hexyl acetate) and terpenes (such as linalool and geraniol) in the fragrant pear wine is significantly increased.
[0018] 2. The sequential fermentation of Meyerozyma caribbica and Saccharomyces cerevisiae, combined with the multi-stage temperature control process, precisely adapts to the metabolic needs of microorganisms, significantly improves the fermentation rate, sugar conversion efficiency, and substrate utilization rate.
[0019] 3. The present application forms a complete brewing process through the synergistic cooperation of each step, finally completely retains the sweet and fruity aroma of the fragrant pear itself without the need for external addition of flavoring agents, solves the problem of weak flavor of pure pear fruit wine in traditional process, and finally obtains a fragrant pear fruit wine with stable quality and excellent flavor. BRIEF DESCRIPTION OF DRAWINGS
[0020] In order to make the technical solutions of the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only need to be drawn based on some embodiments of the present application, and other drawings can be obtained by those of ordinary skill in the art without any creative effort.
[0021] Figure 1 Korla fragrant pear fruit wine provided by the present application; Figure 2 Effects of flavor inducer addition on ester synthetase activity and ester compounds of Korla fragrant pear fruit wine. DETAILED DESCRIPTION
[0022] In order to make the technical solutions of the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only need to be drawn based on some embodiments of the present application, and other drawings can be obtained by those of ordinary skill in the art without any creative effort.
[0023] The various experimental operations involved in the specific embodiments are all conventional techniques in the art, and the parts not specially noted herein can be implemented by referring to various commonly used tool books, scientific and technical literature or related instructions, manuals, etc. before the application date of the present application.
[0024] Strains used in the present application: Kluyveromyces cali cyphicus (K. cali cyphicus) Meyerozyma caribbica CICC 32557, purchased from Hunan Light Industry Research Institute Co., Ltd.; Saccharomyces cerevisiae (S. cerevisiae) Saccharomyces cerevisiae EC1118, purchased from Lallemand Company, Canada.
[0025] The culture medium used in the following embodiments of the present application is as follows: YPD medium: dissolve 10 g of yeast extract, 20 g of peptone in 900 mL of water, and add 20 g of agar powder to prepare a plate; after sterilization at high pressure 121℃ for 20 min, add 100 mL of 20 g of glucose (glucose solution is sterilized and then added).
[0026] Expansion medium: mix Korla fragrant pear fruit pulp and YPD medium according to the volume ratio of 1:1 to obtain.
[0027] Method for activating strains in the following embodiments and comparative examples of the present application: Saccharomyces cerevisiae (S. cerevisiae) Saccharomyces cerevisiae) EC1118 activation: Take the freeze-dried yeast powder of Saccharomyces cerevisiae EC1118 from -80°C and thaw it at room temperature for 10 minutes. Take 0.05 g of the yeast powder and inoculate it into 5 mL of sterilized YPD liquid medium. Incubate it at 28°C for 24 hours. Take 1 mL of the initial activated yeast liquid and inoculate it into 5 mL of fresh YPD medium. Incubate it at 28°C with 150 rpm shaking for 12 hours. Then, inoculate the yeast liquid into the expansion medium at a ratio of 3%-8% (v / v). Incubate it at 28°C with 150 rpm shaking until the yeast number reaches 1×10 6 -2×10 6 cells / mL.
[0028] Kluyveromyces cali-forniensis (K. cali-forniensis) Meyerozyma caribbica ) CICC 32557 activation: Take the bacteria from the slant strain stored at 4°C and inoculate it into 5 mL of YPD medium. Incubate it at 28°C for 48 hours. Take 1 mL of the initial activated yeast liquid and inoculate it into 5 mL of fresh YPD medium. Incubate it at 28°C with 150 rpm shaking for 24 hours. Then, inoculate the yeast liquid into the expansion medium at a ratio of 5%-10% (v / v). Incubate it at 28°C with 150 rpm shaking until the yeast number reaches 1×10 7 -2×10 7 cells / mL.
[0029] Example 1 This example provides a method for brewing pear fruit wine, which consists of the following steps: Step 1: Select Korla pears with a maturity of 95%. After washing them with sterile water three times, immerse them in a 0.1% food-grade citric acid solution for 5 minutes. Crush them to a particle size of 4 mm. Simultaneously, add 0.06 g / kg of D-erythorbic acid. Obtain Korla pear fruit pulp with an initial sugar content of 22°Bx and a pH of 3.5.
[0030] Step 2: Add 0.03% pectinase, 0.015% cellulase, and 0.003% β-glucosidase to the fruit pulp based on its mass. After stirring at 200 rpm for 10 minutes, incubate it at 35°C for 2 hours for enzymatic hydrolysis. After enzymatic hydrolysis, add sodium metabisulfite at a dosage of 40 mg / L. Transfer it to 8°C and incubate it for 2 hours. Obtain the enzymatically hydrolyzed fruit pulp.
[0031] Step 3: Add a specific combination of flavor inducers to the enzymatically hydrolyzed fruit pulp. The addition amount of L-proline is 10 mg / L, the addition amount of linoleic acid is 200 mg / L, the addition amount of manganese sulfate is 0.1 mg / L, and the addition amount of biotin is 0.2 mg / L. Mix well to obtain the fermentation substrate.
[0032] Step 4: Multi-stage temperature-controlled fermentation was performed on the fermentation substrate, the first stage fermentation: inoculate the fermentation substrate with activated yeast solution of Meyerozyma caribbica CICC 32557, the inoculation amount was 5x10 5 cells / mL, 150 rpm stirring for 15 minutes, fermentation at 18°C for 2 days, stirring at 150 rpm for 2 times per day, 5 minutes each time; The second stage: inoculate the fermentation substrate with activated yeast solution of Saccharomyces cerevisiae EC1118, the inoculation amount was 7.5x10 4 cells / mL, the temperature was raised to 26°C for 5 days, stirring 3 times per day, 5 minutes each time, until the alcohol content reached 10% vol and the sugar content decreased to ≤4 g / L; The third stage: the temperature was lowered to 22°C for 3 days, stirring 1 time per day, 100 rpm, 10 minutes.
[0033] Step 5: After the fermentation was completed, the wine and residue were separated by plate and frame press (pressure 0.3 MPa), chitosan (10 mg / L) was added to the wine, 10 minutes later, gelatin (40 mg / L) was added, and it was placed at 12°C for 60 hours, then filtered through a 0.45 μm membrane and aged at 10°C for 4 months, and the barrels were turned over every 20 days.
[0034] Example 2 The present embodiment provides a method for brewing pear fruit wine, which comprises the following steps: Step 1: Select Korla pears with 95% maturity, rinse them with sterile water three times, then immerse them in a 0.1% food-grade citric acid solution for 5 minutes, crush them to 3 mm particle size, and simultaneously add 0.09 g / kg D-erythorbic acid, to obtain Korla pear fruit pulp with an initial sugar content of 24°Bx and a pH of 3.0.
[0035] Step 2: Add 0.02% pectinase, 0.01% cellulase, and 0.005% β-glucosidase to the fruit pulp, stir at 200 rpm for 10 minutes, then place it at 40°C for 1.5 hours for enzymatic hydrolysis; after enzymatic hydrolysis, add sodium pyrosulfite at an amount of 30 mg / L, and transfer it to 6°C for 4 hours to obtain the enzymatically hydrolyzed fruit pulp.
[0036] Step 3: Add a specific flavor inducer combination to the enzymatically hydrolyzed fruit pulp, wherein the addition amount of L-proline is 30 mg / L, the addition amount of linoleic acid is 100 mg / L, the addition amount of manganese sulfate is 0.2 mg / L, and the addition amount of biotin is 0.15 mg / L, mix well to obtain the fermentation substrate.
[0037] Step 4: Multi-stage temperature-controlled fermentation was performed on the fermentation substrate, the first stage fermentation: inoculate the fermentation substrate with activated yeast solution of Meyerozyma caribbica CICC 32557, the inoculation amount was 7.5x105 cells / mL, 150 rpm stirring for 15 minutes, fermentation at 16°C for 3 days, stirring twice a day for 8 minutes each time; Second stage: inoculate the activated Saccharomyces cerevisiae EC1118 bacteria solution, the inoculation amount is 1 x 10 5 cells / mL, increase the temperature to 24°C for fermentation for 6 days, stirring 3 times a day for 10 minutes each time, ferment until the alcohol content reaches 10% vol and the sugar content decreases to ≤4 g / L; Third stage: reduce the temperature to 22°C for fermentation for 3 days, stirring once a day at 100 rpm for 10 minutes.
[0038] Step 5: After fermentation, use a plate and frame press (pressure 0.4 MPa) to separate the wine liquid from the residue, add chitosan (15 mg / L) to the wine liquid, and then add gelatin (40 mg / L) after 8 minutes, stand at 15°C for 48 hours, filter through a 0.45 μm membrane, and then age at 15°C for 2 months, decant the barrels every 15 days.
[0039] Example 3 The present example provides a method for brewing pear fruit wine, which consists of the following steps: Step 1: Select Korla pears with a maturity of 95%, rinse them with sterile water three times, then immerse them in a 0.1% food-grade citric acid solution for 5 minutes, crush them to a particle size of 3 mm, and simultaneously add 0.03 g / kg of D-erythorbic acid to obtain a Korla pear fruit pulp with an initial sugar content of 18°Bx and a pH of 3.8.
[0040] Step 2: Add 0.01% pectinase, 0.005% cellulase, and 0.008% β-glucosidase to the fruit pulp, stir at 200 rpm for 10 minutes, then stand at 45°C for 0.5 hours for enzymatic hydrolysis; after enzymatic hydrolysis, add sodium metabisulfite at an amount of 20 mg / L, transfer to 4°C and stand for 6 hours to obtain the enzymatically hydrolyzed fruit pulp.
[0041] Step 3: Add a specific flavor inducer combination to the enzymatically hydrolyzed fruit pulp, where the addition amount of L-proline is 40 mg / L, the addition amount of linoleic acid is 50 mg / L, the addition amount of manganese sulfate is 0.5 mg / L, and the addition amount of biotin is 0.05 mg / L, mix well to obtain the fermentation substrate.
[0042] Step 4: Perform multi-stage temperature-controlled fermentation on the fermentation substrate, first stage fermentation: inoculate the fermentation substrate with the activated Saccharomycopsis bailii CICC 32557 bacteria solution, the inoculation amount is 1 x 10 6 cells / mL, 150 rpm stirring for 15 minutes, fermentation at 20°C for 1 day, stirring 3 times a day for 10 minutes each time; Second stage: inoculate Saccharomyces cerevisiae EC1118 activated bacteria liquid, the inoculation amount is 5x10 4 cells / mL, the temperature is raised to 28°C for fermentation for 4 days, stirring 2 times a day for 15 minutes each time, and the fermentation is stopped when the alcohol content reaches 8% vol and the sugar content decreases to ≤4 g / L; Third stage: reduce the temperature to 20°C for fermentation for 4 days, stirring 1 time a day for 10 minutes each time.
[0043] Step 5: After the fermentation is completed, a plate and frame press (pressure 0.2 MPa) is used to separate the wine liquid and the residue, chitosan (20 mg / L) is added to the wine liquid, 10 minutes later, gelatin (20 mg / L) is added, 10°C is used for standing for 72 hours, 0.45 μm membrane filtration is carried out, and then 5°C is used for aging for 6 months, and the barrels are poured out every 30 days.
[0044] Comparative Example 1 The present comparative example provides a method for brewing a traditional single-microorganism constant-temperature pear fruit wine, which comprises the following steps: Step 1: select Korla pears with a maturity of 95%, wash them with sterile water for three times, immerse them in a 0.1% food-grade citric acid solution for 5 minutes, crush them to a particle size of 4 mm, and simultaneously add 0.06 g / kg D-erythorbic acid to obtain a Korla pear fruit pulp with an initial sugar content of 22°Bx and a pH of 3.5.
[0045] Step 2: add 0.01% pectinase, 0.015% cellulase and 0.003% β-glucosidase to the fruit pulp according to the mass of the fruit pulp, stir at 200 rpm for 10 minutes, and then stand at 35°C for 2 hours for enzymatic hydrolysis; after the enzymatic hydrolysis, add 60 mg / L sodium metabisulfite, and transfer to 8°C for standing for 2 hours.
[0046] Step 3: inoculate the activated Saccharomyces cerevisiae EC1118 bacteria liquid into the enzymatically hydrolyzed fruit pulp, the inoculation amount is 7.5x10 4 cells / mL, start fermentation after stirring at 150 rpm for 15 minutes, control the temperature at 26°C throughout the fermentation process, stir 2 times a day for 15 minutes each time, and stop the fermentation when the alcohol content reaches 10% vol.
[0047] Step 4: after the fermentation is completed, a plate and frame press (pressure 0.3 MPa) is used to separate the wine liquid and the residue, chitosan (10 mg / L) is added to the wine liquid, 10 minutes later, gelatin (40 mg / L) is added, 12°C is used for standing for 60 hours, 0.45 μm membrane filtration is carried out, and then 10°C is used for aging for 4 months, and the barrels are poured out every 20 days.
[0048] Comparative Example 2 The present comparative example provides a method for brewing a single-microorganism constant-temperature pear fruit wine, which comprises the following steps: Step 1: Select Korla fragrant pears with 95% maturity, rinse with sterile water three times, then immerse in 0.1% food-grade citric acid solution for 5 minutes, crush to 4 mm particle size, and synchronously add 0.06 g / kg D-erythorbic acid to obtain Korla fragrant pear pulp with an initial sugar content of 22 °Bx and a pH of 3.5.
[0049] Step 2: Add 0.01% pectinase, 0.015% cellulase, and 0.003% β-glucosidase according to the mass of the pulp, stir at 200 rpm for 10 minutes, then incubate at 35°C for 2 hours for enzymatic hydrolysis; after enzymatic hydrolysis, add 40 mg / L sodium pyrosulfite, transfer to 8°C and incubate for 2 hours to obtain the enzymatic hydrolysis pulp.
[0050] Step 3: Add a specific flavor inducer combination to the enzymatic hydrolysis pulp, where the addition amount of L-proline is 10 mg / L, the addition amount of linoleic acid is 200 mg / L, the addition amount of manganese sulfate is 0.1 mg / L, and the addition amount of biotin is 0.2 mg / L, and mix well to obtain a fermentation substrate.
[0051] Step 4: Inoculate the activated Saccharomyces cerevisiae EC1118 bacterial solution into the fermentation substrate, with an inoculation amount of 7.5×10 4 cells / mL, stir at 150 rpm for 15 minutes, then start fermentation, control the temperature at 26°C throughout the fermentation process, stir twice a day for 15 minutes each time, and stop fermentation when the alcohol content reaches 10% vol.
[0052] Step 5: After fermentation, separate the wine from the residue using a plate and frame press (pressure 0.3 MPa), add chitosan (10 mg / L) to the wine, then add gelatin (40 mg / L) after 10 minutes, incubate at 12°C for 60 hours, filter through a 0.45 μm membrane, and then age at 10°C for 4 months, with the barrels being turned over every 20 days.
[0053] Comparative Example 3 This comparative example provides a method for brewing fragrant pear wine, which consists of the following steps: Step 1: Select Korla fragrant pears with 95% maturity, rinse with sterile water three times, then immerse in 0.1% food-grade citric acid solution for 5 minutes, crush to 4 mm particle size, and synchronously add 0.06 g / kg D-erythorbic acid to obtain Korla fragrant pear pulp with an initial sugar content of 22 °Bx and a pH of 3.5.
[0054] Step 2: Add 0.03% pectinase, 0.015% cellulase, and 0.003% β-glucosidase according to the mass of the pulp, stir at 200 rpm for 10 minutes, then incubate at 35°C for 2 hours for enzymatic hydrolysis; after enzymatic hydrolysis, add sodium pyrosulfite at an addition amount of 40 mg / L, transfer to 8°C and incubate for 2 hours to obtain the enzymatic hydrolysis pulp.
[0055] Step 3: A specific combination of flavor inducers was added to the enzyme- treated fruit pulp, with L-proline added at 10 mg / L, linoleic acid added at 200 mg / L, manganese sulfate added at 0.1 mg / L, and biotin added at 0.2 mg / L, and the mixture was stirred to obtain a fermentation substrate.
[0056] Step 4: Saccharomyces cerevisiae EC1118 activated bacteria liquid and Meyerozyma caribbica CICC 32557 activated bacteria liquid were inoculated into the fermentation substrate at an inoculation amount of 7.5 x 10 4 cells / mL, and stirred at 150 rpm for 15 minutes, and three-stage fermentation was started. First stage: fermentation at 18°C for 2 days, stirring 2 times per day for 5 minutes each time; second stage: temperature increased to 26°C for 5 days, stirring 3 times per day for 5 minutes each time, until the alcohol content reached 10% vol; third stage: temperature decreased to 22°C for 3 days, stirring 1 time per day for 10 minutes each time.
[0057] Step 5: After fermentation, the wine liquid was separated from the residue using a plate and frame press (pressure 0.3 MPa), and chitosan (10 mg / L) was added to the wine liquid, followed by addition of gelatin (40 mg / L) after 10 minutes, and the mixture was allowed to stand at 12°C for 60 hours, and then filtered through a 0.45 μm membrane and aged at 10°C for 4 months, with the barrels being emptied every 20 days.
[0058] Comparative Example 4 This comparative example provides a method for brewing a pear fruit wine, which consists of the following steps: Step 1: Select Korla pears with a maturity of 95%, wash them three times with sterile water, and then immerse them in a 0.1% food-grade citric acid solution for 5 minutes, and then crush them to a particle size of 4 mm, and simultaneously add 0.06 g / kg of D-erythorbic acid, to obtain a Korla pear fruit pulp with an initial sugar content of 22°Bx and a pH of 3.5.
[0059] Step 2: Add 0.03% pectinase, 0.015% cellulase, and 0.003% β-glucosidase to the fruit pulp based on the mass of the fruit pulp, and stir at 200 rpm for 10 minutes, and then allow the mixture to stand at 35°C for 2 hours for enzyme treatment; after enzyme treatment, add sodium pyrosulfite at an amount of 40 mg / L, and transfer the mixture to 8°C for 2 hours of standing, to obtain enzyme-treated fruit pulp.
[0060] Step 3: Perform multi-stage temperature-controlled fermentation on the enzyme-treated fruit pulp, and in the first stage of fermentation, inoculate the enzyme-treated fruit pulp with Meyerozyma caribbica CICC 32557 activated bacteria liquid at an inoculation amount of 5 x 10 5 cells / mL, and after stirring at 150 rpm for 15 minutes, ferment at 18°C for 2 days, with stirring at 150 rpm for 2 times per day for 5 minutes each time; In the second stage of fermentation, the activated bacterial solution of Saccharomyces cerevisiae EC1118 was inoculated at an inoculation volume of 7.5×10 4 cells / mL, and ferment at 26°C for 5 days, stirring 3 times a day for 5 minutes each time, until the alcohol content reaches 10% vol and the sugar content drops to ≤4g / L; Stage 3: Cool down to 22°C and ferment for 3 days, stirring once a day at 100 rpm for 10 minutes.
[0061] Step 4: After fermentation, the wine and the residue were separated using a plate and frame press (pressure 0.3 MPa), chitosan (10 mg / L) was added to the wine, and gelatin (40 mg / L) was added 10 minutes later. The wine was allowed to stand at 12°C for 60 hours, filtered through a 0.45 μm membrane, and aged at 10°C for 4 months, with barrels being emptied every 20 days.
[0062] Verification Example 1 1. Comparison of fermentation cycle and physical and chemical indicators The basic physical and chemical indicators of the fragrant pear wine prepared in Example 1 and Comparative Examples 1-4 were measured. The alcohol content, total sugar content, total acid content, and volatile acid content of the Korla fragrant pear wine were tested in accordance with GB15038-2006 "General Analytical Methods for Grape and Fruit Wine" and GB2758-2012 "Fermented Wine and Blended Wine." The results are shown in Table 1.
[0063] Table 1 Comparison of the main fermentation cycle and physical and chemical indicators of Korla fragrant pear wine
[0064] As can be seen from Table 1, the Korla pear wine obtained by the brewing method provided by the present invention exhibits significant comprehensive advantages. Although the fermentation cycle takes 10 days, a more thorough sugar conversion is achieved (the residual sugar is only 2.5 g / L, which is 46.8% lower than that of the single-bacteria fermentation in Comparative Example 1); the total acid content (5.8 g / L) is the highest among the four groups. This is because the metabolism of Kalibikerella meyerii yeast produces organic acids such as lactic acid and acetic acid, which form an "acid-alcohol balance" with the ethanol produced by brewer's yeast, thereby improving the roundness of the wine body; the volatile acid content (0.35 g / L) is the lowest among the four groups, indicating that multi-stage temperature control effectively inhibits the metabolism of miscellaneous bacteria such as acetic acid bacteria.
[0065] 2. Comparison of volatile aroma substances Solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) was used to determine the aroma components of Korla fragrant pear wine.
[0066] Table 2 Determination results of volatile aroma compounds in Korla fragrant pear wine (unit: μg / L)
[0067] As can be seen from Table 2, the total amount of esters in the Korla fragrant pear fruit wine of Example 1 reached 845 μg / L, which was 112.3% higher than that of the traditional process (Comparative Example 1). The contents of key esters such as ethyl hexanoate, hexyl acetate, ethyl octanoate and ethyl hexanoate were the highest in each group, indicating that sequential inoculation avoided the nutritional competition between the two bacteria and provided a more optimal metabolic environment for ester synthesis. Terpenes are characteristic substances that reflect the fresh fruit aroma of Korla fragrant pear, and the total amount of terpenes in the fragrant pear fruit wine of Example 1 reached 295 μg / L, which was 2.4 times that of the traditional process (Comparative Example 1); among them, the contents of linalool (70 μg / L) and geraniol (95 μg / L) were significantly improved, which benefited from the preservation and conversion of terpene precursors in the fragrant pear raw materials by multi-stage temperature control fermentation, and at the same time, the combination of flavor inducers promoted the release of bound terpenes by Kloeckera apiculata, strengthening the natural fresh fragrance recognition of fragrant pear.
[0068] Alcohols are key regulating factors for flavor balance in fruit wine, and the content of phenethyl alcohol in the fragrant pear fruit wine of Example 1 was the highest, and was significantly higher than that of other groups, giving the wine body a sweet and smooth feeling; the contents of isoamyl alcohol (heteroalcoholic spicy) and 3-methyl-1-butanol (slightly spicy) were significantly lower than those of other groups, reducing the spiciness of the wine body.
[0069] The total amount of aldehydes in the fragrant pear fruit wine of Example 1 reached 97 μg / L, which was 136.6% higher than that of the traditional brewing process (Comparative Example 1). Benzaldehyde and benzaldehyde gave the wine body a rich aroma, and hexanal preserved the natural fresh bottom note of fragrant pear. The three of them, in cooperation with esters and terpenes, formed a complex aroma network of "fruit aroma-flower aroma-herbal aroma", avoiding the problem of single aroma caused by the lack of aldehydes in the single strain group.
[0070] The total amount of lactic acid and hexanoic acid in the acid substances in the fragrant pear fruit wine of Example 1 was the highest, especially the content of lactic acid was 1.96 times that of the traditional brewing process (Comparative Example 1). The natural organic acids produced by Kloeckera apiculata and the ethanol produced by Saccharomyces cerevisiae formed a stable "acid-alcohol balance", which not only improved the roundness of the wine body, but also avoided the artificial intervention of adding exogenous acid in the single strain group, which was more in line with the flavor demand of "natural fermentation".
[0071] 3. Comparison of ester synthase activity and total ester content The ester synthase activity and total ester content of the fragrant pear fruit wine prepared in Example 1 and Comparative Example 4 were determined, and the results are shown in Table 3. Figure 2 .
[0072] 3.1 Determination method of ester synthase activity (1) Enzyme extraction and purification: Take an appropriate amount of 100 mL of Xiangli wine sample, centrifuge at 8000 r / min for 15 min at 4°C, remove the precipitate such as fruit pulp residue; take the supernatant, add pre-cooled Tris-HCl buffer (pH 8.0, containing 1 mmol / L EDTA, 5 mmol / L DTT) at a ratio of 1:3, pour into dialysis bag, dialyze in the same buffer at 4°C for 12 h (change the solution twice during the period), and the obtained dialysate is the crude enzyme solution, which is stored in ice bath for standby.
[0073] (2) Standard curve drawing: Prepare 0.02-0.1 mmol / L series of p-nitrophenol (p-NP) standard solution, take the blank buffer as reference, measure the absorbance of each concentration at 405 nm wavelength, take p-NP concentration as abscissa and absorbance as ordinate, draw the standard curve and calculate the regression equation.
[0074] (3) Enzymatic reaction and absorbance determination: Construct a 3 mL reaction system containing 2.7 mL Tris-HCl buffer, 0.2 mL 0.2 mmol / L p-nitrophenyl acetate (p-NPA) substrate, preheat at 30°C for 5 min, then add 0.1 mL of crude enzyme solution to start the reaction, immediately record the absorbance at 405 nm every 30 s with a spectrophotometer, and continue to measure for 5 min; at the same time, set up a blank group, inactivate the enzyme solution by boiling for 10 min, and operate in the same way to exclude non-enzymatic reaction interference.
[0075] (4) Enzyme activity calculation: According to the standard curve regression equation, combined with the absorbance difference between the experimental group and the blank group, calculate the amount of p-NP generated per minute, and take "the amount of enzyme required to catalyze the generation of 1 μmol p-NP per minute as 1 enzyme unit (U)" as the specific activity of ester synthase in the sample (U / g).
[0076] 3.2 Determination method of total ester content (1) Sample pretreatment and ester enrichment: Use solid phase microextraction (SPME) method, take 10 mL of Xiangli wine sample into a 20 mL headspace bottle, add 1 g of NaCl and 10 μL of internal standard solution (100 μg / mL of butyl acetate for quantitative correction), seal and equilibrate in a 60°C constant temperature water bath for 15 min; insert the PDMS / DVB material SPME fiber head, adsorb at 60°C for 30 min, desorb at 250°C for 5 min before sample injection, to realize ester enrichment and release.
[0077] (2) GC-MS conditions: Chromatographic conditions: DB-WAX polar capillary column (30 m × 0.25 mm × 0.25 μm); column temperature program: 40°C (3 min) → 5°C / min → 180°C (5 min); carrier gas: He (≥99.999%), flow rate: 1.0 mL / min; split injection (split ratio 10:1). Mass spectrometry conditions: EI ionization source (70 eV), selected ion monitoring (SIM) mode.
[0078] (3) Standard curve and calculation of total esters: Prepare 0.1-10 mg / L mixed ester standards (including ethyl acetate, ethyl octanoate, ethyl decanoate, isoamyl acetate, etc.), add an equal amount of internal standard, and analyze according to the above steps; plot the standard curve for each ester using "standard concentration / internal standard concentration" as the horizontal axis and "standard peak area / internal standard peak area" as the vertical axis, and obtain the regression equation. After sample testing, calculate the concentration of each ester using the corresponding regression equation, and sum them to obtain the total ester content (μg / L).
[0079] Depend on Figure 2 As can be seen, without the addition of an inducer, the ester synthase activity of the sample tested (provided in Comparative Example 4) was 42.3 U / g, indicating low catalytic activity, a total ester content of 400 μg / L, and limited product accumulation. However, after the addition of an inducer, the ester synthase activity of the sample obtained (provided in Example 1) reached over 118.5 U / g, with a total ester content of 502 μg / L, representing a 180.14% increase in enzyme activity and a 25.5% increase in total ester content. These results demonstrate that the addition of an inducer significantly modulates both ester synthase activity and total ester content.
[0080] 4. Sensory evaluation Ten professional wine tasters were invited to conduct a sensory evaluation of Korla fragrant pear wine. The evaluation process was carried out strictly in accordance with the requirements of GB / T10220-2012 “General Theory of Sensory Evaluation Methodology”. The evaluation content included four aspects: appearance (15 points), aroma (35 points), taste (40 points) and typicality (10 points). The scores were given according to the standards in Table 3. The results are shown in Table 4.
[0081] Table 3 Sensory scoring table
[0082] Table 4 Sensory evaluation results of Korla fragrant pear wine
[0083] From Table 4, in the sensory evaluation, the pear fruit wine prepared by using the brewing method provided in the application leads by 95 points, the color is clear, the aroma is rich and complex with pear fruit aroma and rose aroma, the taste is balanced with sweet and sour, the wine body is mellow, and the aftertaste is lasting and pure. It can be seen that the technical scheme significantly improves the sensory quality of Korla pear fruit wine.
[0084] The above merely describes preferred embodiments of the application and is not intended to limit the application, and any modification, equivalent replacement or improvement made within the spirit and principle of the application shall be included in the protection scope of the application.
Claims
1. A method for brewing fragrant pear wine, characterized in that: The following steps are involved: S1: performing a multi-enzyme hydrolysis treatment on Korla fragrant pear pulp to obtain an enzymatic hydrolyzed pulp; the Korla fragrant pear pulp has an initial sugar content of 18-24°Bx and a pH value of 3.0-3.8; S2: adding a flavor inducer combination to the enzymatically hydrolyzed fruit pulp to obtain a fermentation matrix; the inducer combination includes L-proline, linoleic acid, manganese sulfate, and biotin; the amount of L-proline added is 10-40 mg / L; the amount of linoleic acid added is 50-200 mg / L; the amount of manganese sulfate added is 0.1-0.5 mg / L; and the amount of biotin added is 0.05-0.2 mg / L; S3: performing multi-stage temperature-controlled fermentation on the fermentation substrate obtained in S2, wherein the multi-stage temperature-controlled fermentation comprises: In the first stage of fermentation, the yeast Kalibikermeyer yeast ( Meyerozyma caribbica ), ferment at 16-20℃ for 1-3 days, then enter the second stage of fermentation; In the second stage of fermentation, inoculation of brewer's yeast ( Saccharomyces cerevisiae ), continue fermenting at 24-28℃ until the alcohol content reaches 8%-10% vol and the sugar content drops to ≤4g / L, then enter the third stage of fermentation; In the third stage, the temperature is adjusted to 20-24°C for fermentation for 2-4 days, and the fermented material is obtained after the fermentation is completed; S4: The fermented material obtained in S3 is squeezed and separated, and the wine liquid is collected; after the wine liquid is clarified and aged, the fragrant pear wine is obtained.
2. The brewing method according to claim 1, characterized in that The enzymes used in the multi-enzyme hydrolysis treatment in step S1 are pectinase, cellulase and β-glucosidase.
3. The brewing method according to claim 2, characterized in that The specific operation of the multi-enzyme hydrolysis treatment in step S1 is: simultaneously adding pectinase, cellulase and β-glucosidase to the Korla fragrant pear pulp, and letting it stand at a temperature of 35-45°C for 0.5-2 hours; the amount of the pectinase added is 0.01-0.03% (w / w) of the mass of the Korla fragrant pear pulp, the amount of the cellulase added is 0.005-0.015% (w / w) of the mass of the Korla fragrant pear pulp, and the amount of the β-glucosidase added is 0.003-0.008% (w / w) of the mass of the Korla fragrant pear pulp.
4. The brewing method according to claim 2, characterized in that After the multi-enzyme hydrolysis treatment, the method further comprises: adding sodium metabisulfite to the Korla fragrant pear pulp after enzymatic hydrolysis in an amount of 20-60 mg / L, and allowing the pulp to stand at 4-8° C. for 2-6 hours.
5. The brewing method according to claim 1, characterized in that In step S2, the amount of L-proline added is 30 mg / L; the amount of linoleic acid added is 100 mg / L; the amount of manganese sulfate added is 0.2 mg / L; and the amount of biotin added is 0.15 mg / L.
6. The brewing method according to claim 1, characterized in that In step S3, the Kalibiker yeast is Kalibiker yeast CICC 32557, and the inoculation amount is 5×10 5 -1×10 6 cells / mL; the brewer's yeast was Saccharomyces cerevisiae EC1118, and the inoculation size was 5×10 4 -1×10 5 cells / mL.
7. The brewing method according to claim 6, characterized in that The process parameters of the first stage fermentation are: fermentation temperature 16-20°C, stirring 1-2 times a day at a speed of 150 rpm, each stirring time 5-10 minutes, and fermentation time 1-3 days; and / or The process parameters of the second stage fermentation are: fermentation temperature 24-28°C, fermentation time 4-6 days, stirring 2-3 times a day at a speed of 200 rpm, each stirring time 5-15 minutes; and / or The process parameters of the third stage fermentation are: fermentation temperature 20-24° C., fermentation time 2-4 days, stirring once a day at a speed of 100 rpm, and each stirring time for 10 minutes.
8. The brewing method according to claim 1, characterized in that Before step S1, the method further includes a raw material pretreatment step: crushing the Korla fragrant pear into a particle size of 3-5 mm, and adding 0.03-0.09 g / kg of D-isoascorbic acid according to the mass of the pulp during the crushing process to obtain the Korla fragrant pear pulp.
9. The brewing method according to claim 1, characterized in that In S4, chitosan and gelatin are sequentially added to the wine, and the wine is allowed to stand at 10-15°C for 48-72 hours for clarification; the amount of chitosan added is 10-20 mg / L, and the amount of gelatin added is 20-40 mg / L; the clarified wine is membrane filtered and then aged at a temperature of 5-15°C for 2-6 months, and the wine is casked every 15-30 days during the aging process.
10. Pear wine is obtained according to the brewing method according to any one of claims 1 to 9.