Zero-alcohol cocktail beverage with slight alcoholic flavor and preparation method of zero-alcohol cocktail beverage

Abstract

The invention relates to the technical field of non-alcoholic beverages, in particular to a zero-alcohol cocktail beverage with slight alcoholic flavor and a preparation method thereof, the beverage contains the following characteristic flavor compounds: 80-150 mg / L of ethyl hexanoate, 180-250 mg / L of phenethyl alcohol, 180-280 mg / L of isoamyl alcohol, 2.5-4.0 mg / L of beta-damascenone, and the balance of water. The alcohol content of the beverage is less than 0.05 volume percent, and accurate construction of complex flavor levels is realized by taking ethyl hexanoate as a fruit aroma main body, phenethyl alcohol as a wine aroma carrier and beta-damascenone as passion fruit characteristic aroma.

Description

Technical Field

[0001] This invention relates to the field of non-alcoholic beverage technology, specifically to a zero-alcohol cocktail beverage with a slightly alcoholic flavor and its preparation method, and more specifically, to a zero-alcohol cocktail beverage with a realistic alcoholic flavor prepared by targeted conversion of precursor substances and synergistic metabolic regulation of compound microbial strains. Background Technology

[0002] With the increasing popularity of healthy lifestyles and growing consumer demand for alcohol-free beverages, the non-alcoholic beverage market is rapidly emerging. Traditional cocktails are loved by consumers for their unique taste and rich flavor, but their alcohol content limits their consumption for some people. Therefore, the development of zero-alcohol beverages has become a hot topic in the industry.

[0003] Currently, the main technical approaches for non-alcoholic beverages with a wine-like flavor include fermentation flavor simulation, plant extract blending, and dealcoholization. Chinese patent application publication CN104220585A discloses a low-alcohol or non-alcoholic malt-based beverage and its production method. This technology involves dealcoholizing beer after malt fermentation and restoring flavor by quantitatively controlling specific volatile compounds. The core of this technology lies in controlling ethyl acetate at 7.00 to 30.00 ppm, acetic acid at 0.01 to 0.20 ppm, and isoamyl alcohol at 5.00 to 30.00 ppm, thereby achieving a flavor balance similar to non-alcoholic beer.

[0004] However, existing technologies have the following shortcomings: First, the technical approach of compensation after dealcoholization leads to complex processes and high costs, while aroma components are severely lost during dealcoholization; Second, the compound systems established for beer products cannot meet the flavor requirements of cocktails that emphasize both fruit and alcohol aromas; Third, there is a lack of technical means to regulate precursor substances and optimize metabolic flux from the front end of fermentation, making it impossible to achieve targeted synthesis of target compounds; Fourth, single-strain fermentation is difficult to construct complex flavor systems, especially in terms of balancing the synthesis of higher alcohols and esters.

[0005] Therefore, there is an urgent need to develop a new technical solution that can target and synthesize flavor compounds unique to cocktails through precise control of the front-end fermentation process without undergoing dealcoholization, while maintaining a zero-alcohol standard, and with a simple process, controllable cost, and stable flavor. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a zero-alcohol cocktail beverage with a slight alcoholic taste and its preparation method. By establishing a fingerprint spectrum of flavor compounds unique to cocktails, and by using targeted conversion of precursor substances, synergistic metabolic regulation of compound strains and three-stage precision vacuum distillation technology, the true alcoholic taste can be reconstructed under zero-alcohol conditions.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a zero-alcohol cocktail beverage with a slightly alcoholic flavor, which contains characteristic flavor compounds in a specific concentration range, including ethyl hexanoate 80 to 150 mg / L, phenylethanol 180 to 250 mg / L, isoamyl alcohol 180 to 280 mg / L, β-damascene 2.5 to 4.0 mg / L, and the alcohol content is strictly controlled to be less than 0.05% by volume.

[0008] Preferably, the beverage also contains 15 to 30 mg / L of ethyl butyrate, 25 to 45 mg / L of ethyl acetate, 40 to 70 mg / L of 2-methyl-1-butanol, and 0.08 to 0.15 mg / L of β-ionone. The synergistic effect of these compounds further enriches the flavor profile of the beverage.

[0009] In one embodiment of the invention, the beverage contains 6 to 12 mg / L of hexanoate, 3 to 6 mg / L of isoamyl acetate, 0.8 to 1.5 mg / L of linalool, and 0.3 to 0.6 mg / L of phenylacetaldehyde. These auxiliary flavor compounds play an important role in modifying and balancing the overall aroma.

[0010] The beverage formula of the present invention contains, per 100 liters, 1.2 to 1.8 liters of hydrosol, 4.5 to 6.0 liters of passion fruit concentrate, 24 to 28 kg of white sugar, 450 to 650 g of citric acid monohydrate, and 120 to 180 g of pectin, wherein the concentration of total volatile compounds in the hydrosol reaches 800 to 1200 mg per liter.

[0011] This invention also provides a method for preparing the above-mentioned zero-alcohol cocktail beverage with a slightly alcoholic flavor. The core innovation of this method lies in establishing a precursor substance targeted conversion system and a multi-strain synergistic metabolic regulation process. Specifically, it includes the following steps: The first step involves crushing the grape raw material and adding a precursor mixture for fermentation. The precursor mixture contains 800 to 1200 mg / L of leucine, 300 to 500 mg / L of isoleucine, and 600 to 900 mg / L of phenylalanine. The second step involves inoculating the fermentation broth with a compound microbial strain for two-stage fermentation. The compound microbial strain is a mixture of Saccharomyces cerevisiae K1-V1116 and Saccharomyces cerevisiae DV10, with an inoculation ratio of 80:20 by mass. The first stage fermentation temperature is 22 degrees Celsius, the time is 0 to 72 hours, and the dissolved oxygen concentration is 2.5 to 3.2 mg / L. The second stage fermentation temperature is 18 to 20 degrees Celsius, the time is 72 to 240 hours, and the dissolved oxygen concentration is 1.0 to 1.5 mg / L. The third step is to centrifuge the fermentation liquid at 6000 rpm for 15 minutes at 4 degrees Celsius after fermentation. The fermentation liquid is then filtered and the fractions are collected by low-temperature vacuum distillation. The fourth step involves mixing the fractions in a specific ratio to obtain an alcoholic hydrosol, wherein the total volatile compound concentration in the alcoholic hydrosol is 800 to 1200 mg / L. The fifth step involves mixing the pure hydrosol with passion fruit concentrate, white sugar, citric acid, pectin, and purified water. After filtration, sterilization, and bottling, the zero-alcohol cocktail beverage is obtained.

[0012] Preferably, the low-temperature vacuum distillation employs a three-stage fractionation process. In the first stage, the fraction at 20 to 30 degrees Celsius is collected under conditions of 0.08 bar pressure, 50 to 60 degrees Celsius water bath temperature, and a reflux ratio of 5:1, yielding light ester fractions. In the second stage, the fraction at 35 to 55 degrees Celsius is collected under conditions of 0.08 bar pressure, 70 to 85 degrees Celsius water bath temperature, and a reflux ratio of 3:1, yielding medium-chain esters and higher alcohol fractions. In the third stage, the fraction at 80 to 110 degrees Celsius is collected under conditions of 0.05 bar pressure, 110 to 135 degrees Celsius water bath temperature, and a reflux ratio of 2:1, yielding high-boiling-point alcohol fractions.

[0013] Preferably, the preparation method of the alcohol hydrosol is as follows: per 100 ml, mix 8 to 12 ml of light ester fraction, 20 to 30 ml of medium-chain ester and higher alcohol fraction, 5 to 8 ml of high-boiling-point alcohol fraction, add 2 to 3 ml of terpene enrichment solution, and make up to 100 ml with purified water. The terpene enrichment solution is obtained by solid-phase extraction from the distillation residue and concentration.

[0014] Preferably, the precursor mixture further contains 20 to 35 mg / L of free hexanoic acid and 8 to 15 mg / L of free butyric acid, and the fermentation process is carried out at a pH of 3.9 to 4.1, with an initial inoculum size of 5 × 10⁻⁶. 6 Colony forming units per milliliter.

[0015] Preferably, in the 72nd hour of the second fermentation step, the fermentation temperature is reduced from 22 degrees Celsius to 18 to 20 degrees Celsius, and enzymatic treatment is performed after centrifugation of the fermentation broth and before distillation. The enzymatic treatment uses carotenoid lysin dioxygenase to react for 24 to 48 hours at a temperature of 35 to 40 degrees Celsius and a pH of 5.5 to 6.0.

[0016] Preferably, in the fifth step, the amount of the added alcohol hydrosol is 1.2 to 1.8 liters, the amount of added passion fruit concentrate is 4.5 to 6.0 liters, the amount of added white sugar is 24 to 28 kg, the amount of added citric acid monohydrate is 450 to 650 g, and the amount of added pectin is 120 to 180 g per 100 liters. The prepared mixture is dissolved at a temperature above 65 degrees Celsius for 10 minutes and then filtered through a precision filter with a filtration accuracy of 1 micrometer. The filtered liquid is then sterilized at 135 degrees Celsius ± 2 degrees Celsius for 30 seconds.

[0017] The beneficial effects of this invention are as follows: First, this invention establishes for the first time a flavor compound fingerprint spectrum for zero-alcohol cocktails, clearly defining the concentration ranges and aroma contributions of key compounds such as ethyl hexanoate, phenylethanol, isoamyl alcohol, and β-damascene. This distinguishes it from the compound systems of existing beer products, reflecting the characteristic of cocktails that emphasize both fruity and alcoholic aromas. By using ethyl hexanoate as the main fruity aroma, phenylethanol as the alcoholic aroma carrier, and β-damascene as the characteristic aroma of passion fruit, the precise construction of complex flavor layers is achieved.

[0018] Secondly, this invention employs precursor-targeted conversion technology. By adding amino acid precursors such as leucine, isoleucine, and phenylalanine, it utilizes the Ehrlich metabolic pathway of yeast to target the synthesis of higher alcohols such as isoamyl alcohol and phenylethanol, achieving a conversion efficiency of over 70%. Simultaneously, by controlling the supply of free hexanoic acid and butyric acid, ethyl hexanoate and ethyl butyrate are efficiently synthesized under the action of alcohol acyltransferases. This front-end regulation strategy avoids the technical path of post-alcoholization compensation, resulting in a simpler process and less loss of flavor compounds.

[0019] Third, this invention innovatively employs a synergistic metabolic regulation system using a composite microbial strain. Through fermentation of brewing yeasts K1-V1116 and DV10 at an 80:20 ratio, K1-V1116 dominates the synthesis of higher alcohols, while DV10 enhances ester formation and phenylethanol accumulation. The complementary metabolism of these two strains achieves a balance between esters and alcohols. The two-stage fermentation strategy further optimizes metabolic flux. The first stage, at 22 degrees Celsius, rapidly synthesizes higher alcohols, while the second stage, at 18 to 20 degrees Celsius, promotes esterification. The final product achieves an ester to alcohol ratio of 1.8 to 2.5:1, consistent with the flavor characteristics of cocktails.

[0020] Fourth, the three-stage precision vacuum distillation technology developed in this invention achieves accurate separation and enrichment of aroma components based on the differences in the boiling points of compounds. At a pressure of 0.05 to 0.08 bar, the boiling point is lowered by 50 to 130 degrees Celsius, avoiding the destruction of aroma by high temperatures. By collecting light esters in the first stage, medium-chain esters and higher alcohols in the second stage, and high-boiling-point alcohols in the third stage, the resulting alcohol hydrosol has a total volatile compound concentration of 800 to 1200 mg / L, which is 3 to 5 times that of conventional distillation.

[0021] Fifth, the product of this invention has a high degree of alcohol flavor reproduction. Sensory evaluation shows an alcohol flavor intensity of 7.8 points, close to the 8.5 points level of alcoholic cocktails, a fruit aroma intensity of 8.2 points, and an overall acceptability of 8.0 points. The product has an alcohol content of less than 0.05% by volume, meeting the zero-alcohol standard, and is suitable for consumers with limited alcohol tolerance. Furthermore, the product uses natural fermentation and extraction processes, without the use of artificial flavorings, aligning with the trend towards clean labeling.

[0022] Sixth, the product of this invention exhibits excellent stability, with a shelf life of up to 12 months at room temperature and a retention rate of over 85% for key compounds. By adding ascorbic acid, β-cyclodextrin, and other protective agents, and employing nitrogen sealing and low-temperature sterilization technologies, the oxidation and volatilization of volatile compounds are effectively inhibited. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0024] Example 1 This embodiment prepares a zero-alcohol passion fruit cocktail beverage with a slightly alcoholic flavor. The beverage contains ethyl hexanoate at 80 mg / L, phenylethanol at 180 mg / L, isoamyl alcohol at 180 mg / L, β-damascene at 2.5 mg / L, and an alcohol content of less than 0.05% by volume.

[0025] The preparation method includes the following steps: The first step is the preparation of the precursor mixture. Accurately weigh 52.48 g of leucine, 19.65 g of isoleucine, and 39.30 g of phenylalanine, dissolve them in purified water, and bring the volume to 65.5 liters. Add 1.31 g of free hexanoic acid and 0.52 g of free butyric acid to this solution, and stir thoroughly to dissolve them, obtaining the precursor mixture with the following concentrations: leucine 800 mg / L, isoleucine 300 mg / L, phenylalanine 600 mg / L, hexanoic acid 20 mg / L, and butyric acid 8 mg / L.

[0026] The second step is the processing of grape raw materials. 50 kg of fresh, ripe grapes are selected, and after ultrasonic cleaning to remove surface impurities, they are crushed. The crushed grape pulp is sterilized at 65 degrees Celsius for 15 minutes, cooled to room temperature, and then mixed evenly with the aforementioned precursor mixture to obtain the fermentation substrate.

[0027] The third step is the activation and inoculation of the mixed bacterial strain. Saccharomyces cerevisiae K1-V1116 and DV10 were activated and cultured separately in YPD medium for 24 hours, then mixed at a mass ratio of 80:20, centrifuged to collect the cells, and resuspended in physiological saline. After counting, the cells were cultured at 5 × 10⁻⁶ cells / day.6 The colony-forming units are inoculated into the fermentation substrate at a rate of one inoculation per milliliter.

[0028] The fourth step is two-stage fermentation. The inoculated fermentation substrate is placed in a fermenter. The first stage of fermentation is carried out at a temperature of 22°C and an initial pH of 3.9, with dissolved oxygen maintained at 2.5 mg / L by nitrogen purging, for 72 hours. At the 72-hour mark, the fermentation temperature is lowered to 18°C, the dissolved oxygen concentration is adjusted to 1.0 mg / L, and the pH is naturally maintained within the range of 3.9 to 4.0, continuing fermentation for 240 hours. During fermentation, residual sugar, ethanol concentration, and pH are monitored every 24 hours to ensure normal fermentation progress.

[0029] Step 5: Separation and treatment of the fermentation broth. After fermentation, the broth is cooled to 4 degrees Celsius and then centrifuged at 6000 rpm for 15 minutes to remove the yeast cells, resulting in a clear fermentation broth. The broth is then filtered through a 200-mesh filter for precision filtration.

[0030] Step 6: Low-temperature vacuum distillation. A three-stage precision distillation was performed using a rotary evaporator. The first stage was conducted at a pressure of 0.08 bar and a water bath temperature of 50°C, with a reflux ratio of 5:1 and a distillation rate controlled at 2 mL / min. Approximately 5.24 L of the fraction from 20 to 30°C was collected, yielding a light ester fraction rich in ethyl acetate and ethyl butyrate. The second stage maintained a pressure of 0.08 bar, with the water bath temperature increased to 70°C. The reflux ratio was adjusted to 3:1, and the distillation rate was 3 mL / min. Approximately 13.1 L of the fraction from 35 to 55°C was collected, yielding a medium-chain ester and higher alcohol fraction rich in ethyl hexanoate and isoamyl alcohol. The third stage reduced the pressure to 0.05 bar, increased the water bath temperature to 110°C, with a reflux ratio of 2:1 and a distillation rate of 2 mL / min. Approximately 3.28 L of the fraction from 80 to 110°C was collected, yielding a high-boiling-point alcohol fraction rich in phenylethyl alcohol. The distillation residue was used to enrich terpenoid compounds by C18 solid-phase extraction column, eluted with methanol-water at a ratio of 80:20 and concentrated 10 times to obtain a terpenoid-rich solution.

[0031] Step 7: Preparation of the alcohol hydrosol. For every 100 ml, mix 8 ml of light ester fraction, 20 ml of medium-chain ester and higher alcohol fraction, 5 ml of high-boiling-point alcohol fraction, and 2 ml of terpene enrichment. Add purified water to a final volume of 100 ml to obtain a standardized alcohol hydrosol. Add 200 mg / L of ascorbic acid as an antioxidant to the hydrosol, pasteurize at 65°C for 30 minutes, cool to room temperature, and seal for storage under nitrogen protection.

[0032] Step 8: Final Product Preparation. Prepare a 100-liter mixing container. First, add approximately 70 liters of purified water and heat to above 65 degrees Celsius. Add 24 kg of granulated sugar and 450 g of citric acid monohydrate, stirring thoroughly until completely dissolved. Then add 4.5 liters of passion fruit concentrate, 120 g of pectin, 8 g of sucralose, and 200 g of β-cyclodextrin, and continue stirring for 10 minutes. Once the temperature drops to 50 degrees Celsius, add 1.2 liters of the above-mentioned hydrosol, and bring the volume to 100 liters with purified water, mixing thoroughly.

[0033] Step nine: Precision filtration and sterilization. The prepared mixture is filtered through a 1-micron precision filter to remove tiny particles and microorganisms. The filtered liquid is then subjected to ultra-high temperature instantaneous sterilization at 135 degrees Celsius ± 2 degrees Celsius for 30 seconds, followed by rapid cooling to below 35 degrees Celsius.

[0034] Step 10: Filling and Packaging. The sterilized beverage is filled into cleaned and sterilized PET bottles, each with a capacity of 500 ml. The filling process is carried out in a clean environment, with the filling temperature controlled between 30 and 35 degrees Celsius. Immediately after filling, the bottles are capped, the production date is printed on them, and after labeling, they are boxed to obtain the finished product.

[0035] Example 2 This embodiment prepares a slightly alcoholic, zero-alcohol passion fruit cocktail beverage. The beverage contains 150 mg / L ethyl hexanoate, 250 mg / L phenylethanol, 280 mg / L isoamyl alcohol, 4.0 mg / L β-damascene, and has an alcohol content of less than 0.05% by volume.

[0036] In the first step of the preparation method, when preparing the precursor mixture, accurately weigh 78.72 g of leucine, 32.75 g of isoleucine, and 58.95 g of phenylalanine, dissolve them in purified water, and bring the volume to 65.5 liters. Add 2.29 g of free hexanoic acid and 0.98 g of free butyric acid to the solution to obtain the precursor mixture, wherein the concentration of leucine is 1200 mg / L, the concentration of isoleucine is 500 mg / L, the concentration of phenylalanine is 900 mg / L, the concentration of hexanoic acid is 35 mg / L, and the concentration of butyric acid is 15 mg / L. The second to fifth steps are the same as in Example 1, but in the first stage of fermentation in the fourth step, the dissolved oxygen concentration is controlled at 3.2 mg / L, and the fermentation temperature in the second stage is set at 20 degrees Celsius with a dissolved oxygen concentration of 1.5 mg / L. In the sixth step, low-temperature vacuum distillation, the first fractionation is carried out at a water bath temperature of 60 degrees Celsius, with a distillation rate of 3 ml / min, and approximately 7.86 liters of distillate are collected. In the second fractionation, the water bath temperature is raised to 85 degrees Celsius, with a distillation rate of 4 ml / min, and approximately 19.65 liters of distillate are collected. In the third fractionation, the water bath temperature is raised to 135 degrees Celsius, with a distillation rate of 3 ml / min, and approximately 5.24 liters of distillate are collected. In the seventh step, the preparation of the alcohol hydrosol involves mixing 12 ml of light esters, 30 ml of medium-chain esters and higher alcohols, 8 ml of high-boiling-point alcohols, and 3 ml of terpene enrichment per 100 ml, and then adding purified water to bring the volume to 100 ml. In step eight, during the final product preparation, add 28 kg of white sugar, 650 g of citric acid monohydrate, 6.0 L of passion fruit concentrate, 180 g of pectin, 12 g of sucralose, and 300 g of β-cyclodextrin to the mixing tank, followed by 1.8 L of alcoholic hydrosol. Steps nine and ten are the same as in Example 1.

[0037] Example 3 This embodiment prepares a slightly alcoholic, zero-alcohol passion fruit cocktail beverage, which contains 115 mg / L ethyl hexanoate, 215 mg / L phenylethanol, 230 mg / L isoamyl alcohol, and 3.2 mg / L β-damascene.

[0038] In the first step of the preparation method, when preparing the precursor mixture, accurately weigh 65.60 g of leucine, 26.20 g of isoleucine, and 49.13 g of phenylalanine, dissolve them in purified water, and bring the volume to 65.5 liters. Add 1.80 g of free hexanoic acid and 0.75 g of free butyric acid to this solution to obtain the precursor mixture, in which the concentrations are 1000 mg / L for leucine, 400 mg / L for isoleucine, 750 mg / L for phenylalanine, 27.5 mg / L for hexanoic acid, and 11.5 mg / L for butyric acid. In the fourth step of the two-stage fermentation, the first stage fermentation temperature is 22 degrees Celsius, and the dissolved oxygen concentration is controlled at 2.8 mg / L; the second stage fermentation temperature is lowered to 19 degrees Celsius, and the dissolved oxygen concentration is 1.2 mg / L. In step six, low-temperature vacuum distillation, the first fractionation water bath temperature was 55°C, and approximately 6.55 liters of distillate were collected; the second fractionation water bath temperature was 78°C, and approximately 16.38 liters of distillate were collected; the third fractionation water bath temperature was 123°C, and approximately 4.26 liters of distillate were collected. In step seven, during the preparation of the alcoholic hydrosol, per 100 ml, 10 ml of light ester fraction, 25 ml of medium-chain ester and higher alcohol fraction, 6.5 ml of high-boiling-point alcohol fraction, and 2.5 ml of terpene enrichment were mixed. In step eight, during the final product blending, 26 kg of white sugar, 550 g of citric acid monohydrate, 5.2 liters of passion fruit concentrate, 150 g of pectin, 10 g of sucralose, 250 g of β-cyclodextrin, and 1.5 liters of alcoholic hydrosol were added. The remaining steps were the same as in Example 1.

[0039] Example 4 This embodiment prepares a slightly alcoholic, zero-alcohol passion fruit cocktail beverage, which also contains 22 mg / L ethyl butyrate, 35 mg / L ethyl acetate, 55 mg / L 2-methyl-1-butanol, and 0.11 mg / L β-ionone.

[0040] The first seven steps of the preparation method are basically the same as in Example 3, but enzymatic enhancement was performed at the 72nd hour of the fourth fermentation process. Specifically, before cooling down at the 72nd hour, a portion of the fermentation broth was centrifuged, and carotenoid lysing dioxygenase was added to the supernatant at a rate of 50 units per liter of fermentation broth. The pH was adjusted to 5.8, the temperature was set to 37 degrees Celsius, and the reaction was carried out for 36 hours under nitrogen protection. After the enzymatic reaction was completed, the treated broth was remixed with the main fermentation broth, and then the second stage of fermentation continued. Furthermore, 65 ml of natural alcoholic flavoring was added during the final product blending in the eighth step to further enhance the aroma and complexity of the product.

[0041] Example 5 This embodiment prepares a zero-alcohol passion fruit cocktail beverage with a slightly alcoholic flavor. The beverage contains 9 mg / L hexanoate, 4.5 mg / L isoamyl acetate, 1.1 mg / L linalool, and 0.45 mg / L phenylacetaldehyde.

[0042] In the preparation method, during the first step of preparing the precursor mixture, the concentrations were: leucine 900 mg / L, isoleucine 350 mg / L, phenylalanine 700 mg / L, hexanoic acid 25 mg / L, and butyric acid 10 mg / L. The fourth step, fermentation, employed a temperature gradient control strategy. In the first stage, the temperature was maintained at 22°C for the first 36 hours, gradually decreasing to 20°C from 36 to 72 hours, with dissolved oxygen maintained at 2.6 mg / L. In the second stage, the temperature was stabilized at 19°C, with dissolved oxygen at 1.3 mg / L, and fermentation was extended to 260 hours to promote the formation of more ester compounds. The sixth step, distillation, employed more precise temperature control. The first fraction collection temperature was precisely controlled between 23 and 28°C, the second between 40 and 50°C, and the third between 90 and 105°C, improving the purity of the target compounds. In the seventh step of preparing the alcoholic hydrosol, 50 mg / L of tocopherol and 30 mg / L of EDTA-Na2 were added as compound antioxidants, which significantly improved the stability of the hydrosol. The remaining steps were the same as in Example 3.

[0043] Comparative Example 1 This comparative example prepared a zero-alcohol passion fruit cocktail beverage, but without adding a precursor mixture during fermentation, to verify the importance of precursors for the synthesis of target flavor compounds.

[0044] The first step of the preparation method is omitted. In the second step, the grape raw material is simply mixed with purified water without the addition of amino acid precursors. Steps three through ten are the same as in Example 1. Due to the lack of exogenous amino acid precursors, the yeast can only utilize endogenous amino acids in the grape pulp for Ehrlich metabolism during fermentation, resulting in a significant reduction in the yield of higher alcohols. Simultaneously, ester synthesis is also limited due to insufficient free fatty acid precursors.

[0045] Comparative Example 2 This comparative example prepared a zero-alcohol passion fruit cocktail beverage, but only used a single strain of brewer's yeast K1-V1116 during the fermentation process, without using a compound strain for synergistic fermentation, in order to verify the superiority of the compound strain system.

[0046] In the third step of the preparation method, only Saccharomyces cerevisiae K1-V1116 is inoculated, with an inoculation amount of 5 × 10⁻⁶. 6Colony formation units per milliliter. The remaining steps are the same as in Example 1. Although single-strain fermentation can produce high levels of isoamyl alcohol and ethyl butyrate, it is insufficient in the synthesis of phenylethanol and ethyl hexanoate, resulting in a thin flavor profile and an imbalance in the ratio of esters to alcohols.

[0047] Comparative Example 3 This comparative example prepared a zero-alcohol passion fruit cocktail beverage, but conventional atmospheric distillation was used instead of three-stage precision vacuum distillation during the fermentation broth treatment to verify the necessity of the fractional distillation technique.

[0048] The sixth step of the preparation method was changed to simple distillation under normal pressure, with the distillation temperature gradually increased from 70 degrees Celsius to 150 degrees Celsius. All fractions were collected, mixed, and used directly as a hydrosol. Due to the high-temperature distillation, some heat-sensitive aroma compounds decomposed or oxidized, especially phenylethanol and terpenes, which suffered significant losses. Furthermore, because fractional collection and precise separation were not performed, the concentration ratios of various compounds were difficult to control accurately. The remaining steps were the same as in Example 1.

[0049] Comparative Example 4 This comparative example prepared a zero-alcohol passion fruit cocktail beverage, but used a one-stage isothermal fermentation instead of a two-stage variable-temperature fermentation to verify the advantages of the two-stage fermentation strategy.

[0050] The fourth step of the preparation method, fermentation, was carried out for 240 hours at 22 degrees Celsius and dissolved oxygen of 2.5 mg / L without any temperature or dissolved oxygen gradient adjustments. Due to the lack of low-temperature promotion in the second stage, the esterification reaction efficiency was low, resulting in insufficient ester content in the final product. Simultaneously, although the higher alcohols were fully synthesized, their ratio with the esters was unbalanced. The remaining steps were the same as in Example 1.

[0051] To comprehensively evaluate the quality and superiority of the products of this invention, systematic testing and analysis were conducted on the above-mentioned embodiments and comparative examples. The testing items included three aspects: determination of key flavor compound content, sensory evaluation, and stability evaluation.

[0052] The content of key flavor compounds was quantitatively analyzed using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-MS). The specific method was as follows: 5 mL of beverage sample was placed in a 20 mL headspace vial, and 1.5 g of sodium chloride was added. A 50 / 30 μm DVB / CAR / PDMS three-phase fiber extraction head was activated at 250°C for 60 minutes and then inserted into the headspace portion of the sample vial. Extraction was performed at 40°C for 30 minutes. The extracted fiber head was then inserted into the gas chromatograph inlet and desorbed at 250°C for 5 minutes. The gas chromatographic conditions were: DB-WAX capillary column, helium as carrier gas, flow rate 1.2 mL / min, programmed temperature increase from 40°C for 3 minutes, then increased at 5°C / min to 120°C for 2 minutes, and finally increased at 8°C / min to 230°C for 10 minutes. Mass spectrometry conditions were as follows: electron impact ion source, ion source temperature 230°C, and scan range 35 to 350 mass-to-charge ratio. External standard method was used for quantification; the purity of the standards was greater than 98%, and the linear correlation coefficient of the standard curve was greater than 0.995.

[0053] Sensory evaluation was conducted by a panel of 15 trained professional tasters, using a 10-point scoring system to assess the product across four dimensions: intensity of flavor, fruitiness, body of taste, and overall palatability. The scoring criteria were: 1-3 points for poor, 4-6 points for average, 7-8 points for good, and 9-10 points for excellent. Each sample was scored independently by one taster, and the average score was used as the final score.

[0054] Stability evaluation employed accelerated stability testing, where samples were stored in a 40°C incubator for 3 months. Key flavor compound content was periodically measured, and retention rates were calculated. The retention rate was calculated as follows: retention rate = post-storage content divided by initial content multiplied by 100%.

[0055] The results of the determination of key flavor compounds in each example and comparative example are shown in Table 1. As can be seen from Table 1, the contents of key compounds such as ethyl hexanoate, phenethyl alcohol, isoamyl alcohol, and β-damascene in Examples 1 to 5 all meet the expected target range, and the proportions of each compound are harmonious. In particular, Examples 3 and 5 achieved an optimal balance of flavor compound concentrations due to optimized process parameters. In contrast, Comparative Example 1, lacking precursor supplementation, had a phenethyl alcohol content of only 95 mg / L and an isoamyl alcohol content of 108 mg / L, representing reductions of 47.2% and 40.0% respectively compared to Example 1. Comparative Example 2, using a single-strain fermentation, achieved an isoamyl alcohol content of 165 mg / L, but ethyl hexanoate was only 52 mg / L and phenethyl alcohol was 142 mg / L, resulting in an incomplete flavor compound profile. Comparative Example 3, due to the high-temperature degradation during atmospheric distillation, suffered significant losses of phenethyl alcohol and β-damascene, decreasing to 128 mg / L and 1.3 mg / L respectively. The content of ester compounds in Comparative Example 4 was significantly lower, with ethyl hexanoate at only 61 mg / L, indicating that single-stage fermentation is not conducive to the esterification reaction.

[0056] Table 1. Content of key flavor compounds in each example and comparative example. Sample number Ethyl hexanoate (mg / L) Phenylacetyl alcohol (mg / L) Isoamyl alcohol (mg / L) β-Damasteone (mg / L) Ethyl butyrate (mg / L) Ethyl acetate (mg / L) Example 1 82 183 182 2.6 16 26 Example 2 148 247 276 3.9 29 44 Example 3 113 212 228 3.2 22 35 Example 4 116 218 235 3.4 23 37 Example 5 119 221 238 3.3 24 36 Comparative Example 1 38 95 108 1.8 9 15 Comparative Example 2 52 142 165 2.3 28 31 Comparative Example 3 75 128 171 1.3 14 22 Comparative Example 4 61 176 192 2.5 11 25 The sensory evaluation results are shown in Table 2. The sensory scores of Examples 1 to 5 were generally excellent. Examples 3 and 5, due to their most reasonable flavor compound ratios, achieved high scores across all indicators. Example 3 achieved an alcoholic intensity of 8.1, a fruity aroma intensity of 8.5, a body of mouth of 7.8, and an overall acceptability of 8.3. Example 5, due to the addition of a compound antioxidant and extended fermentation time, exhibited a richer aroma profile and achieved an overall acceptability of 8.4. In contrast, the sensory scores of the comparative examples were significantly lower. Comparative Example 1 had an alcoholic intensity of only 4.2 and a fruity aroma intensity of 5.1; tasters generally commented that the product had a thin flavor and lacked alcoholic characteristics. Comparative Example 2, while possessing a certain alcoholic intensity, lacked sufficient fruity aroma and overall harmony. Comparative Example 3 suffered from insufficient overall flavor intensity due to aroma loss during distillation. Comparative Example 4 had a low ester content, resulting in insufficient fruity aroma and affecting its body of mouth.

[0057] Table 2 Sensory evaluation results of each embodiment and comparative example Sample number Intensity of alcohol Fruity aroma Rich and mellow taste Overall acceptance Example 1 7.5 7.8 7.2 7.6 Example 2 8.3 8.8 8.1 8.5 Example 3 8.1 8.5 7.8 8.3 Example 4 8.2 8.6 7.9 8.4 Example 5 8.3 8.7 8 8.4 Comparative Example 1 4.2 5.1 4.8 4.5 Comparative Example 2 6.8 5.9 6.5 6.2 Comparative Example 3 6.2 6.5 6.1 6.3 Comparative Example 4 7.1 6.3 6.8 6.7 The stability evaluation results are shown in Table 3. After storage at 40°C for 3 months, Examples 1 to 5 all showed a retention rate of over 80% for the key flavor compounds. Example 5, due to the addition of a compound antioxidant, achieved a phenethyl alcohol retention rate of 96% and a β-damascene retention rate of 83%, demonstrating excellent stability. The comparative examples generally showed poor stability, especially Comparative Example 3, which, due to insufficient antioxidant addition in the initial hydrosol, had a phenethyl alcohol retention rate of only 71% and a β-damascene retention rate of 62%.

[0058] Table 3. Stability evaluation results (3-month retention rate %) of each embodiment and comparative example Sample number Ethyl hexanoate Phenylacetyl alcohol Isoamyl alcohol β-Damasteone Example 1 85 92 94 75 Example 2 88 94 95 78 Example 3 87 95 95 79 Example 4 89 95 96 81 Example 5 91 96 96 83 Comparative Example 1 82 88 91 70 Comparative Example 2 84 90 92 72 Comparative Example 3 78 71 89 62 Comparative Example 4 83 91 93 74 The test results above demonstrate that this invention successfully reconstructs the slightly alcoholic flavor of zero-alcohol cocktails through the synergistic effects of key technologies such as precursor material targeted conversion, synergistic metabolic regulation by compound microbial strains, and three-stage precision vacuum distillation. The addition of precursor materials provides ample substrates for the biosynthesis of target compounds. Through the Ehrlich metabolic pathway and acyltransferase catalysis of yeast, amino acid precursors are efficiently converted into higher alcohols, and fatty acid precursors are converted into ester compounds. The compound microbial strain system fully leverages the advantages of K1-V1116 in higher alcohol synthesis and the strengths of DV10 in ester synthesis and phenylethanol accumulation; their complementary metabolism achieves a balanced production of flavor compounds. The two-stage fermentation strategy optimizes the temporal distribution of metabolic flux through gradient regulation of temperature and dissolved oxygen. The first stage rapidly synthesizes higher alcohols, providing ethanol substrates for subsequent esterification reactions, while the second stage, with its low temperature, promotes esterification and increases ester yield. Three-stage precision vacuum distillation technology achieves accurate separation based on the differences in boiling points of compounds, maximizing the preservation of heat-sensitive aroma components under low-temperature conditions. The resulting hydrosol has a high concentration of volatile compounds in a controllable proportion. The flavor compound composition of the final product conforms to the characteristics of a cocktail, exhibits excellent sensory evaluation, and demonstrates good stability, making it a novel zero-alcohol beverage with broad market prospects.

[0059] The preferred embodiment of the present invention is Example 3 or Example 5, and its key process parameters are as follows: the concentration of leucine in the precursor material is 900 to 1000 mg / L, the concentration of isoleucine is 350 to 400 mg / L, and the concentration of phenylalanine is 700 to 750 mg / L; the compound strains K1-V1116 and DV10 are mixed and inoculated at a mass ratio of 80:20; the first stage fermentation is carried out at 22 degrees Celsius and dissolved oxygen of 2.6 to 2.8 mg / L for 72 hours; the second stage fermentation is carried out at 19 degrees Celsius and dissolved oxygen of 1.2 to 1.3 mg / L for 168 to 188 hours; the collection temperatures of the three-stage vacuum distillation are 23 to 28 degrees Celsius, 40 to 50 degrees Celsius, and 90 to 105 degrees Celsius, respectively; and the volume ratio of light ester fraction to medium chain ester and higher alcohol fraction to high boiling point alcohol fraction in the alcohol hydrosol is 10:25:6.5. The product produced under these conditions has an ethyl hexanoate content of 113 to 119 mg / L, a phenylethanol content of 212 to 221 mg / L, an isoamyl alcohol content of 228 to 238 mg / L, a β-damascene content of 3.2 to 3.3 mg / L, a taste intensity of 8.1 to 8.3, a fruit aroma intensity of 8.5 to 8.7, and an overall acceptability of 8.3 to 8.4. All indicators have reached the optimal level.

[0060] The superior technical effects of this invention stem from the following synergistic mechanism: First, the precursor-targeted conversion mechanism is based on the Ehrlich metabolic pathway in yeast. Leucine is converted to α-ketoisocaproic acid by aminotransferases, then decarboxylated by branched-chain α-keto acid decarboxylases to generate isopentanal, and finally reduced to isopentanol by alcohol dehydrogenases. Phenylalanine is converted to phenylpyruvate by aromatic amino acid transaminases ARO8 / ARO9, then decarboxylated by pyruvate decarboxylase PDC to generate phenylacetaldehyde, which is then reduced to phenylethanol by alcohol dehydrogenase ADH. By adding a high concentration of exogenous amino acid precursors, the metabolic flux was significantly increased, raising the biosynthesis efficiency of higher alcohols to over 70%.

[0061] Secondly, the synergistic metabolic mechanism of the two strains is reflected in the metabolic complementarity of the two yeast strains, K1-V1116 and DV10. Strain K1-V1116 exhibits a strong branched-chain amino acid metabolism capacity, efficiently synthesizing isoamyl alcohol and 2-methylbutanol, while its high ATF enzyme activity promotes the synthesis of esters such as ethyl butyrate. Strain DV10, on the other hand, excels in the aromatic amino acid metabolic pathway, particularly its high expression of the ARO8 gene, which significantly improves the conversion efficiency of phenylalanine to phenylethanol. The synergistic fermentation of the two strains at an 80:20 ratio ensures both the total yield of higher alcohols and the sufficient accumulation of phenylethanol, a key aroma compound.

[0062] Furthermore, the two-stage fermentation process, with its temperature and dissolved oxygen gradient control, achieved temporal optimization of metabolic flux. In the first stage, at 22°C and with relatively high dissolved oxygen, the yeast was in its logarithmic growth phase, exhibiting high activity of amino acid transaminases and decarboxylases, which facilitated the rapid synthesis of higher alcohols. In the second stage, the temperature was lowered to 18-20°C, and dissolved oxygen levels decreased. While yeast growth slowed, the activity of alcohol acyltransferase (ATF) increased. The higher alcohols accumulated in the earlier stage and the organic acids produced during fermentation were efficiently synthesized into esters under ATF catalysis. Hexanoic acid combined with ethanol to form ethyl hexanoate, and butyric acid combined with ethanol to form ethyl butyrate, achieving a dynamic balance between esters and alcohols.

[0063] Furthermore, low-temperature vacuum distillation significantly lowers the boiling points of compounds by reducing system pressure, thus avoiding the damage to heat-sensitive aroma components caused by high temperatures. Terpenes such as β-ionone and linalool contain unsaturated double bonds, which are prone to oxidation and polymerization at high temperatures, but can be completely preserved under low-temperature vacuum conditions. The three-stage fractionation process achieves precise separation based on the differences in the boiling points of the compounds. The first stage enriches low-boiling-point ethyl acetate and ethyl butyrate, the second stage collects medium-boiling-point ethyl hexanoate and isoamyl alcohol, and the third stage specifically collects high-boiling-point phenylethanol. The purity and concentration of each fraction are significantly higher than those of conventional distillation, providing high-quality aroma bases for the final product.

[0064] In summary, this invention achieves the precise reconstruction of the subtle alcoholic flavor in zero-alcohol cocktails by organically combining multiple technical steps, including precursor substance targeted conversion, synergistic metabolism of compound microorganisms, two-stage fermentation regulation, and low-temperature vacuum precision distillation, providing an innovative technical solution for the non-alcoholic beverage industry.

[0065] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A non-alcoholic cocktail beverage with a slightly alcoholic flavor, characterized in that, The beverage contains the following characteristic flavor compounds: ethyl hexanoate 80 to 150 mg / L, phenylethanol 180 to 250 mg / L, isoamyl alcohol 180 to 280 mg / L, and β-damascene 2.5 to 4.0 mg / L, and the beverage has an alcohol content of less than 0.05% by volume.

2. The zero-alcohol cocktail beverage with a slightly alcoholic flavor according to claim 1, characterized in that, The beverage also contains the following flavor compounds: ethyl butyrate 15 to 30 mg / L, ethyl acetate 25 to 45 mg / L, 2-methyl-1-butanol 40 to 70 mg / L, and β-ionone 0.08 to 0.15 mg / L.

3. The zero-alcohol cocktail beverage with a slightly alcoholic flavor according to claim 1 or 2, characterized in that, The beverage contains 6 to 12 mg / L hexanoate, 3 to 6 mg / L isoamyl acetate, 0.8 to 1.5 mg / L linalool, and 0.3 to 0.6 mg / L phenylacetaldehyde.

4. The zero-alcohol cocktail beverage with a slightly alcoholic flavor according to any one of claims 1 to 3, characterized in that, The beverage contains, per 100 liters, 1.2 to 1.8 liters of hydrosol, 4.5 to 6.0 liters of passion fruit concentrate, 24 to 28 kg of white sugar, 450 to 650 g of citric acid monohydrate, and 120 to 180 g of pectin, wherein the concentration of total volatile compounds in the hydrosol is 800 to 1200 mg per liter.

5. A method for preparing a zero-alcohol cocktail beverage with a slightly alcoholic flavor as described in any one of claims 1 to 4, characterized in that, Includes the following steps: The first step involves crushing the grape raw material and adding a precursor mixture for fermentation. The precursor mixture contains 800 to 1200 mg / L of leucine, 300 to 500 mg / L of isoleucine, and 600 to 900 mg / L of phenylalanine. The second step involves inoculating the fermentation broth with a compound microbial strain for two-stage fermentation. The compound microbial strain is a mixture of Saccharomyces cerevisiae K1-V1116 and Saccharomyces cerevisiae DV10, with an inoculation ratio of 80:20 by mass. The first stage fermentation temperature is 22 degrees Celsius, the time is 0 to 72 hours, and the dissolved oxygen concentration is 2.5 to 3.2 mg / L. The second stage fermentation temperature is 18 to 20 degrees Celsius, the time is 72 to 240 hours, and the dissolved oxygen concentration is 1.0 to 1.5 mg / L. The third step is to centrifuge the fermentation liquid at 6000 rpm for 15 minutes at 4 degrees Celsius after fermentation. The fermentation liquid is then filtered and the fractions are collected by low-temperature vacuum distillation. The fourth step involves mixing the fractions in a specific ratio to obtain an alcoholic hydrosol, wherein the total volatile compound concentration in the alcoholic hydrosol is 800 to 1200 mg / L. The fifth step involves mixing the pure hydrosol with passion fruit concentrate, white sugar, citric acid, pectin, and purified water. After filtration, sterilization, and bottling, the zero-alcohol cocktail beverage is obtained.

6. The preparation method according to claim 5, characterized in that, The low-temperature vacuum distillation employs a three-stage fractionation process. In the first stage, the fraction at 20 to 30 degrees Celsius is collected under conditions of 0.08 bar pressure, 50 to 60 degrees Celsius water bath temperature, and a reflux ratio of 5:1, yielding light ester fractions. In the second stage, the fraction at 35 to 55 degrees Celsius is collected under conditions of 0.08 bar pressure, 70 to 85 degrees Celsius water bath temperature, and a reflux ratio of 3:1, yielding medium-chain esters and higher alcohol fractions. In the third stage, the fraction at 80 to 110 degrees Celsius is collected under conditions of 0.05 bar pressure, 110 to 135 degrees Celsius water bath temperature, and a reflux ratio of 2:1, yielding high-boiling-point alcohol fractions.

7. The preparation method according to claim 6, characterized in that, The preparation method of the alcohol hydrosol is as follows: per 100 ml, mix 8 to 12 ml of light ester fraction, 20 to 30 ml of medium-chain ester and higher alcohol fraction, and 5 to 8 ml of high-boiling-point alcohol fraction, then add 2 to 3 ml of terpene enrichment solution, and make up to 100 ml with purified water. The terpene enrichment solution is obtained by solid-phase extraction from the distillation residue and concentration.

8. The preparation method according to claim 7, characterized in that, The precursor mixture also contains 20 to 35 mg / L of free hexanoic acid and 8 to 15 mg / L of free butyric acid. The fermentation process is carried out at a pH of 3.9 to 4.1, with an initial inoculum size of 5 × 10⁻⁶. 6 Colony forming units per milliliter.

9. The preparation method according to claim 8, characterized in that, In the 72nd hour of the second fermentation step, the fermentation temperature was reduced from 22 degrees Celsius to 18 to 20 degrees Celsius. After centrifugation of the fermentation broth and before distillation, enzymatic treatment was performed. The enzymatic treatment used carotenoid lysin dioxygenase to react for 24 to 48 hours at a temperature of 35 to 40 degrees Celsius and a pH of 5.5 to 6.

0.

10. The preparation method according to claim 9, characterized in that, In the fifth step, the amount of the added alcohol hydrosol is 1.2 to 1.8 liters, the amount of the added passion fruit concentrate is 4.5 to 6.0 liters, the amount of added white sugar is 24 to 28 kilograms, the amount of added citric acid monohydrate is 450 to 650 grams, and the amount of added pectin is 120 to 180 grams per 100 liters. After the mixed liquid is dissolved at a temperature above 65 degrees Celsius for 10 minutes, it is filtered through a precision filter with a filtration accuracy of 1 micrometer. The filtered liquid is then sterilized at 135 degrees Celsius ± 2 degrees Celsius for 30 seconds.