Method for preparing high ester flavor liquor with yellow water

Abstract

The application discloses a method for preparing a high-ester flavor liquor of the strong-flavor type by using yellow water, and belongs to the technical field of liquor brewing. The method uses yellow water as a fermentation substrate, inoculates a functional bacterial group constructed into the yellow water, and ferments under an anaerobic environment and at 35-38 DEG C for 5 days to obtain a fermentation liquor with a content of caproic acid greater than 15 g / L. Then, a proper amount of strong-flavor liquor head or base liquor and 0.2‰ (w / v) lipase are added into the fermentation liquor, and esterification is carried out at 30 DEG C for 3 days. Finally, multi-stage vacuum rectification treatment is carried out to obtain the strong-flavor liquor with a content of ethyl caproate greater than 14 g / L. The application can effectively increase the content of ethyl caproate in the base liquor, regulate the phenomenon of the strong-flavor liquor that the ratio of ethyl caproate to caproic acid is inverted, enhance the typical style and flavor coordination of the liquor body, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention discloses a method for preparing a strong-aroma, high-ester flavoring liquor using yellow water, belonging to the field of liquor brewing technology. Background Technology

[0002] Strong-aroma baijiu, as a major aroma type of traditional Chinese baijiu, relies heavily on the content of ethyl hexanoate and a reasonable ester ratio for its typical style. However, the industry currently faces two major technical bottlenecks: firstly, the ethyl hexanoate content in the base liquor is too low, making it difficult to present a rich cellar aroma; secondly, the ethyl acetate to ethyl hexanoate ratio is unbalanced (i.e., an "inverted ethyl-to-hexanoate ratio"), resulting in a heavy astringent taste, a lack of prominent aroma, and insufficient typicality. Traditional processes often improve flavor by extending the fermentation cycle or adding exogenous esters, but these methods suffer from long cycles (60-90 days), high costs, and the potential to introduce off-flavors. On the other hand, the solid-state fermentation process of strong-aroma baijiu produces a large amount of yellow water byproducts (annual output reaching millions of tons). This yellow water is rich in organic acids, residual starch, and other nutrients, possessing high reuse potential. However, it is currently mostly treated as low-value feed or wastewater, failing to achieve effective resource utilization and exacerbating the environmental burden.

[0003] Flavoring spirits, as functional spirits prepared through special fermentation, distillation, or post-processing techniques, play a crucial role in enhancing the typicality, harmony, and stability of baijiu (Chinese white liquor) flavor. Their core value lies in precisely "modifying" and improving the quality of the spirit by enriching specific skeletal components or key flavor substances (such as the characteristics of extra-strong and extra-mild spirits), synergistically regulating the ester ratio (such as correcting the "inverted ethyl-hexyl ratio"), and compensating for deficiencies in the aroma or taste of the base spirit (such as rare flavors like aged / fermented aroma). In the current context of the baijiu industry's trend towards high-end and standardized development, the systematic construction and application of flavoring spirit technology has become a key path to improving the quality of defective spirits and promoting the high-quality development of strong-aroma baijiu.

[0004] Currently, in the field of strong-aroma flavored liquor preparation, CN116515574A discloses a method for preparing strong-aroma high-ester flavored liquor, CN110283681A discloses a strong-aroma Daqu liquor and its brewing process, and CN102321516A discloses a secondary utilization processing technology of organic matter in the solid-state fermentation of strong-aroma baijiu. The above schemes have made positive explorations in improving the quality of flavored liquor. However, the related technologies have the following problems: (1) The preparation process requires a long fermentation time, resulting in a long production cycle and low efficiency; (2) The utilization of by-products such as yellow water is insufficient, causing resource waste; (3) The ethyl acetate content is too high, while the ethyl hexanoate content is too low, resulting in insufficient aroma coordination; (4) It relies on the addition of a large amount of exogenous enzyme preparations, which increases the production cost; (5) The ecological structure of the microbial community used is unstable, leading to product quality fluctuations and poor stability in large-scale production; (6) Non-fractional distillation affects the hydrolysis of esters under high temperature conditions and the production of Maillard reaction, affecting the style of the liquor.

[0005] Therefore, a brewing process with high ethyl hexanoate content and low ethyl acetate content is needed to improve the quality of flavored wines. Summary of the Invention

[0006] To address the shortcomings of the existing technology, this invention provides a method for preparing a strong-aroma high-ester flavored liquor using yellow water, which can effectively solve the problems of wasted yellow water by-products of strong-aroma liquor, high production costs, and the phenomena of "inverted ethyl-to-hexyl ratio" and uncoordinated aroma.

[0007] To achieve the above technical objectives, the following technical solution is proposed.

[0008] A method for preparing a strong-aroma high-ester flavored liquor using yellow water includes: using yellow water as a substrate to produce high levels of hexanoic acid under the action of functional microorganisms; performing a first vacuum distillation on the fermentation broth to extract the hexanoic acid solution and remove off-flavors; then adding esterification enzyme for esterification; and finally performing vacuum distillation on the esterified liquor to obtain a strong-aroma high-ester flavored liquor with an ethyl hexanoate content greater than 14 g / L.

[0009] This invention provides a method for preparing a strong-aroma, high-ester flavoring liquor using yellow water, comprising the following steps:

[0010] (1) The functional bacterial group was inoculated into mCGM medium and cultured stepwise to the third-level seed liquid. The prepared third-level seed liquid was inoculated into a glucose system with added yellow water and fermented under anaerobic conditions at 35-37℃ for 4-5 days to obtain a fermentation liquid with a hexanoic acid concentration greater than 15g / L.

[0011] (2) The fermentation broth obtained in step (1) is subjected to a first distillation to obtain a concentrated hexanoic acid solution;

[0012] (3) Add the hexanoic acid concentrate obtained in step (2) to the base wine, and add at least 0.2‰ of enzyme activity of 10,000 U / g to lipase according to the mass fraction, and esterify at 30-35℃ for 2-3 days to obtain esterified liquid;

[0013] (4) The esterified liquid obtained in step (3) is subjected to a second distillation, and the distilled components are collected to obtain the strong-aroma high-ester flavored wine.

[0014] In one embodiment of the present invention, the method for preparing the functional microbial community is as follows: the pit mud is inoculated into mCGM medium for stepwise culture, the inoculation amount of seed liquid in each step is 10-12%, and the culture conditions are 35-37℃ for 3-5 days.

[0015] In one embodiment of the present invention, the method for enriching the functional bacterial community includes:

[0016] Weigh 8 g / L of pit mud into 80 mL of mCGM medium and culture for 4 days to obtain the first generation of enriched microorganisms. Then, inoculate the enriched microorganisms into another 80 mL mCGM medium at a 10% inoculation ratio and culture for 4 days to obtain the second generation of enriched microorganisms. Repeat this process for four generations.

[0017] The mCGM medium (g / L) consists of 8-10g yeast extract, 8-10g peptone, 5-6g sodium acetate, 8-10g glucose, 0.5-1g sodium dihydrogen phosphate, 1-2g dimethyl hydrogen phosphate, 0.02-0.05g magnesium sulfate heptahydrate, and 1-2g ammonium sulfate.

[0018] In one embodiment of the present invention, the fermentation conditions include:

[0019] The functional microbial consortium's third-level seed culture was inoculated at 10% onto LY medium and fermented anaerobically at 35-37℃ for 5 days. The fermentation broth was analyzed using liquid chromatography; further distillation was initiated when the hexanoic acid yield reached greater than 15 g / L.

[0020] The seed culture is prepared by activating the functional bacterial community in mCGM medium and culturing it for 3 days, which is the first-level seed culture. Then, it is inoculated into fresh mCGM at a 10% inoculation ratio and cultured for 36-48 hours, which is the second-level seed culture. Finally, it is inoculated into fresh mCGM medium at a 10% inoculation ratio and cultured for 36-48 hours, which is the third-level seed culture.

[0021] The LY medium (g / L) is composed of yellow water to water in a ratio of 1:3 (lactate content is 30%) and glucose 10.

[0022] The liquid chromatography conditions included: Carbomix H-NP 10 column (10 μm, 8% crosslinking, 7.8 × 300 mm), 2.5 mM concentrated sulfuric acid as the mobile phase, flow rate 0.6 mL / min, injection volume 10 μL, 55 °C, 45 min.

[0023] In one embodiment of the present invention, the esterification conditions are as follows: esterification enzyme is added at a ratio of 0.2‰, the temperature is 30-35℃, and 50% of the fermentation liquid is added with base wine or distillate heads for esterification for 3 days.

[0024] In one embodiment of the present invention, the conditions for the first distillation are: a vacuum of 50 mbar and a temperature of 80°C.

[0025] In one embodiment of the present invention, the second vacuum distillation is to perform a three-stage distillation on the first distillate and the esterified liquid after esterification.

[0026] In one embodiment of the present invention, the second distillation is a three-stage distillation:

[0027] First stage distillation: The esterified liquid is distilled, with the distillation vacuum set at 350-400 mbar and the top temperature controlled at 70-80℃. After distillation for 1.5-2 hours, the second stage distillation is carried out.

[0028] Second stage distillation: Set the distillation vacuum to 250-300 mbar and control the top temperature at 70-80℃ for 3-4 hours, then proceed with the third stage distillation;

[0029] The third stage of distillation: Set the distillation vacuum to 150-200 mbar and control the top temperature at 70-80℃ for distillation for 2-3 hours;

[0030] Collect the components from the second-stage and third-stage distillation.

[0031] In one embodiment of the present invention, the functional bacterial community is inoculated into mCGM medium and cultured stepwise to a three-stage seed culture. The inoculation amount of each seed culture is 10-12%, and the culture conditions are 35-37℃ and anaerobic culture for 3-5 days.

[0032] In one embodiment of the present invention, the mCGM culture medium has the following (g / L) composition: yeast extract 8-10, peptone 8-10, sodium acetate 5-6, glucose 8-10, sodium dihydrogen phosphate 0.5-1, dimethyl hydrogen phosphate 1-2, magnesium sulfate heptahydrate 0.02-0.05, ammonium sulfate 1-2, pH 6.7.

[0033] In one embodiment of the present invention, the glucose system with added yellow water is: yellow water 300-500g / L, glucose 8-10g / L;

[0034] In one embodiment of the present invention, the yellow water (g / l) contains 60-80 lactic acid, 5-7 acetic acid, 8-10 propionic acid, 4-6 butyric acid, 4-6 glucose, and has a pH of 3-4.

[0035] In one embodiment of the present invention, the lipase is: an immobilized lipase derived from Candida porapsilosis, an AY lipase derived from Candida rugosa, a MER lipase derived from Rhizomucor miehei, or an esterase HLP-0100.

[0036] In one embodiment of the present invention, the inoculation amount of the pit mud is 5-10 (m / v, g / 100ml) according to the mass-volume ratio.

[0037] In one embodiment of the present invention, the three-stage distillation is as follows:

[0038] First stage distillation: The esterified liquid is distilled, with the distillation vacuum set at 400 mbar and the top temperature controlled at 80℃. After distillation for 1.5-2 hours, the second stage distillation is carried out.

[0039] Second stage distillation: Set the distillation vacuum to 300 mbar and control the top temperature at 80℃ for 3-4 hours, then proceed with the third stage distillation.

[0040] Third stage distillation: Set the distillation vacuum to 200 mbar and control the top temperature at 80℃ for distillation for 2-3 hours.

[0041] The three-stage distillation conditions are as follows: 400 mbar at 80°C, 300 mbar at 80°C, and 200 mbar at 80°C. The first stage (400 mbar, 80°C): Under this lower vacuum, the components with the lowest boiling points will preferentially distill out. This mainly includes residual ethanol, water, and trace amounts of low-boiling-point substances such as acetaldehyde, ethyl formate, and ethyl acetate. Once these low-boiling-point impurities are largely distilled off, the concentration of the target product (ethyl hexanoate) in the distillate begins to rise significantly. This point in concentration change is the optimal time for conversion. The second stage (300 mbar, 80°C): Further increasing the vacuum lowers the boiling point of ethyl hexanoate, allowing it to distill efficiently at 80°C. This stage collects the core fraction rich in ethyl hexanoate. The third stage (200mbar, 80℃): After most of the ethyl hexanoate has been distilled out, the vacuum level is further increased to distill out substances with higher boiling points, such as a small amount of ethyl lactate, ethyl butyrate, and some high-boiling-point acids. The presence of a small amount of these substances can increase the complexity of the wine, but too much will produce off-flavors.

[0042] The present invention also provides a strong-aroma, high-ester flavored wine prepared by the above method.

[0043] The present invention also provides the application of the method in the preparation of high-fat flavored wines.

[0044] Beneficial effects

[0045] 1) In this invention, the raw materials, such as the by-product yellow water of strong-aroma baijiu, the heads of strong-aroma baijiu or the base liquor with an inverted ethyl-hexanoate ratio, high-efficiency lipase, and functional bacterial liquid from cellar mud, are readily available and inexpensive. The process involved is simple, has a short cycle, and can produce high-ester strong-aroma baijiu with ethyl hexanoate content greater than 14 g / L, making it suitable for standardized production.

[0046] 2) In this invention, the prepared strong-aroma high-ester liquor has a typical strong-aroma style, with prominent ester aroma and a pure body. It contains ethyl hexanoate at a concentration greater than 14 g / L, ethyl butyrate and ethyl lactate at concentrations less than 1 g / L, and low acid content.

[0047] 3) In this invention, the functional bacterial liquid of cellar mud, strong-aroma base liquor, lipase and other substances have stable effects, which ensures the stability, continuity and standardization of this process. Attached Figure Description

[0048] Figure 1 Process flow diagram.

[0049] Figure 2 Sensory evaluation of the wine. Detailed Implementation

[0050] The present invention will now be described in detail with reference to specific embodiments.

[0051] The lipase HLP-0100 involved in the following examples was purchased from Jiangsu Boli Biological Products Co., Ltd., and its enzyme activity was 10000 U / g.

[0052] The culture media involved in the following examples are as follows:

[0053] mCGM medium (g / L): yeast extract 8-10, peptone 8-10, sodium acetate 5-6, glucose 8-10, sodium dihydrogen phosphate 0.5-1, dimethyl hydrogen phosphate 1-2, magnesium sulfate heptahydrate 0.02-0.05, ammonium sulfate 1-2, pH 6.7, autoclaved at 115℃ for 30 min.

[0054] CY medium ( / L): 30% yellow water (v / v); 10g glucose; pH 6.5, autoclave at 115℃ for 30min.

[0055] The yellow liquid mentioned in the following examples refers to the yellowish-brown slurry seeping from the bottom of the fermentation pits during the brewing of strong-aroma baijiu. It mainly originates from the dissolution, hydrolysis, and accumulation of microbial metabolic products during grain fermentation within the pits. Its typical characteristics include being rich in organic acids (hexanoic acid, acetic acid, lactic acid, butyric acid, etc.), residual sugars, amino acids, humic substances, and various anaerobic microbial communities. Specific indicators of the yellow liquid are shown in Table 1.

[0056] Table 1: Yellow Water Parameters

[0057]

[0058] The pit mud involved in the following embodiments:

[0059] The pit mud used for inoculation: The selected pit mud is generally over 30 years old and comes from old pits that have been in continuous and normal use. The physical properties of high-quality pit mud are: grayish-black or jet black, with a delicate and soft feel, obvious viscosity and ester aroma. Its physicochemical indicators need to meet the following requirements: pH value maintained between 6.8 and 7.5; its organic acid content is determined by high performance liquid chromatography (HPLC) or gas chromatography (GC), and pit mud samples with high hexanoic acid content (usually greater than 5 g / kg dry mud) and relatively low lactic acid content (usually less than 20 g / kg dry mud) are comprehensively screened to ensure that functional bacteria such as hexanoic acid bacteria dominate the pit mud microbial community and serve as seed pit mud inoculation sources for subsequent experiments.

[0060] The detection methods involved in the following embodiments:

[0061] The hexanoic acid content in the enrichment solution was determined by high-performance liquid chromatography (HPLC), and the solution with the highest hexanoic acid content was selected for next-generation enrichment. The HPLC method was as follows: Carbomix H-NP 10 column (10 μm, 8% crosslinking, 7.8 × 300 mm), 2.5 mM concentrated sulfuric acid as the mobile phase, flow rate 0.6 mL / min, injection volume 10 μL, 55℃, 45 min.

[0062] Detection of high-ester wine flavor

[0063] GC-FID was used for detection. 10 mL of wine sample was pipetted into a volumetric flask and diluted to volume. 100 μL of a mixed internal standard (amyl acetate 438 mg / L; 4-octanol 41.10 mg / L; 2-ethylbutyric acid 460 mg / L) was added, and 1 μL was taken for analysis. The temperature program was set as follows: initial temperature 35℃, stable operation for 3 min, increasing to 100℃ at 3℃ / min, then increasing to 200℃ at 7℃ / min, and maintaining this temperature for 16 min. A split injection ratio of 30:1 was used, with the injection port and detector temperature set at 250℃. Nitrogen (>99.999%) was used as the carrier gas at a flow rate of 0.7 mL / min.

[0064] Flavor Evaluation

[0065] A panel of 12 highly trained baijiu sensory experts evaluated the aroma characteristics of the optimized samples. The panel members underwent a three-month training program (two hours of classes per week) focusing on aroma identification, intensity perception, and descriptive terminology.

[0066] Sensory analysis was performed using 15 mL samples in tulip-shaped glasses. A five-point scale (0, none; 5, strong) was used to evaluate 12 attributes (sweetness, fruitiness, esters, alcohol, acidity, toastiness, cellar aroma, acidity, sweetness, bitterness, body, and finish). Descriptive assessments were integrated into a radar chart ( Figure 2 In the quantitative score, the average score is taken among the group members.

[0067] Example 1: Enrichment of Functional Microbial Communities

[0068] The specific steps are as follows:

[0069] 1. Selection of pit mud

[0070] Select cellar mud for strong-aroma baijiu with a cellar age of more than 30 years, hexanoic acid greater than 5g / kg, and lactic acid less than 20g / kg.

[0071] 2. Preparation of culture medium:

[0072] mCGM medium (g / L): glucose 10; tryptone 5; yeast extract 5; ammonium sulfate 2; sodium dihydrogen phosphate 1; dipotassium hydrogen phosphate 1; magnesium sulfate heptahydrate 0.05; ferrous sulfate 0.02; manganese sulfate 0.01; cobalt chloride 0.01; calcium chloride 0.01; pH 6.7; autoclave at 115℃ for 30 min.

[0073] 3. Enrichment of functional microbiota

[0074] (1) The selected pit mud was inoculated into 80 mL of the mCGM medium obtained in step 2 at an inoculation ratio of 10% (m / v, g / 100 ml) and cultured for 4 days under anaerobic conditions at 37 °C to obtain the first generation of enriched microbial community.

[0075] (2) The first generation of enriched bacteria were inoculated into 80 mL of mCGM medium at an inoculation ratio of 10% (v / v) and cultured for 4 days under anaerobic conditions at 37°C to obtain the second generation of functional bacteria.

[0076] (3) The second generation of functional bacteria was inoculated into 80 mL of mCGM medium at an inoculation ratio of 10% (v / v) and cultured for 4 days under anaerobic conditions at 37°C to obtain the third generation of functional bacteria.

[0077] (4) The third generation functional bacteria were inoculated into 80 mL of mCGM medium at an inoculation ratio of 10% (v / v) and cultured for 4 days under anaerobic conditions at 37°C to obtain the fourth generation functional bacteria.

[0078] The fourth-generation functional microbiota was used as the enriched functional microbiota for subsequent experiments.

[0079] Example 2: Laboratory fermentation of functional bacterial culture

[0080] The specific steps are as follows:

[0081] 1) Seed activation:

[0082] Glycerol tubes containing the fourth-generation functional bacterial group prepared in Example 1, which were frozen at -80℃, were rapidly inoculated into 10 mL of mCGM medium and cultured strictly anaerobically at 37℃ for 3 days to obtain activated seed culture.

[0083] 2) Secondary seed solution:

[0084] The activated seed solution prepared in step 1) was inoculated into mCGM medium at an inoculation ratio of 10% (v / v) and cultured under anaerobic conditions at 37°C for 36 h to obtain secondary seed solution.

[0085] 3) Third-level seed liquid:

[0086] The secondary seed culture prepared in step 2) was inoculated into mCGM medium at an inoculation ratio of 10% (v / v) and cultured statically at 37°C for 36 hours under anaerobic conditions to obtain the tertiary seed culture.

[0087] 4) Fermentation:

[0088] Preparation of LY medium (g / L): Yellow water 400; glucose 10; The preparation method is as follows: Fresh yellow water taken from a traditional solid-state fermentation pit is coarsely filtered through double-layer gauze, allowed to stand / centrifuge for clarification, sterilized at 115℃ for 20 minutes to remove impurities, and then mixed with 10g / L glucose solution sterilized at 115℃ for 15 minutes at a ratio of 400:10 (g / L). The pH is adjusted to 4.0-4.5, and the mixture is dispensed and sterilized to obtain LY medium.

[0089] The tertiary seed culture prepared in step 3) was inoculated into LY medium at an inoculation ratio of 10% (v / v) and cultured under anaerobic conditions at 37°C for 5 days, with samples taken daily for testing.

[0090] Samples were taken at 0, 1, 2, 3, 4 and 5 days, and the content of organic acids (lactic acid, acetic acid, butyric acid and hexanoic acid) was determined by HPLC.

[0091] The results are shown in Table 2.

[0092] Table 2: Metabolic kinetics under laboratory conditions

[0093] 0 26.84±1.75 4.18±0.05 0.00±0.00 0.00±0.00 1 19.97±0.09 3.89±0.09 0.09±0.02 3.05±0.24 2 5.38±0.08 4.22±0.12 0.65±0.09 6.22±0.10 3 0.83±0.03 4.50±0.24 1.59±0.23 7.90±0.98 4 0.72±0.12 4.94±1.39 2.07±0.01 9.48±0.25 5 0.58±0.07 4.09±0.67 2.16±0.01 10.32±0.07

[0094] The results showed that, through this fermentation, after 5 days of fermentation, the fourth-generation functional bacterial group prepared in Example 1 could effectively utilize 26.84±1.75 g / L lactic acid in the yellow water substrate, and produce 2.16±0.01 g / L butyric acid and 10.32±0.07 g / L hexanoic acid.

[0095] Example 3: Fermentation of functional bacterial culture under pilot-scale conditions

[0096] The specific steps are as follows:

[0097] 1) Seed activation:

[0098] Glycerol tubes containing the fourth-generation functional bacterial group prepared in Example 1, which were frozen at -80℃, were rapidly inoculated into 10 mL of mCGM medium and cultured strictly anaerobically at 37℃ for 3 days to obtain activated seed culture.

[0099] 2) Secondary seed solution:

[0100] The activated seed solution prepared in step 1) was inoculated into mCGM medium at an inoculation ratio of 10% (v / v) and cultured under anaerobic conditions at 37°C for 36 h to obtain secondary seed solution.

[0101] 3) Third-level seed liquid:

[0102] The secondary seed culture prepared in step 2) was inoculated into mCGM medium at an inoculation ratio of 10% (v / v) and cultured under anaerobic conditions at 37°C for 36 h to obtain the tertiary seed culture.

[0103] 4) Fermentation:

[0104] Preparation of LY medium (g / L): 400 g of yellow water; 10 g of glucose.

[0105] The method is as follows: Fresh yellow water taken from a traditional solid-state fermentation pit is coarsely filtered through double-layer gauze, allowed to stand / centrifuge for clarification, and sterilized at 115℃ for 20 minutes to remove impurities. Then, it is mixed with a 10g / L glucose solution sterilized at 115℃ for 15 minutes at a ratio of 400:10 (g / L), the pH is adjusted to 4.0-4.5, and the mixture is dispensed and sterilized to obtain LY culture medium.

[0106] The tertiary seed culture prepared in step 3) was inoculated into LY medium at an inoculation ratio of 10% (v / v) and cultured for 5 days under anaerobic conditions of 37°C, 100 r / min, and 4L fermenter. Samples were taken and tested daily.

[0107] Samples were taken at 0, 1, 2, 3, 4 and 5 days, and the content of organic acids (lactic acid, acetic acid, butyric acid and hexanoic acid) was determined by HPLC.

[0108] The results are shown in Table 3.

[0109] Table 3: Metabolic kinetics under pilot-scale conditions

[0110] 0 25.72±0.06 6.40±0.02 1.31±0.00 0.012±0.000 1 22.96±0.23 6.10±0.05 1.40±0.02 5.32±0.04 2 12.09±0.90 5.77±0.16 2.05±0.02 7.64±0.57 3 2.00±0.03 4.44±0.07 3.04±0.14 12.05±0.27 4 1.11±0.01 5.02±0.10 3.61±0.14 13.52±0.45 5 0.40±0.01 4.47±0.09 4.08±0.05 14.80±0.78

[0111] The results showed that, under pilot-scale conditions, through this fermentation for 5 days, the functional microbial community of the present invention could utilize 25.72±0.06 g / L lactic acid in the yellow water substrate to produce 4.08±0.05 g / L butyric acid and 14.80±0.78 g / L hexanoic acid.

[0112] The functional bacterial culture prepared after 5 days of cultivation was used for subsequent experiments.

[0113] Example 4: A method for preparing a strong-aroma, high-ester flavoring wine

[0114] 1. Preparation of low-ester base spirit for strong-aroma baijiu

[0115] The base liquor used in the preparation method comes from high-quality strong-aroma daqu liquor brewed using the traditional solid-state fermentation method at a factory in Sichuan. Specifically, the first part of the distillate, with an alcohol content of over 75% vol and rich in specific flavor compounds, is selected.

[0116] The specific steps are as follows:

[0117] (1) Raw material processing: Sorghum is used as raw material. It is crushed into 4-6 pieces (60%-70% pass through a 20-mesh sieve) and mixed with mother mash (the fermented mash on the upper side, with a grain-to-mash ratio of 1:4-5). It is then fermented in a mud pit according to the traditional continuous mash feeding process.

[0118] (2) Fermentation management: The original pit layering process is adopted, the pit is a mud pit, and the fermentation cycle is 60-90 days.

[0119] (3) Distillation: After the fermented mash is removed from the cellar, it is distilled slowly according to the principle of layered distillation and quality-based distillation, and the head portion with an alcohol content of ≥75% vol is collected (the distillate at this stage is rich in aldehydes, esters and high-boiling-point flavor precursors, but the total ester content is relatively low, which provides a basis for subsequent esterification and enrichment).

[0120] The content of flavor compounds in the base liquor was tested and found to be as follows:

[0121] Ethyl hexanoate (main ester): 180 mg / L;

[0122] Ethyl lactate: 120 mg / L;

[0123] Ethyl butyrate: 30 mg / L;

[0124] Ethyl acetate: 150 mg / L;

[0125] Total acid (calculated as acetic acid): 65 mg / L.

[0126] 2. Preparation of strong-aroma, high-ester flavoring wine

[0127] 1) Preparation of fermentation broth

[0128] The fermentation broth was prepared after 5 days of cultivation according to the method in Example 3.

[0129] 2) First vacuum distillation

[0130] Hexanoic acid was separated from the fermentation broth by distillation in a distillation column with a vacuum of 50 mbar and a temperature of 80°C for 2-3 hours to obtain hexanoic acid solution.

[0131] 3) Esterification

[0132] Add the low-ester base liquor of strong-aroma baijiu prepared in step 1 to the hexanoic acid solution obtained in step 2) at a ratio of 60% (v / v) to adjust the alcohol content to 40% vol. Then, add esterification enzyme (HLP-0100, enzyme activity: 10,000 U / g, Jiangsu Boli Biological Products Co., Ltd.) at a dosage of 0.2‰, and catalyze the esterification reaction at 30℃ for 2 days to prepare the esterified solution.

[0133] 4) Second vacuum distillation

[0134] The esterified liquid obtained in step 3) was distilled using a distillation column. The distillation vacuum was set to 400 mbar and the top temperature was controlled at 80℃ (to remove low-boiling-point impurities). After distillation for 1.5-2 hours, the distilled components were discarded. The distillation vacuum was set to 300 mbar and the top temperature was controlled at 80℃ (to collect the main aroma fraction). After distillation for 3-4 hours, the components were collected. The distillation vacuum was set to 200 mbar and the top temperature was controlled at 80℃ (to collect high-boiling-point flavor substances). After distillation for 2-3 hours, the components were collected.

[0135] After the collected components are mixed, a strong-aroma, high-ester flavoring liquor base is obtained.

[0136] 3. The obtained strong-aroma high-ester flavoring liquor base was tested.

[0137] 1) Food safety testing

[0138] The high-ester flavoring base liquor prepared in step 2 was subjected to the following food safety index tests to ensure that the product complies with the provisions of GB 2757-2012 "National Food Safety Standard for Distilled Spirits and Their Blended Spirits":

[0139] The results showed that the prepared strong-aroma high-ester flavoring liquor base contained:

[0140] Methanol: ≤0.6g / L (detected by gas chromatography, results are far below the national standard limit); Cyanide: Not detected; Heavy metals such as lead and manganese: Not detected (meets national standard limits); Plasticizers (phthalates): Not detected (stainless steel equipment was used throughout the process to avoid contact with plastics, in compliance with GB 5009.271-2016 requirements);

[0141] Conclusion: All safety indicators of this product are superior to national standards, making it safe and reliable.

[0142] 2) Quantitative detection and analysis of flavor components

[0143] Qualitative and quantitative analyses of the finished product were performed using gas chromatography-mass spectrometry (GC-MS) and gas chromatography-FID (GC-FID). The results are shown in Table 4.

[0144] Table 4: Content of major flavor compounds and their sensory contribution

[0145]

[0146] 3) Sensory evaluation results of the final product

[0147] Appearance: Colorless and transparent, clear and transparent, free of suspended matter and sediment.

[0148] Aroma: The aroma is characterized by prominent cellar and ester notes, rich and pure. The complex fruity aroma of ethyl hexanoate is dominant, accompanied by a pleasant cellar and grain aroma, creating a harmonious fragrance without any off-odors.

[0149] Taste: The initial taste is mellow, smooth, sweet, and refreshing, with a full and harmonious body and a long finish, exhibiting typical characteristics of high-quality strong-aroma baijiu. It has low astringency and a smooth mouthfeel.

[0150] Style: The strong aroma is a typical characteristic.

[0151] Example 5: Effect of different esterifying enzyme types on esterification efficiency

[0152] The specific implementation method is the same as in Example 4, except that the esterifying enzyme in step 3) is adjusted as follows:

[0153] CPL: Immobilized lipase (derived from Candida porapsilosis), a commonly used nonspecific lipase, purchased from Novozymes, catalog number: 435, enzyme activity: ≥10,000 U / g.

[0154] Lipase AY: Lipase AY (derived from Candida rugosa), has good activity for medium and long chain fatty acids (such as caproic acid), purchased from Amano Enzyme, catalog number: Amano 30SD, enzyme activity: ≥30,000U / g.

[0155] Lipase MER: Lipase MER (derived from Rhizomucor miehei), commonly used in the synthesis of food flavor esters, purchased from DuPont, catalog number: MER, enzyme activity: ≥15,000 U / g.

[0156] Meanwhile, the esterase used in Example 4 was used as a positive control (HLP-0100, Jiangsu Boli).

[0157] Following the method in Example 4, esterification reactions were carried out under the same enzyme dosage conditions. After the reaction was completed, the esterified liquids of each group were subjected to a second vacuum distillation, the finished products were collected and analyzed by GC-MS to determine the content of key ester substances.

[0158] The results are shown in Table 5 below.

[0159] Table 5: Effect of esterification enzyme type on esterification efficiency

[0160]

[0161] The results show:

[0162] (1) Esterification efficiency:

[0163] All enzyme preparations catalyzed the esterification reaction, but their efficiencies varied significantly. The HLP-0100 group showed significantly higher yields of ethyl hexanoate (13.08 g / L) and total ester content (15.13 g / L) than the other comparative groups, demonstrating that it has the highest catalytic activity and substrate specificity for this reaction system.

[0164] (2) Product specificity:

[0165] The nonspecificity of CPL enzymes leads to the formation of a high concentration of ethyl acetate (0.35 g / L), which introduces unwanted solvent flavor and affects flavor purity. HLP-0100, while efficiently catalyzing hexanoate esterification, strongly inhibits the formation of ethyl acetate, ensuring the purity of the target flavor.

[0166] (3) Flavor harmony:

[0167] While Lipase MER and Lipase AY have acceptable total ester content, their proportions of ethyl lactate and ethyl butyrate are higher than the control group. HLP-0100, however, achieves ultra-high ethyl hexanoate content while maintaining a stable ethyl lactate to ethyl butyrate ratio of approximately 1:1, a crucial factor in constructing a harmonious and mellow strong-aroma baijiu flavor.

[0168] Comparative Example 1: Effect of different hexanoic acid concentrations on esterification efficiency

[0169] The specific implementation method is the same as in Example 4, except that the fermentation broth preparation and distillation steps are omitted. Instead, a simulated solution is prepared directly using purchased hexanoic acid and edible alcohol to eliminate the interference of complex factors such as fermentation by-products and to examine the effect of hexanoic acid concentration alone.

[0170] The specific steps are as follows:

[0171] (1) Ethanol solution was added to solutions with hexanoic acid content of 5 g / L, 8 g / L, 10 g / L, 12 g / L, 15 g / L, 18 g / L, 20 g / L, 23 g / L, 25 g / L and 30 g / L respectively, so that the final ethanol concentration was 20% vol; hexanoic acid / ethanol aqueous solutions with different hexanoic acid concentrations were prepared respectively.

[0172] (2) Add the same biological esterifying enzyme (HLP-0100) to the solutions prepared in step (1) at a rate of 0.2‰, and incubate them in a constant temperature incubator at 30℃ for 4 days. After esterification, heat the esterified solution for distillation (atmospheric pressure distillation apparatus, 120-125℃), collect the distillate, and determine the content of ethyl hexanoate in the distillate by gas chromatography (GC).

[0173] The results are shown in Table 6 below.

[0174] Table 6: Effect of hexanoic acid concentration on esterification efficiency

[0175] 5 3.82 18 10.48 8 5.98 20 10.25 10 7.45 23 9.87 12 8.91 25 9.53 15 10.52 30 8.76

[0176] The results showed that the initial concentration of hexanoic acid had a significant impact on the efficiency of enzymatic esterification to synthesize ethyl hexanoate. When the concentration was too low, the reaction rate was substrate-limited; when the concentration was too high, a substrate inhibition effect was triggered.

[0177] This experiment determined that, under the given enzyme preparation and reaction conditions (20% vol ethanol, esterification reaction conditions: 30℃, 4d), the optimal initial hexanoic acid concentration was 15 g / L.

[0178] Comparative Example 2: Effect of different alcohol concentrations on esterification efficiency

[0179] The specific implementation method is the same as in Example 4, except that: instead of fermentation broth preparation and distillation steps, a simulated solution is prepared directly using purchased hexanoic acid and edible alcohol to investigate the effect of ethanol concentration on the reaction.

[0180] The specific steps are as follows:

[0181] (1) Take an appropriate amount of hexanoic acid concentrate, add anhydrous ethanol and distilled water to prepare an appropriate amount of hexanoic acid / ethanol aqueous solution with a fixed hexanoic acid content of 10 g / L and ethanol volume concentration (%vol) of 5%, 10%, 15%, 18%, 20%, 22%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and 60%.

[0182] (2) Add the same biological esterifying enzyme (HLP-0100) to the solutions prepared in step (1) at a rate of 0.2‰, and incubate them in a constant temperature incubator at 30℃ for 4 days. After esterification, heat the esterified solution for distillation (atmospheric pressure distillation apparatus, 120-125℃), collect the distillate, and determine the content of ethyl hexanoate in the distillate by gas chromatography (GC).

[0183] The results are shown in Table 7 below.

[0184] Table 7: Effect of different ethanol concentrations on the amount of ethyl hexanoate produced

[0185] 5 1.25 30 8.94 10 2.78 35 9.48 15 4.62 40 9.82 18 5.91 45 9.45 20 6.85 50 8.13 22 7.54 55 6.02 25 8.26 60 3.87

[0186] The results showed that ethanol concentration is a key factor affecting the efficiency of enzymatic esterification synthesis of ethyl hexanoate. Too low a concentration shifts the reaction equilibrium to the left, resulting in low conversion; too high a concentration leads to enzyme denaturation and inactivation, severely inhibiting the reaction. This experiment determined that, under the given enzyme preparation and reaction conditions (10 g / L hexanoate, esterification reaction conditions: 30℃, 4 days), the optimal ethanol concentration was 40% vol.

[0187] Comparative Example 3: Effect of different enzyme dosages on esterification efficiency

[0188] The specific implementation method is the same as in Example 4, except that the fermentation broth preparation and distillation steps are omitted. Instead, a simulated solution is prepared directly using purchased hexanoic acid and edible alcohol to investigate the effect of the enzyme addition amount on the reaction.

[0189] The specific steps are as follows:

[0190] (1) Prepare an appropriate amount of hexanoic acid / ethanol aqueous solution with a hexanoic acid content of 10 g / L and an ethanol content of 20% vol.

[0191] (2) The same biological esterifying enzyme (HLP-0100) was added to each group of solutions at concentrations of 0.05‰, 0.1‰, 0.15‰, 0.2‰, 0.25‰, 0.3‰, 0.35‰, and 0.4‰, respectively, and the solutions were placed in a constant temperature incubator at 30℃ for 4 days. After esterification, the esterified solution was heated and distilled (using an atmospheric pressure distillation apparatus at 120-125℃), and the distillate was collected. The content of ethyl hexanoate in the distillate was determined by gas chromatography (GC).

[0192] The results are shown in Table 8 below.

[0193] Table 8: Effect of different enzyme addition amounts on the production of ethyl hexanoate

[0194] 0.05 3.21 0.25 9.62 0.10 5.87 0.30 9.58 0.15 7.92 0.35 9.60 0.20 9.56 0.40 9.55

[0195] The results showed that the amount of esterifying enzyme added significantly affected the synthesis efficiency of ethyl hexanoate. When the enzyme amount was insufficient, the reaction rate was limited by the catalyst concentration; when the enzyme amount was sufficient, the reaction entered a plateau phase, and the yield no longer increased. This experiment determined that under the reaction conditions (hexanoic acid 10 g / L, ethanol 20% vol, esterification reaction conditions: 30℃, 4d), the optimal enzyme addition amount was 0.20‰.

[0196] Comparative Example 4: Effect of different esterification temperatures on esterification effect

[0197] The specific implementation method is the same as in Example 4, except that: instead of fermentation broth preparation and distillation steps, a simulated solution is prepared directly using purchased hexanoic acid and edible alcohol to investigate the effect of reaction temperature variable on enzyme activity.

[0198] The specific steps are as follows:

[0199] (1) Prepare an appropriate amount of hexanoic acid / ethanol aqueous solution with a hexanoic acid content of 10 g / L (i.e., 1000 mg / 100 mL) and an ethanol content of 20% vol.

[0200] (2) The same biological esterifying enzyme (HLP-0100) was added to each group of solutions at a dosage of 0.2‰, and then placed in constant temperature incubators at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃ for 4 days. After esterification, the esterified solution was heated and distilled (atmospheric pressure distillation apparatus, 120-125℃), and the distillate was collected. The content of ethyl hexanoate in the distillate was determined by gas chromatography (GC).

[0201] The results are shown in the table below.

[0202] Table 9: Effect of different reaction temperatures on the yield of ethyl hexanoate

[0203]

[0204]

[0205] The results showed that reaction temperature is a crucial factor affecting enzymatic esterification. At excessively low temperatures, enzyme activity is not fully activated, resulting in a slow reaction; at excessively high temperatures, the enzyme rapidly denatures and becomes inactive. This experiment determined that under the given enzyme preparation and reaction conditions (10 g / L hexanoic acid, 20% vol ethanol, 0.2‰ enzyme concentration, and esterification reaction conditions of 4 days), the optimal reaction temperature was 30℃. This temperature represents the optimum temperature for this specific esterifying enzyme, maximizing its catalytic efficiency.

[0206] Comparative Example 5: Effect of different esterification times on esterification effect

[0207] The specific implementation method is the same as in Example 4, except that: instead of fermentation broth preparation and distillation steps, a simulated solution is prepared directly using purchased hexanoic acid and edible alcohol to examine the effect of the reaction time variable on the reaction process.

[0208] The specific steps are as follows:

[0209] (1) Prepare an appropriate amount of hexanoic acid / ethanol aqueous solution with a hexanoic acid content of 10 g / L (i.e., 1000 mg / 100 mL) and an ethanol content of 20% vol.

[0210] (2) The same biological esterifying enzyme (HLP-0100) was added to each group of solutions at a dosage of 0.2‰, and the solutions were placed in a constant temperature incubator at 30℃ for 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 days, respectively. At each time point, the esterified solution of the corresponding group was taken and subjected to heating distillation (atmospheric pressure distillation apparatus, 120-125℃). The distillate was collected, and the content of ethyl hexanoate in the distillate was determined by gas chromatography (GC). The results are shown in the table below.

[0211] Table 10: Effect of different reaction times on the amount of ethyl hexanoate produced

[0212] 1.0 3.28 3.5 9.50 1.5 5.12 4.0 9.52 2.0 7.05 4.5 9.51 2.5 8.41 5.0 9.49 3.0 9.48 - -

[0213] The results showed that reaction time significantly affected the progress of the enzymatic esterification reaction. Too short a time resulted in incomplete reaction and low yield; too long a time meant the reaction had reached equilibrium, making it impossible to further increase the yield and instead increasing production cycle time and energy costs. This experiment determined that under the given reaction conditions (10 g / L hexanoic acid, 20% vol ethanol, esterification reaction conditions: enzyme 0.2‰, 30℃), the optimal reaction time was 3 days.

[0214] Example 6: The effect of pressure and temperature changes during the first vacuum distillation on the esterification effect

[0215] The specific implementation method is the same as in Example 4, except that the conditions for the first vacuum distillation are adjusted, that is: different combinations of pressure and temperature (100mbar, 80℃; 50mbar, 70℃; 50mbar, 90℃; 100mbar, 60℃) are used for treatment, and the hexanoic acid concentrate under each condition is collected.

[0216] Subsequently, all other variables were kept constant (using the same batch and volume of hexanoic acid concentrate, adding 60% v / v of the same low-ester base spirit, adjusting the alcohol content to 40% vol, adding the same enzyme at a rate of 0.2‰, and esterifying at 30°C for 3 days). After esterification, the esterified liquid was subjected to a second vacuum distillation (operated under the optimal conditions of Example 4), and the final product was collected and the ethyl hexanoate content was determined by gas chromatography (GC).

[0217] The results are shown in the table below.

[0218] Table 11: Effect of First Distillation Conditions on Final Ethyl Hexanoate Yield

[0219] control group 50 mbar, 80℃ Pale yellow transparent liquid with a distinct ester aroma 14.06 Experimental group 1 100mbar, 80℃ Colorless and transparent liquid 13.15 Experimental group 2 50 mbar, 70℃ pale yellow transparent liquid 12.78 Experimental group 3 50 mbar, 90℃ Dark yellow liquid with a slight burnt smell 9.85 Experimental group 4 100mbar, 60℃ A pale yellow, semi-transparent liquid with flocculent matter. 8.41

[0220] The results showed that, through comparative analysis of the appearance of hexanoic acid solution and the content of ethyl hexanoate in the final product obtained under different vacuum distillation conditions, the hexanoic acid solution obtained by the 50mbar, 80℃ process (control group) was a pale yellow transparent liquid with a distinct ester aroma, and corresponded to the highest yield of ethyl hexanoate (14.06g / L).

[0221] In contrast, the products from other experimental groups all exhibited different defects:

[0222] Although a colorless and transparent liquid was obtained under conditions of 100 mbar and 80 °C, the product content decreased to 13.15 g / L;

[0223] At 50 mbar and 70 °C, the liquid remained pale yellow and transparent, but the product yield further decreased to 12.78 g / L;

[0224] At 50 mbar and 90°C, the liquid was dark yellow with a burnt odor, and the product content was significantly reduced to 9.85 g / L.

[0225] The worst performing group was at 100 mbar and 60°C, where the hexanoic acid solution was light yellow and semi-transparent with flocculent matter, and the final yield of ethyl hexanoate was the lowest, at only 8.41 g / L.

[0226] Example 7: The effect of pressure and temperature changes during the second vacuum distillation on the esterification effect

[0227] The specific implementation method is the same as in Example 4 (control group), except that the conditions for the second vacuum distillation are adjusted. Specifically, during the second vacuum distillation, different pressure and temperature combinations as shown in the table below are used for segmented distillation. The results are shown in Table 12.

[0228] Table 12: The impact of the second vacuum distillation conditions on the overall product quality

[0229]

[0230] The results showed that, under different distillation conditions, the content of the main esters and the yield of ethyl hexanoate in each experimental group were significantly different from those in the control group (100%).

[0231] The yield of ethyl hexanoate in experimental group 1 (70℃) was 85.31%;

[0232] The yield of ethyl hexanoate in experimental group 2 (90℃) was 98.27%;

[0233] The yield of ethyl hexanoate in experimental group 3 (60℃) was significantly reduced to 66.31%;

[0234] The yield of experimental group 4 (600→400→200mbar) was slightly higher than that of the control group, with an ethyl hexanoate yield of 102.59%.

[0235] The yield of ethyl hexanoate in experimental group 5 (200→150→100mbar) was 89.99%.

[0236] The yield of ethyl hexanoate in experimental group 6 (500→400→300mbar) was 99.35%.

[0237] Under constant pressure conditions, the yields of ethyl hexanoate in experimental groups 7 (400 mbar), 8 (300 mbar), and 9 (200 mbar) were 96.47%, 94.67%, and 87.11%, respectively.

[0238] The content of esters in each group also varied accordingly. For example, the content of ethyl acetate, ethyl lactate, and ethyl butyrate in experimental group 4 was higher than that in the control group, while the content of these esters in experimental group 3 was significantly lower.

[0239] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a strong-aroma, high-ester flavoring liquor using yellow water, characterized in that, Includes the following steps: (1) The pit mud microbial agent is inoculated into mCGM medium and cultured stepwise to the third-level seed liquid. The prepared third-level seed liquid is inoculated into a glucose system with added yellow water and fermented under anaerobic conditions at 35-37℃ for 4-5 days to obtain a fermentation liquid with a hexanoic acid concentration greater than 15 g / L. The pit mud microbial agent is prepared by inoculating pit mud into mCGM medium and culturing stepwise to the fourth-level microbial agent. The inoculation amount of each level of seed liquid is 10-12%, and the culture conditions are 35-37℃ for 3-5 days. The glucose system with added yellow water is: 300-500 g / L yellow water and 8-10 g / L glucose. The cellar mud is: cellar mud for strong-aroma baijiu with a cellar age of more than 30 years, a hexanoic acid content of more than 5g / kg and a lactic acid content of less than 20g / kg. The yellow water contains: lactic acid 60-80 g / L, acetic acid 5-7 g / L, propionic acid 8-10 g / L, butyric acid 4-6 g / L, glucose 4-6 g / L, and pH 3-4. (2) The fermentation broth obtained in step (1) is subjected to a first distillation to obtain a concentrated hexanoic acid solution. The distillation conditions are: vacuum degree of 45-50 mbar and temperature of 75-80℃. (3) Add the hexanoic acid concentrate obtained in step (2) to the base wine, and add at least 0.2‰ of lipase with an enzyme activity of 10000 U / g. Esterify at 30-35℃ for 2-3 days to obtain esterified liquid. The lipase is Jiangsu Boli Biotechnology's esterase HLP-0100. (4) The esterified liquid obtained in step (3) is subjected to a second distillation, and the distilled components are collected to obtain the strong-aroma high-ester flavored wine. The second distillation is a three-stage distillation: First stage distillation: The esterified liquid is distilled, with the distillation vacuum set at 400 mbar and the top temperature controlled at 80℃. After distillation for 1.5-2 hours, the second stage distillation is carried out. Second stage distillation: Set the distillation vacuum to 300 mbar and control the top temperature at 80℃. After distilling for 3-4 hours, proceed to the third stage distillation. The third stage of distillation: set the distillation vacuum to 1200 mbar and control the top temperature at 80℃ for 2-3 hours. Collect the components from the second-stage and third-stage distillation.

2. The method according to claim 1, characterized in that, The cellar mud microbial agent was inoculated into mCGM medium and cultured stepwise to three levels of seed culture. The inoculation amount of each level of seed culture was 10-12%, and the culture conditions were 35-37℃ and anaerobic culture for 3-5 days.

3. The method according to claim 2, characterized in that, The mCGM medium consists of: 8-10 g / L yeast extract, 8-10 g / L peptone, 5-6 g / L sodium acetate, 8-10 g / L glucose, 0.5-1 g / L sodium dihydrogen phosphate, 1-2 g / L dipotassium hydrogen phosphate, 0.02-0.05 g / L magnesium sulfate heptahydrate, 1-2 g / L ammonium sulfate, and pH 6.

7.

4. The method according to claim 3, characterized in that, The inoculation amount of the pit mud is 5g / 100 ml - 10g / 100 ml by mass-volume ratio.

5. The strong-aroma, high-ester flavored wine prepared by the method according to any one of claims 1 to 4.

6. The application of the method according to any one of claims 1 to 4 in the preparation of high-fat flavored wine.

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