A method for reducing the ratio of ethyl acetate to ethyl caproate in base liquor of Luzhou-flavor liquor

Abstract

The present invention relates to a method for reducing the content ratio of ethyl acetate to ethyl caproate in the base liquor of Luzhou-flavor liquor, and the method comprises the following steps: S1, preparation of bacterial liquid A; S2, preparation of bacterial liquid B; S3, preparation of bacterial liquid C; S4, preparation of composite biological fortification bacterial agent D; S5, fermentation in a pool. The beneficial effects of the present invention are: the content of ethyl caproate in the cellar can be increased, the generation of ethyl acetate can be reduced, and the composition of flavor substances in the upper layer of lees can be improved; the wine body is uniform and mellow, and the Luzhou-flavor style is prominent.

Description

Technical Field

[0001] The invention relates to the technical field of winemaking, and in particular to a method for reducing the content ratio of ethyl acetate to ethyl caproate in base wine of Luzhou-flavor liquor. Background Art

[0002] Chinese liquor is a pearl of the wisdom of the ancient people. It has a brewing history of thousands of years and is one of the representative symbols of my country's traditional culture. It is known as the "six major distilled spirits in the world" together with whiskey, brandy, vodka, gin and rum. Among the twelve major flavor liquors, the strong-flavor liquor is famous at home and abroad for its characteristics of "colorless and transparent, elegant cellar fragrance, soft and sweet, soft and coordinated, long tail and typical style", and occupied half of the national liquor market with a large advantage in 2022. Esters in strong-flavor liquor account for about 60% of the aromatic components, among which ethyl caproate is the main aroma component of strong-flavor liquor. Together with appropriate amounts of ethyl acetate, ethyl lactate, ethyl butyrate, etc., it creates the unique style characteristics of strong-flavor liquor, such as strong cellar fragrance, soft and sweet, and clean and refreshing tail. If the composition ratio of the relevant ester substances is seriously unbalanced, the strong-flavor liquor will lose its style characteristics.

[0003] In the entire brewing process of liquor, ester substances are generated by esterification between acids and alcohols. There are three main formation pathways reported so far: first, ester-producing microorganisms (such as yeast) directly carry out biosynthesis in cells, which involves two key intracellular enzymes: acyl-CoA synthetase and alcohol acetyltransferase, which is the main formation pathway of acetate in liquor; second, esterification enzymes secreted by enzyme-producing microorganisms catalyze the corresponding ester synthesis reaction with acids and alcohols as substrates. Ethyl hexanoate, ethyl butyrate and ethyl lactate mainly rely on this pathway for production; third, a slow pathway that occurs in the late fermentation and storage of liquor, that is, free acid-alcohol molecules generate esters through simple organic chemical reactions. Therefore, the microbial system in the lees has a vital influence on the flavor of liquor. In recent years, many researchers have made a lot of explorations and attempts to accelerate and guide liquor brewing using biofortification. The feasibility of using biofortification to improve the quality of liquor products has been confirmed, and it has been found that liquor production can be better predicted and controlled by adding biofortification agents with specific functions.

[0004] In recent years, the ester ratio relationship in Luzhou-flavor liquor, especially that produced by small and medium-sized liquor companies, has gradually become unbalanced, and the ethyl acetate content has begun to show a trend of surpassing ethyl caproate, resulting in the blurring of the typical style characteristics of Luzhou-flavor liquor, lack of comfort in taste, and decreased coordination of the wine body. The increasingly serious inverted phenomenon of the "ethyl caproate to ethyl hexanoate ratio" has also become a top technical problem that needs to be overcome in the production process of today's liquor companies.

[0005] Therefore, it is particularly important to invent a method for reducing the ratio of ethyl acetate to ethyl caproate in the base liquor of Luzhou-flavor liquor. Summary of the invention

[0006] The purpose of the present invention is to provide a method for reducing the content ratio of ethyl acetate to ethyl caproate in base liquor of Luzhou-flavor liquor, by increasing the content of ethyl caproate in a cellar through the action of a composite biological strengthening bacterial agent, reducing the generation of ethyl acetate, and improving the composition of flavor substances in the upper lees; by using the method to brew Luzhou-flavor liquor, the content ratio of ethyl acetate to ethyl caproate in the base liquor produced is significantly reduced, the liquor body is uniform and mellow, and the Luzhou-flavor style is prominent.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] A method for reducing the ratio of ethyl acetate to ethyl caproate in a base liquor of a Luzhou-flavor liquor comprises the following steps:

[0009] S1. Preparation of bacterial solution A

[0010] The seed liquid of Bacillus subtilis YTPW-105 is transferred to a container filled with a spore-forming medium at an inoculation rate of 8% to 12% to obtain a bacterial liquid A;

[0011] S2. Preparation of bacterial solution B

[0012] The powdered Miller yeast YTPW-213 seed solution is transferred to a container filled with yeast proliferation medium at an inoculation rate of 8%-12% to obtain bacterial solution B;

[0013] S3. Preparation of bacterial solution C

[0014] The seed solution of Staphylococcus epidermidis YTPW-140 is transferred to a container containing a bacterial proliferation medium at an inoculation rate of 8% to 12% to obtain a bacterial solution C;

[0015] S4. Preparation of composite bio-enhancing agent D

[0016] The bacterial suspension of bacterial solution A, the bacterial suspension of bacterial solution B and the bacterial suspension of bacterial solution C are mixed to obtain a composite bio-enhanced bacterial agent D;

[0017] S5, Fermentation in the tank

[0018] The mash was fermented with composite bio-enhanced bacterial agent D.

[0019] The powdered Miller yeast (YTPW-213), the classification name of its biological material is: Millerozymafarinose, the preservation number is: CCTCC No.M2022633, the preservation date is: May 16, 2022, the preservation unit is: China Center for Type Culture Collection, the preservation address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province;

[0020] The Bacillus subtilis (YTPW-105) is named as Bacillus subtilis, with a deposit number of CCTCC No. M2022631, a deposit date of May 16, 2022, a depository of China Center for Type Culture Collection, and a deposit address of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province;

[0021] The Staphylococcus epidermidis (YTPW-140) is classified and named as Staphylococcus epidermidis, with a deposit number of CCTCC No. M2022632, a deposit date of May 16, 2022, a depository of China Center for Type Culture Collection, and a deposit address of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province;

[0022] Powdered Saccharomyces milleri (YTPW-213), Bacillus subtilis (YTPW-105), and Staphylococcus epidermidis (YTPW-140) were all isolated from the Luzhou-flavor medium-high temperature Daqu produced by Chengdu Shuzhiyuan Winery.

[0023] In one embodiment of the present invention, the step S5 of fermentation in a tank is specifically as follows:

[0024] A portion of the composite biological fortification agent D is sprayed on the four walls of the cellar, and the other portion is mixed in at a ratio of 0.4%-0.5% of the volume of the mash, spread on the upper layer of the cellar, and then fermented. After the fermentation is completed, it is taken out of the cellar and distilled to obtain a Luzhou-flavor base wine. The Luzhou-flavor base wine is a Luzhou-flavor base wine with low ethyl acetate and high ethyl caproate.

[0025] In one embodiment of the present invention, the spore production medium comprises the following raw materials in parts by weight: 90-100 parts of bran powder, 4-6 parts of glucose, 2-3 parts of corn flour, 1.5-2 parts of ammonium sulfate, 1.5-2 parts of dipotassium hydrogen phosphate, 3-3 parts of sodium chloride, 0.3-0.5 parts of magnesium sulfate, 0.008-0.016 parts of zinc acetate, 0.2-0.3 parts of manganese sulfate, 1000 parts of distilled water, and natural pH.

[0026] In one embodiment of the present invention, the yeast proliferation medium comprises the following raw materials in parts by weight: 1-2 parts of glucose, 4-5 parts of molasses, 7-8 parts of bran, 7-8 parts of dried distiller's grains, 1-2 parts of cottonseed meal, 1-2 parts of yeast extract, 0.5-1 parts of ammonium sulfate, 2.5-3 parts of potassium dihydrogen phosphate, 2.5-3 parts of magnesium sulfate, 1-1.5 parts of calcium chloride, 0.075-0.1 parts of inositol, 1000 parts of distilled water, and the pH is 6.5-6.8.

[0027] In one embodiment of the present invention, the bacterial proliferation medium comprises the following raw materials by weight: 4-5 parts of peptone, 3-4 parts of glucose, 1-1.5g of potassium dihydrogen phosphate, 0.5-1 part of dipotassium hydrogen phosphate, 0.5-0.7 part of magnesium sulfate, 0.1-0.2 part of calcium chloride, 1-2 parts of glycerol, 1000mL of distilled water, and a pH of 6.0-6.5.

[0028] In one embodiment of the present invention, the culture conditions of the bacterial solution A are 37-39°C, 150r / min, 36-48h, and the number of viable cells detected is 10 8 -10 9 CFU / mL.

[0029] In one embodiment of the present invention, the culture conditions of the bacterial solution B are 29-31°C, 150r / min, 24-36h, and the number of viable cells detected is 10 7 -10 8 CFU / mL.

[0030] In one embodiment of the present invention, the culture conditions of the bacterial solution C are 36-38°C, 150r / min, 36-48h, and the number of viable cells detected is 10 8 -10 9 CFU / mL.

[0031] In one embodiment of the present invention, in step S4, the mixing volume ratio of the bacterial suspension of bacterial solution A, the bacterial suspension of bacterial solution B and the bacterial suspension of bacterial solution C is 1-1.4:2-2.3:0.9-1.1.

[0032] In a preferred embodiment of the present invention, in step S4, the mixing volume ratio of the bacterial suspension of bacterial solution A, the bacterial suspension of bacterial solution B and the bacterial suspension of bacterial solution C is 1:2:1.

[0033] The preparation of the bacterial suspension is as follows: after centrifuging the bacterial solutions A, B and C, the bacterial bodies are collected, and then an equal volume of sterile physiological saline is added to resuspend the bacterial suspension. The number of viable bacteria in the composite bio-enhanced bacterial agent D is 10 7 -10 8 CFU / mL.

[0034] In one embodiment of the present invention, the thickness of the mash mixed with the composite biological enhancement agent D spread on the upper layer of the pit in step S5 is 0.5-0.9 m.

[0035] In the present invention, the fortifying bacteria powdered Miller's yeast (Millerozymafarinose), Bacillus subtilis (Bacillus subtilis) and Staphylococcus epidermidis (Staphylococcus epidermidis) in the composite biological fortifying bacterial agent are all separated from the finished product of Luzhou-flavor medium-high temperature Daqu, have high temperature resistance, can safely pass the high-fire stage of Daqu culture and fermentation, and can still survive at the highest koji making temperature of 60°C.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The powdered Millerozyma farinose fortified strain selected by the present invention not only has excellent wine production ability, but also can metabolize more flavor substances. Bacillus subtilis and Staphylococcus epidermidis are both high-yield esterification enzyme strains that use caproic acid and ethanol as substrates to synthesize ethyl caproate. The three together can not only improve the starch conversion rate of winemaking raw materials, provide more substrates for self-fermentation and wine production, but also increase the yield of ethyl caproate in liquor. Compared with the raw liquor produced by traditional production, the ratio of ethyl acetate to ethyl caproate in the raw liquor produced by using the biological fortified bacterial agent is significantly reduced from 1.03-1.33 to 0.53-0.74;

[0038] (2) The present invention adds a composite biological strengthening agent to the upper layer of the lees, and the microorganisms slowly penetrate into the lower layer of the lees along with the fermentation liquid, thereby avoiding the problem of microorganisms being enriched in the middle and lower layers of the lees in the traditional fermentation and the lower yield of flavor substances in the upper layer of the lees; compared with the traditional method, the total concentration of flavor compounds in the upper, middle and lower layers of the original wine produced by this method is significantly increased by 42.22%, 26.89% and 18.58% respectively, and the flavor content of each layer of the original wine is closer, and the wine body is more uniform;

[0039] At the same time, this scheme identified a total of 118 flavor compounds from all mash samples, including 16 alcohols, 13 aldehydes and ketones, 8 acids, 55 esters, 5 phenols, 21 hydrocarbons and other substances. Biofortification had no significant effect on the total concentration of flavor compounds in mash, but significantly increased the content of alcohols, aldehydes and ketones, esters and phenols in the mash of Luzhou-flavor liquor fermentation, and significantly reduced the content of acids and hydrocarbons and other substances. Therefore, the biofortification agent can adjust the proportion of flavor components without changing the total amount of flavor substances in the mash.

[0040] (3) The present invention significantly reduces the concentration of n-butanol and isoamyl alcohol in the original liquor. Among alcohol substances, n-butanol is one of the main components of higher alcohols in liquor, and its taste is bitter and slightly astringent. When the content is too high, it will affect the drinking taste. Isoamyl alcohol is the key higher alcohol that causes discomfort after drinking liquor and has a negative impact on human health. The present invention significantly reduces the concentration of n-butanol and isoamyl alcohol in the original liquor by 18.24%-48.83% and 19.90-51.22% respectively through biofortification, making the taste of the liquor softer and helping to improve its drinking safety.

[0041] (4) In the present invention, when preparing Bacillus subtilis liquid, corn flour and ammonium sulfate are used as dual nitrogen sources, and the spore conversion rate is much higher than that of a single nitrogen source. + Has osmotic regulating effect, Mg 2+ It is an important enzyme activator of EMP and TCA pathways. The invention increases the growth rate of bacteria and promotes spore production by adding sodium chloride and magnesium sulfate to the culture medium.

[0042] (5) In the preparation of the powdered Miller yeast liquid, the present invention uses dried distiller's grains and cottonseed meal as nitrogen sources, which can not only provide nutrition for the growth of yeast and promote the production of active substances, but also turn the dried distiller's grains into valuables, thereby reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 A comparison chart of the total amount of flavor compounds in the base liquor of Luzhou-flavor liquor fermented by the present invention and by the traditional method;

[0044] Figure 2 This is a comparison chart of the detection results of the six major flavor substances in each layer of the fermented lees using the present invention and the traditional fermentation method;

[0045] Figure 3 Graph showing the ethyl caproate content of each layer of raw wine fermented by the present invention and by the traditional method;

[0046] Figure 4 It is a graph showing the ethyl acetate content of each layer of raw liquor fermented by the present invention and the traditional method;

[0047] Figure 5 The graph is a ratio of ethylhexyl ratio of each layer of raw liquor fermented by the present invention and the traditional method;

[0048] Figure 6 It is a diagram of the pairwise interaction relationship between the three fortified bacterial strains (in the figure, Bs: Bacillus subtilis YTPW-105; Se: Staphylococcus epidermidis YTPW-140; Mf: Millerozyma farinose YTPW-213);

[0049] Figure 7 This is a graph showing the effect of the interaction between the three enhanced bacterial strains on their growth diameter. DETAILED DESCRIPTION

[0050] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings, but the protection scope of the present invention is not limited to the following.

[0051] Example 1

[0052] A method for reducing the ratio of ethyl acetate to ethyl caproate in a base liquor of a Luzhou-flavor liquor comprises the following steps:

[0053] (1) Preparation of bacterial solution A: 10% of the seed solution of Bacillus subtilis was transferred to a 250 mL Erlenmeyer flask containing 100 mL of spore-forming medium and cultured at 39°C and 150 rpm for 48 h. The number of viable cells was detected to be 10 9 CFU / mL.

[0054] (2) Preparation of bacterial solution B: 10% of the seed solution of powdered Millerozyma farinose was transferred to a 250 mL Erlenmeyer flask containing 100 mL of yeast proliferation medium and cultured at 31°C and 150 rpm for 36 h. The viable count was detected to be 10 8 CFU / mL.

[0055] (3) Preparation of bacterial solution C: 10% of the seed solution of Staphylococcus epidermidis was transferred to a 250 mL Erlenmeyer flask containing 100 mL of spore-forming medium and cultured at 38°C and 150 rpm for 54 h. The number of viable cells was detected to be 10 9 CFU / mL.

[0056] (4) Preparation of composite bio-enhanced bacterial agent D: After centrifuging bacterial solutions A, B and C, the bacterial bodies were collected and resuspended in equal volumes of sterile physiological saline to prepare bacterial suspensions. Then, bacterial suspensions of Bacillus subtilis, Millerozyma farinose and Staphylococcus epidermidis were mixed in a volume ratio of 1:2:1 to obtain composite bio-enhanced bacterial agent D. The viable bacterial count was detected to be 10 8 CFU / mL, prepare 50L in total.

[0057] (5) Fermentation in the cellar: The volume of the cellar is 3m×2m×2.1m. Take 20L of the composite biological enhancement agent D in step (4) and mix it with 4.2m 3Volume of cooled mash is mixed evenly, and the rest is sprayed on the four walls of the cellar. The mash mixed with composite biological fortification agent D is evenly spread on the upper layer of the cellar, and then fermented. The fermentation cycle is 60 days. After the fermentation is completed, it is taken out of the cellar and distilled to obtain a Luzhou-flavor original liquor with low ethyl acetate and high ethyl caproate.

[0058] Furthermore, various flavor components were detected and compared using a traditionally produced Luzhou-flavor raw liquor as a control group. The control group followed the preparation steps of Example 1, and the only difference between the control group and Example 1 was that the control group did not use the composite bio-enhancer D, and the traditional Daqu and the cooled mash were mixed and then directly put into the pool for fermentation, and the fermentation cycle was 60 days.

[0059] The various flavor components of the base liquor produced by the method of the present invention (Example 1) and the Luzhou-flavor base liquor produced by the traditional method were detected, and the comparison results were as follows: Figure 1 , Figure 2 As shown in Table 1, TBJ represents the original wine produced by the traditional method; BBJ represents the original wine produced by the present invention; U, M, and B represent the original wine distilled from the upper, middle, and lower layers of fermented grains. For example, TBJ-U represents the original wine produced by the upper layer of fermented grains using the traditional production method.

[0060] Depend on Figure 1 It can be seen that the total concentrations of flavor compounds in the original liquor distilled from the control group without the addition of biofortification agents were 8.29 g / L (upper layer), 9.26 g / L (middle layer) and 11.79 g / L (lower layer), while the total concentrations of flavor compounds in the original liquor distilled from the biofortification group (Example 1) were 11.75 g / L (upper layer), 11.65 g / L (middle layer) and 13.68 g / L (lower layer). Compared with the control group, it was observed that the total concentrations of flavor compounds in the upper, middle and lower layers of the original liquor in the biofortification group increased significantly by 42.22%, 26.89% and 18.58%, respectively. It can be seen that the original liquor distilled from the lower layer of mash in the biofortification group has the highest content of flavor compounds, but biofortification has the most significant effect on improving the flavor of the original liquor distilled from the upper layer of mash. Figure 2 It can be seen that biofortification significantly increased the content of ethyl esters in the upper and lower raw wines.

[0061] It can be seen from Table 1 that among the increased alcohol substances, ethanol, n-butanol and isoamyl alcohol are the three flavor compounds with the highest content, accounting for as much as 91.26% of the alcohol substances. Biofortification significantly reduced the concentrations of n-butanol and isoamyl alcohol in the original wine by 18.24%-48.83% and 19.90-51.22%, respectively, indicating that biofortification can make the taste of the wine softer and help improve its drinking safety.

[0062] Table 1 Contents of main alcohol substances in base liquor of Luzhou-flavor liquor

[0063]

[0064] Furthermore, the ratio of ethyl acetate to ethyl caproate in the base liquor produced by the method of the present invention and the base liquor of the traditional method was tested, and the comparison results were as follows: Figure 3 to Figure 5 shown.

[0065] Existing studies have shown that the ratio of ethyl acetate to ethyl caproate (ethyl hexanoate ratio) in Luzhou-flavor base liquor is preferably less than 1, and the ethyl hexanoate ratio of high-quality Luzhou-flavor base liquor is about 0.5. However, in recent years, the ethyl acetate content of base liquor produced by enterprises is relatively high, and the ethyl hexanoate ratio of base liquors of different grades is even greater than 1, resulting in the misalignment of the base liquor flavor and poor typicality. Similar problems also occurred in the base liquor of the control group of the present invention, such as Figure 5 As shown, the ethyl hexanoate ratios in the upper, middle and lower layers of the raw wine in the control group were 1.21, 1.33 and 1.03, respectively, all greater than 1, indicating that the ethyl hexanoate ratio has become imbalanced. The ethyl hexanoate ratios in the upper, middle and lower layers of the raw wine in the bio-fortified group were 0.54, 0.74 and 0.53, respectively, which showed a significant decrease. Therefore, the combined action of Millerozyma farinose, Bacillus subtilis and Staphylococcus epidermidis can not only improve the starch conversion rate of brewing raw materials, provide more substrates for its own fermentation and wine production, but also increase the production of ethyl hexanoate in the raw wine. Compared with the traditionally produced raw wine, the ratio of ethyl acetate to ethyl hexanoate in the raw wine produced using the bio-fortified bacterial agent was significantly reduced from 1.03-1.33 to 0.53-0.74. In addition, Figure 3 and Figure 4 It can also be clearly seen that the present invention shows the positive effect of biofortification on "increasing alcohol and reducing ethyl" in Luzhou-flavor base liquor.

[0066] Example 2

[0067] The present embodiment provides a method for reducing the ratio of ethyl acetate to ethyl caproate in a base liquor of a Luzhou-flavor liquor, which has the same steps as those of Example 1, and the only difference from Example 1 is that in step S4, the mixing volume ratio of the bacterial suspension of bacterial solution A, the bacterial suspension of bacterial solution B, and the bacterial suspension of bacterial solution C is 1.2:2.1:0.9.

[0068] After testing, the ethyl-hexyl ratios in the upper, middle and lower layers of the raw liquor prepared in this embodiment were 0.51, 0.69 and 0.55 respectively, indicating that the biological fortification agent prepared in this embodiment has a positive effect on "increasing ethyl and reducing ethyl" in Luzhou-flavor raw liquor.

[0069] Example 3

[0070] The present embodiment provides a method for reducing the ratio of ethyl acetate to ethyl caproate in a base liquor of a Luzhou-flavor liquor, which has the same steps as those of Example 1, and the only difference from Example 1 is that in step S4, the mixing volume ratio of the bacterial suspension of bacterial solution A, the bacterial suspension of bacterial solution B, and the bacterial suspension of bacterial solution C is 1.4:2.3:1.1.

[0071] After testing, the ethyl-hexyl ratios in the upper, middle and lower layers of the raw liquor prepared in this embodiment were 0.63, 0.69 and 0.60 respectively, indicating that the biological fortification agent prepared in this embodiment has a positive effect on "increasing ethyl and reducing ethyl" in Luzhou-flavor raw liquor.

[0072] Experimental Example 1

[0073] In this experimental example, before Bacillus subtilis YTPW-105, Staphylococcus epidermidis YTPW-140 and Millerozyma farinose YTPW-213 were prepared into a bio-fortified bacterial agent, the three fortified bacterial strains were first mixed and cultured in an enzyme-producing fermentation medium according to different inoculation ratios in Table 2, and then the optimal inoculation ratio was determined by measuring the esterase activity of the fermentation broth.

[0074] Enzyme fermentation medium: soluble starch 2%, yeast extract powder 2%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, ferrous sulfate 0.001%, potassium chloride 0.05%, manganese sulfate 0.03%, olive oil 2%, pH 7.4.

[0075] During the experiment, the optimal bacterial inoculation ratio (Bs:Se) was first determined according to the results of experimental groups 1 to 5 in Table 2, and then Bacillus subtilis YTPW-105 and Staphylococcus epidermidis YTPW-140 were regarded as a bacterial whole, and the bacteria and yeast (Millerozyma farinose YTPW-213) were inoculated into the enzyme fermentation medium with different initial inoculation concentrations (10% of the volume of the enzyme fermentation medium). The cell concentration ratios of bacteria and yeast in the enzyme fermentation medium after inoculation were 1:0.2, 1:0.5, 1:1, 1:2, and 1:5, respectively. In all the ratio groups after inoculation, the cell concentration of bacteria remained the same. Subsequently, the fermentation was carried out at 37°C, 150r / min, and the esterase enzyme activity of the fermentation broth was determined according to the conventional determination method. The determination results are shown in Table 2.

[0076] Table 2 Esterase activity at different inoculation ratios

[0077]

[0078] In Table 2, Bs: Bacillus subtilis; Se: Staphylococcus epidermidis; Mf: Millerozyma farinose.

[0079] As shown in experimental groups 1 to 5 in Table 2, when the cell concentration ratio of Bacillus subtilis YTPW-105 and Staphylococcus epidermidis YTPW-140 in the enzyme fermentation medium was 1:1, the crude enzyme solution had the strongest ability to synthesize ethyl hexanoate, with a content of 645.09 mg / 100 mL, and the esterase activity of the crude enzyme solution was 2.36 U / mL; therefore, 1:1 was used as the optimal inoculation ratio of Bacillus subtilis YTPW-105 and Staphylococcus epidermidis YTPW-140, and Millerozyma farinose YTPW-213 was inoculated for co-culture again. The results are shown in experimental groups 5 to 10 in Table 2. When Bacillus subtilis YTPW-105, Staphylococcus epidermidis YTPW-140 and Millerozyma farinose were inoculated in the enzyme fermentation medium, the inoculation ratio of Bacillus subtilis YTPW-105, Staphylococcus epidermidis YTPW-140 and Millerozyma farinose When the ratio of YTPW-213 was 1:1:2, that is, when the ratio of bacteria to yeast was 1:1, the content of ethyl hexanoate synthesized by the crude enzyme solution was the highest, reaching 763.71 mg / 100 mL, and the esterase activity was 2.79 U / mL. Based on the above results, the ratio of Bacillus subtilis YTPW-105:Staphylococcusepidermidis YTPW-140:Millerozyma farinose YTPW-213=1:1:2 was finally adopted as the preferred inoculation ratio for the preparation of bio-enhanced bacterial agents.

[0080] As can be seen from Table 2, when the inoculation ratio (Bs:Se:Mf) of Millerozyma farinose, Bacillus subtilis and Staphylococcus epidermidis is 1:1:2, the activity of esterase is significantly enhanced and the content of synthesized ethyl hexanoate is significantly increased; this indicates that the combined effect of the three can not only improve the starch conversion rate of brewing raw materials, provide more substrates for its own fermentation and wine production, but also increase the yield of ethyl hexanoate in the original wine.

[0081] Experimental Example 2

[0082] This experimental example is to verify whether three fortifying strains of Bacillus subtilis YTPW-105, Staphylococcus epidermidis YTPW-140 and Millerozyma farinose YTPW-213 can be inoculated together to prepare a composite biofortifying agent.

[0083] In this experiment, three strains of enhanced bacteria were co-cultured in pairs on a PDA plate. Figure 6 As shown in the figure, Bacillus subtilis YTPW-105, Staphylococcus epidermidis YTPW-140 and Millerozyma farinose YTPW-213 can grow together on PDA plates, and there is no obvious antagonism between the three. However, the presence of Staphylococcus epidermidis YTPW-140 and Millerozyma farinose YTPW-213 seems to curb the trend of Bacillus subtilis YTPW-105 to diverge from the edge of the colony to the surrounding of the plate. In addition, Bacillus subtilis YTPW-105 and Millerozyma farinose YTPW-213 also slightly slowed down the growth of Staphylococcus epidermidis YTPW-140.

[0084] In order to more intuitively illustrate the degree of mutual influence between the enhanced strains, the growth diameter of the enhanced strains on the PDA plate was further measured. Figure 7The results show that there was no significant difference in the growth diameters of the three fortified strains on the PDA plate, whether cultured alone or in pairs (P>0.05). When co-cultured with the other two fortified strains, the growth diameter of Bacillus subtilis decreased by 0.19 cm at most, and the growth diameter of Staphylococcus epidermidis decreased by 0.15 cm at most. However, Millerozyma farinose only decreased its growth diameter by 0.12 cm when co-cultured with Bacillus subtilis, but slightly increased its growth diameter by 0.06 cm when co-cultured with Staphylococcus epidermidis. Our results showed that the three fortified strains Bacillus subtilis YTPW-105, Staphylococcus epidermidis YTPW-140 and Millerozyma farinose YTPW-213 did not produce obvious growth inhibition effects, and Staphylococcus epidermidis YTPW-140 could even stimulate the growth of Millerozyma farinose YTPW-213 to a certain extent. Therefore, it is feasible to select these three fortified strains to prepare a composite biofortified agent.

[0085] The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not deviate from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. A method for reducing the ratio of ethyl acetate to ethyl caproate in a base liquor of a Luzhou-flavor liquor, characterized in that: The following steps are involved: S1. Preparation of bacterial solution A The seed solution of Bacillus subtilis YTPW-105 is transferred to a container containing a spore-forming medium at an inoculation rate of 8% to 12% to obtain a bacterial solution A; S2. Preparation of bacterial solution B The powdered Millerozyma farinose YTPW-213 seed solution is transferred to a container containing yeast proliferation medium at an inoculation rate of 8%-12% to obtain bacterial solution B; S3. Preparation of bacterial solution C The seed solution of Staphylococcus epidermidis YTPW-140 is transferred to a container containing a bacterial proliferation medium at an inoculation rate of 8% to 12% to obtain a bacterial solution C; S4. Preparation of composite bio-enhancing agent D The bacterial suspension of bacterial solution A, the bacterial suspension of bacterial solution B and the bacterial suspension of bacterial solution C are mixed to obtain a composite bio-enhanced bacterial agent D; S5, Fermentation in the tank The mash was fermented with composite biological fortification agent D; The spore production medium comprises the following raw materials by weight: 90-100 parts of bran powder, 4-6 parts of glucose, 2-3 parts of corn flour, 1.5-2 parts of ammonium sulfate, 1.5-2 parts of dipotassium hydrogen phosphate, 3-3 parts of sodium chloride, 0.3-0.5 parts of magnesium sulfate, 0.008-0.016 parts of zinc acetate, 0.2-0.3 parts of manganese sulfate, 1000 parts of distilled water, and natural pH; The yeast proliferation medium comprises the following raw materials by weight: 1-2 parts of glucose, 4-5 parts of molasses, 7-8 parts of bran, 7-8 parts of dried distiller's grains, 1-2 parts of cottonseed meal, 1-2 parts of yeast extract, 0.5-1 parts of ammonium sulfate, 2.5-3 parts of potassium dihydrogen phosphate, 2.5-3 parts of magnesium sulfate, 1-1.5 parts of calcium chloride, 0.075-0.1 parts of inositol, 1000 parts of distilled water, and a pH of 6.5-6.8; The bacterial proliferation medium includes the following raw materials by weight: 4-5 parts of peptone, 3-4 parts of glucose, 1-1.5 g of potassium dihydrogen phosphate, 0.5-1 part of dipotassium hydrogen phosphate, 0.5-0.7 parts of magnesium sulfate, 0.1-0.2 parts of calcium chloride, 1-2 parts of glycerol, 1000 mL of distilled water, and a pH of 6.0-6.5; The culture conditions of the bacterial solution A are 37-39°C, 150r / min, 36-48h, and the number of viable bacteria detected is 10 8 -10 9 CFU / mL; The culture conditions of the bacterial solution B are 29-31°C, 150r / min, 24-36h, and the number of viable bacteria detected is 10 7 -10 8 CFU / mL; In step S4, the mixing volume ratio of the bacterial suspension of bacterial solution A, the bacterial suspension of bacterial solution B and the bacterial suspension of bacterial solution C is 1-1.4:2-2.3:0.9-1.

1.

2. The method for reducing the ratio of ethyl acetate to ethyl caproate in the base liquor of Luzhou-flavor liquor according to claim 1, characterized in that: The step S5 of fermentation in the tank is specifically as follows: A portion of the composite biological fortification agent D is sprayed on the four walls of the cellar, and the other portion is mixed in at a ratio of 0.4%-0.5% of the volume of the mash, spread on the upper layer of the cellar, and then fermented. After the fermentation is completed, it is taken out of the cellar and distilled to obtain the Luzhou-flavor original liquor.

3. The method for reducing the content of ethyl acetate and ethyl caproate in the base liquor of Luzhou-flavor liquor according to claim 2, characterized in that: In step S5, the thickness of the mash mixed with the composite biological enhancement agent D spread on the upper layer of the cellar is 0.5-0.9 m.

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