A compound bacterial liquid and a method for brewing Luzhou-flavor liquor by using the same

Abstract

The present invention discloses a compound bacterial liquid and a method for brewing Luzhou-flavor liquor by using the same, belonging to the technical field of microbial materials. The compound bacterial liquid comprises Aspergillus fisheri, Acinetobacter baylyi and Saccharomyces cerevisiae. The present invention screens three microbial strains to form a compound bacterial liquid, which is mixed in equal proportion and used for fermentation in combination with Daqu. The Luzhou-flavor liquor produced by using the bacterial liquid and process of the present invention has an increased alcohol production amount and an increased acidity content; the characteristic flavor substances in the base liquor related to the quality of the base liquor are also improved to a certain extent. The obtained liquor has a clear and transparent color, a harmonious and strong aroma, a mellow and sweet taste, a lasting fragrance and a heavy body, meeting the taste and quality requirements of Luzhou-flavor liquor.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial materials, and particularly relates to a composite bacterial liquid and a method for brewing Luzhou-flavor liquor using the same. Background Art

[0002] There are two ways to make wine with grain raw materials in various countries around the world. One is to use the enzymes produced when grains germinate to catalyze the raw materials into sugars and then convert them into alcohol by yeast, and the other is to use the enzymes contained in the koji to saccharify and ferment grains into wine. Most liquors are brewed with koji. "Koji is the soul of wine". Koji is the driving force of liquor brewing and fermentation, and is an indispensable part of liquor. "Koji determines the type of liquor". Daqu is divided into Daqu, Xiaoqu, and Fuqu. Daqu is a "multi-bacteria and multi-enzyme" mixture made of wheat (or barley, peas, etc.) as raw materials, which is crushed, mixed with water, pressed into shape (usually a rectangular block), and cultivated in the koji room. Due to different classification methods, Daqu has different classifications. Medium-high temperature Daqu is a special koji for brewing Luzhou-flavor liquor. In the brewing process of liquor, Daqu plays a role in providing bacteria sources, enzymes, aroma and aroma precursors, raw materials, etc. The type of Daqu determines the flavor of liquor and is one of the core indicators of winemaking quality control. Optimizing the composition and functional properties of microorganisms in Daqu is of great significance to improving the quality of Daqu, increasing liquor production and improving the quality of liquor.

[0003] Daqu plays a vital role in the production of Luzhou-flavor liquor, and its microbial species have a significant impact on the flavor and quality of liquor. The microorganisms in Daqu mainly include four categories: molds, yeasts, bacteria and actinomycetes. These microorganisms provide a variety of enzymes and flavor precursors for the fermentation of liquor through their metabolic activities, thus determining the unique flavor of liquor.

[0004] At present, in the brewing of liquor, the microorganisms used are mostly self-selected microbial strains, such as the Chinese patent application: CN 201310220126.5, which discloses an acetic acid bacteria (Acetobacter sp.) HJB-012 that can improve the flavor of liquor, belonging to the field of liquor brewing technology. The strain has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on December 12, 2012, with the deposit number CGMCC NO.6982. The HJB-012 strain is a heteroanaerobic fermentation, which produces both acetic acid and lactic acid, has good acid and ester production performance, and can effectively improve the quality of liquor and improve the flavor of liquor. The strain HJB-012 is applied to liquor brewing, which supplements important functional bacteria for liquor brewing and strengthens liquor making. It can provide theoretical basis and technical support for the improvement of modern automated liquor making technology, and provide technical guidance for promoting the upgrading of traditional industries. The promotion of this technology is also of great practical significance for the production of Lao Baigan flavor and other flavor liquors.

[0005] However, the strains used are self-selected, the production process is complicated, the cost of use is high, it is difficult to be effectively promoted and utilized, and the actual application scope is narrow. Summary of the invention

[0006] In view of the problems existing in the prior art, the invention selects and compounds three highly active microbial strains, applies them to the preparation of medium-high temperature Daqu, and applies them to the production of Luzhou-flavor liquor. The prepared Luzhou-flavor liquor has high ethyl caproate content and good wine quality.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0008] A composite bacterial liquid, comprising Aspergillus Fischer ( Aspergillus fischeri )、Acinetobacter beijerinckii( Acinetobacter beijerinckii ) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae ).

[0009] Furthermore, the strain number of the Aspergillus fisheri is CGMCC No.3.15369, and the original preservation time is September 3, 2013; the strain number of the Acinetobacter beijerinckii is CGMCC No.1.10392, and the original preservation time is March 23, 2010; the strain number of the Saccharomyces cerevisiae is CGMCC No.2.3871, and the original preservation time is June 5, 2008.

[0010] The Aspergillus Fischerii, Acinetobacter beijerinii and Saccharomyces cerevisiae selected in the present invention are all purchased from the General Microbiological Center of China National Committee for the Preservation of Microbiological Cultures and can be purchased through public catalog inquiries without repeated biological preservation.

[0011] Furthermore, the volume ratio of Aspergillus Fischer, Acinetobacter beijerinckii and Saccharomyces cerevisiae is 1:1:1.

[0012] The method for brewing Luzhou-flavor liquor using the composite bacterial liquid of the present application comprises the following steps:

[0013] (1) Strain activation: Use an inoculation loop to pick up a loop of Aspergillus Fischerii, Acinetobacter beijerinckii, and Saccharomyces cerevisiae, respectively, and inoculate it into beef extract peptone slant culture medium, and culture it in a constant temperature incubator at 25-30° C. for 20-24 h to obtain three activated strains;

[0014] (2) Seed solution preparation: Pick one ring of activated strains of Aspergillus Fischerii, Acinetobacter beijerinii and Saccharomyces cerevisiae, respectively, and inoculate them into 100 mL of beef extract peptone liquid culture medium, and culture them in a shaking incubator at 25-30° C. and 180-200 r / min for 24 h to obtain seed solutions of the three bacteria;

[0015] (3) Fermentation culture: Inoculate the seed liquid of Aspergillus Fischerii, Acinetobacter beijerinii and Saccharomyces cerevisiae into a fermentation tank at an inoculum rate of 5% respectively, and ferment; culture temperature is 25-30° C., culture for 60-72 hours, and fermentation is completed to obtain three kinds of bacterial liquid;

[0016] (4) Collecting bacterial cells: The three bacterial liquids were mixed at a volume ratio of 1:1:1 to obtain a mixed bacterial liquid, and the mixed bacterial liquid was inoculated into a fermentation tank at an inoculum amount of 8%, and fermented at a temperature of 25-30 degrees for 48-72 hours to obtain a mixed fermentation liquid, which was centrifuged, the supernatant was discarded, and the bacterial cells after centrifugation were collected;

[0017] (5) Preparation of inorganic nutrient solution: MgSO 4 , L-phenylalanine, tartaric acid, KH 2 PO 4 , water in a mass ratio of (2-4): (1-2): (1-2): (1-2): 10000, stirred evenly, fully dissolved, allowed to stand and cool, to obtain an inorganic nutrient solution;

[0018] (6) Preparation of composite bacterial solution: Add the bacterial cells obtained in step (4) to the inorganic nutrient solution obtained in step (5) to obtain an effective viable bacterial count of (1-3)×10 8 cfu / mL of composite bacterial solution;

[0019] (7) Raw material cooking: Soak the sorghum in water at 70-75°C for 24 hours, first cook it at 98°C for 2 hours, then stew it at 75°C for 8 hours, drain the stewing water, and then spread the steamed sorghum to cool to 35°C;

[0020] (8) Adding the koji: adding crushed Luzhou-flavor Daqu that has passed through a 30-mesh sieve to the cooled sorghum in an amount of 20% of the mass of the sorghum before steaming, and mixing evenly to obtain a koji-sorghum mixture;

[0021] (9) Fermentation: Spray the microbial liquid evenly on the sorghum mixture obtained in step (8), stir and mix evenly, and ferment at 28° C.-35° C. for 32-36 days to obtain fermented grains;

[0022] (10) Distilling the liquor: Distill the fermented mash in a distillation steamer to obtain Luzhou-flavor liquor.

[0023] Furthermore, the composition of the culture medium in the fermentation tank in step (3) is: 10 g glucose, 10 g yeast extract, 10 g calcium carbonate, 1000 mL distilled water, pH 7.5, and sterilization at 121° C. for 20 min.

[0024] Furthermore, in step (8), the Luzhou-flavor Daqu is a mixture of medium-high temperature Daqu and high temperature Daqu in a ratio of (2-3):1.

[0025] Furthermore, in step (9), the solid-to-liquid ratio of the microbial liquid to the sorghum mixture is 5-10 mL / kg.

[0026] Beneficial effects:

[0027] (1) The present invention selects three microbial strains to form a composite bacterial liquid, mixes them in equal proportions and uses them in combination with Daqu for fermentation. During the fermentation process, the three strains show a significant synergistic effect. The three strains of the present invention all have a high alcohol tolerance and can maintain a high activity during the fermentation process. Among them, Aspergillus Fischer efficiently secretes saccharifying enzymes and proteases. These bioactive substances and metabolites have high saccharification and esterification power, making the flavor of the liquor fuller, thereby improving the quality of the liquor. In addition, Aspergillus Fischer can also promote the formation of flavor components such as alcohols, pyrazines, esters and aromatic compounds during the fermentation process.

[0028] (2) Acinetobacter beijerinii not only produces high levels of saccharifying enzymes and proteases, but also secretes a variety of hydrolytic enzymes during the fermentation process, such as breaking down proteins into small molecules such as amino acids, which then react to produce pyrazines, organic acids, and aromatic substances, which play an important role in the flavor of liquor.

[0029] (3) The selected brewer's yeast can reproduce rapidly and start the fermentation process, converting sugars into ethanol and carbon dioxide, providing power for the production of alcohol. Secondly, brewer's yeast can secrete extracellular enzymes such as amylase, acid protease and β-glucosidase, which help the degradation and saccharification of starch, thereby promoting the production of alcohol in the later stage. When it is mixed with Acinetobacter beijerinii and Aspergillus fishermanii in equal proportions for co-fermentation, it can affect the structure and composition of the microbial community in the mash, reducing the niche width of the microbial community, which helps to form a more stable fermentation environment and makes an important contribution to the aroma and taste of liquor.

[0030] (4) The Luzhou-flavor liquor produced by the bacterial liquid and process of the present invention has an increased alcohol production and acidity content; the characteristic flavor substances (alcohols, acids, esters) in the original liquor related to the quality of the original liquor are also increased to a certain extent. The obtained liquor has a clear and transparent color, a coordinated and rich aroma, a mellow and sweet taste, a long-lasting fragrance, and a thick body, which meets the taste quality requirements of Luzhou-flavor liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] 图1 It is a test diagram of saccharifying enzyme production of Aspergillus Fischer, Acinetobacter beijerinckii and Saccharomyces cerevisiae of the present invention;

[0032] 图2 It is a test diagram of protease production of Aspergillus Fischer, Acinetobacter beijerinckii and Saccharomyces cerevisiae of the present invention;

[0033] 图3 This is the result of the alcohol tolerance test of Aspergillus Fischer;

[0034] 图4 This is the alcohol tolerance test result of Acinetobacter beijerinii;

[0035] 图5 This is the alcohol tolerance test result of Saccharomyces cerevisiae;

[0036] 图6 This is the temperature tolerance test result of Aspergillus Fischer;

[0037] 图7 This is the temperature tolerance test result of Acinetobacter beijerinckii;

[0038] 图8 These are the results of the temperature tolerance test of Saccharomyces cerevisiae. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

[0040] Example 1

[0041] A composite bacterial liquid, comprising Aspergillus Fischer ( Aspergillus fischeri )、Acinetobacter beijerinckii( Acinetobacter beijerinckii ) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae ).

[0042] The strain number of the Aspergillus fisheri is CGMCC No.3.15369, and the original preservation time is September 3, 2013; the strain number of the Acinetobacter beijerinckii is CGMCC No.1.10392, and the original preservation time is March 23, 2010; the strain number of the Saccharomyces cerevisiae is CGMCC No.2.3871, and the original preservation time is June 5, 2008.

[0043] The Aspergillus Fischerii, Acinetobacter beijerinii and Saccharomyces cerevisiae selected in this example were all purchased from the General Microbiological Center of China National Microbiological Culture Collection Administration and can be purchased through public catalog inquiries without repeated biological deposits.

[0044] The volume ratio of Aspergillus Fischl, Acinetobacter beijerinckii and Saccharomyces cerevisiae is 1:1:1.

[0045] The method for brewing Luzhou-flavor liquor using the composite bacterial liquid of this embodiment comprises the following steps:

[0046] (1) Strain activation: Use an inoculation loop to pick up a loop of Aspergillus Fischerii, Acinetobacter beijerinckii, and Saccharomyces cerevisiae, respectively, and inoculate it into beef extract peptone slant culture medium, and culture it in a constant temperature incubator at 25-30°C for 20 hours to obtain three activated strains;

[0047] (2) Seed solution preparation: Pick one ring of activated strains of Aspergillus Fischerii, Acinetobacter beijerinckii, and Saccharomyces cerevisiae, respectively, and inoculate them into 100 mL of beef extract peptone liquid culture medium, and culture them in a shaking incubator at 25-30° C. and 180 rpm for 24 h to obtain seed solutions of the three bacteria;

[0048] (3) Fermentation culture: The seed liquids of Aspergillus Fischerii, Acinetobacter beijerinii and Saccharomyces cerevisiae were inoculated into a fermentation tank at an inoculum rate of 5% respectively, and fermentation was carried out; the culture temperature was 25-30° C., the culture time was 60 hours, and the fermentation was completed to obtain three bacterial liquids;

[0049] (4) Collection of bacterial cells: The three bacterial liquids were mixed at a volume ratio of 1:1:1 to obtain a mixed bacterial liquid, and the mixed bacterial liquid was inoculated into a fermentation tank at an inoculum amount of 8%, and fermented at a temperature of 25-30 degrees for 72 hours to obtain a mixed fermentation liquid, which was centrifuged, the supernatant was discarded, and the bacterial cells after centrifugation were collected;

[0050] (5) Preparation of inorganic nutrient solution: MgSO 4 , L-phenylalanine, tartaric acid, KH 2 PO 4 , water in a mass ratio of 2:1:1:1:10000, stir evenly, fully dissolve, stand and cool, to obtain an inorganic nutrient solution;

[0051] (6) Preparation of composite bacterial solution: Add the bacterial cells obtained in step (4) to the inorganic nutrient solution obtained in step (5) to obtain an effective viable bacterial count of (1-3)×10 8 cfu / mL of composite bacterial solution;

[0052] (7) Raw material cooking: Soak the sorghum in water at 70-75°C for 24 hours, first cook it at 98°C for 2 hours, then stew it at 75°C for 8 hours, drain the stewing water, and then spread the steamed sorghum to cool to 35°C;

[0053] (8) Adding the koji: adding crushed Luzhou-flavor Daqu that has passed through a 30-mesh sieve to the cooled sorghum in an amount of 20% of the mass of the sorghum before steaming, and mixing evenly to obtain a koji-sorghum mixture;

[0054] (9) Fermentation: Spray the microbial liquid evenly on the sorghum mixture obtained in step (8), stir and mix evenly, and ferment at 28° C.-35° C. for 32 days to obtain fermented grains;

[0055] (10) Distilling the liquor: Distill the fermented mash in a distillation steamer to obtain Luzhou-flavor liquor.

[0056] Step (3) The composition of the culture medium in the fermentation tank is: 10 g glucose, 10 g yeast extract, 10 g calcium carbonate, 1000 mL distilled water, pH 7.5, and sterilized at 121°C for 20 min.

[0057] In step (8), the Luzhou-flavor Daqu is prepared by mixing the medium-high temperature Daqu and the high temperature Daqu in a ratio of 2:1.

[0058] In step (9), the solid-liquid ratio of the microbial liquid and the sorghum mixture is 5 mL / kg.

[0059] Example 2

[0060] A composite bacterial liquid, comprising Aspergillus Fischer ( Aspergillus fischeri )、Acinetobacter beijerinckii( Acinetobacter beijerinckii ) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae ).

[0061] The strain number of the Aspergillus fisheri is CGMCC No.3.15369, and the original preservation time is September 3, 2013; the strain number of the Acinetobacter beijerinckii is CGMCC No.1.10392, and the original preservation time is March 23, 2010; the strain number of the Saccharomyces cerevisiae is CGMCC No.2.3871, and the original preservation time is June 5, 2008.

[0062] The Aspergillus Fischerii, Acinetobacter beijerinii and Saccharomyces cerevisiae selected in this example were all purchased from the General Microbiological Center of China National Microbiological Culture Collection Administration and can be purchased through public catalog inquiries without repeated biological deposits.

[0063] The volume ratio of Aspergillus Fischl, Acinetobacter beijerinckii and Saccharomyces cerevisiae is 1:1:1.

[0064] The method for brewing Luzhou-flavor liquor using the composite bacterial liquid of this embodiment comprises the following steps:

[0065] (1) Strain activation: Use an inoculation loop to pick up a loop of Aspergillus Fischerii, Acinetobacter beijerinii, and Saccharomyces cerevisiae, respectively, and inoculate it into beef extract peptone slant culture medium, and culture it in a constant temperature incubator at 25-30°C for 22 hours to obtain three activated strains;

[0066] (2) Seed solution preparation: Pick one ring of activated strains of Aspergillus Fischerii, Acinetobacter beijerinckii, and Saccharomyces cerevisiae, respectively, and inoculate them into 100 mL of beef extract peptone liquid culture medium, and culture them in a shaking incubator at 25-30° C. and 200 rpm for 24 h to obtain seed solutions of the three bacteria;

[0067] (3) Fermentation culture: The seed liquids of Aspergillus Fischerii, Acinetobacter beijerinii and Saccharomyces cerevisiae were inoculated into a fermentation tank at an inoculum rate of 5% respectively, and fermentation was carried out; the culture temperature was 25-30° C., the culture time was 65 hours, and the fermentation was completed to obtain three bacterial liquids;

[0068] (4) Collection of bacterial cells: The three bacterial liquids were mixed at a volume ratio of 1:1:1 to obtain a mixed bacterial liquid, and the mixed bacterial liquid was inoculated into a fermentation tank at an inoculum amount of 8%, and fermented at a temperature of 25-30 degrees for 55 hours to obtain a mixed fermentation liquid, which was centrifuged, the supernatant was discarded, and the bacterial cells after centrifugation were collected;

[0069] (5) Preparation of inorganic nutrient solution: MgSO 4 , L-phenylalanine, tartaric acid, KH 2 PO 4 , water in a mass ratio of 3:1:2:1:10000, stir evenly, fully dissolve, stand and cool, and obtain an inorganic nutrient solution;

[0070] (6) Preparation of composite bacterial solution: Add the bacterial cells obtained in step (4) to the inorganic nutrient solution obtained in step (5) to obtain an effective viable bacterial count of (1-3)×10 8 cfu / mL of composite bacterial solution;

[0071] (7) Raw material cooking: Soak the sorghum in water at 70-75°C for 24 hours, first cook it at 98°C for 2 hours, then stew it at 75°C for 8 hours, drain the stewing water, and then spread the steamed sorghum to cool to 35°C;

[0072] (8) Adding the koji: adding crushed Luzhou-flavor Daqu that has passed through a 30-mesh sieve to the cooled sorghum in an amount of 20% of the weight of the sorghum before steaming, and mixing evenly to obtain a koji-sorghum mixture;

[0073] (9) Fermentation: Spray the microbial liquid evenly on the sorghum mixture obtained in step (8), stir and mix evenly, and ferment at 28° C.-35° C. for 34 days to obtain fermented grains;

[0074] (10) Distilling the liquor: Distill the fermented mash in a distillation steamer to obtain Luzhou-flavor liquor.

[0075] The composition of the culture medium in the fermentation tank in step (3) is: 10 g glucose, 10 g yeast extract, 10 g calcium carbonate, 1000 mL distilled water, pH 7.5, and sterilized at 121°C for 20 min.

[0076] In step (8), the Luzhou-flavor Daqu is prepared by mixing the medium-high-temperature Daqu and the high-temperature Daqu in a ratio of 2.5:1.

[0077] In step (9), the solid-to-liquid ratio of the microbial liquid and the sorghum mixture is 7 mL / kg.

[0078] Example 3

[0079] A composite bacterial liquid, comprising Aspergillus Fischer ( Aspergillus fischeri )、Acinetobacter beijerinckii( Acinetobacter beijerinckii ) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae ).

[0080] The strain number of the Aspergillus fisheri is CGMCC No.3.15369, and the original preservation time is September 3, 2013; the strain number of the Acinetobacter beijerinckii is CGMCC No.1.10392, and the original preservation time is March 23, 2010; the strain number of the Saccharomyces cerevisiae is CGMCC No.2.3871, and the original preservation time is June 5, 2008.

[0081] The Aspergillus Fischerii, Acinetobacter beijerinii and Saccharomyces cerevisiae selected in this example were all purchased from the General Microbiological Center of China National Microbiological Culture Collection Administration and can be purchased through public catalog inquiries without repeated biological deposits.

[0082] The volume ratio of Aspergillus Fischl, Acinetobacter beijerinckii and Saccharomyces cerevisiae is 1:1:1.

[0083] The method for brewing Luzhou-flavor liquor using the composite bacterial liquid of this embodiment comprises the following steps:

[0084] (1) Strain activation: Use an inoculation loop to pick up a loop of Aspergillus Fischerii, Acinetobacter beijerinckii, and Saccharomyces cerevisiae, respectively, and inoculate it into beef extract peptone slant culture medium, and culture it in a constant temperature incubator at 25-30°C for 24 hours to obtain three activated strains;

[0085] (2) Seed solution preparation: Pick one ring of activated strains of Aspergillus Fischerii, Acinetobacter beijerinckii, and Saccharomyces cerevisiae, respectively, and inoculate them into 100 mL of beef extract peptone liquid culture medium, and culture them in a shaking incubator at 25-30° C. and 200 rpm for 24 h to obtain seed solutions of the three bacteria;

[0086] (3) Fermentation culture: The seed liquids of Aspergillus Fischerii, Acinetobacter beijerinii and Saccharomyces cerevisiae were inoculated into a fermentation tank at an inoculum rate of 5% respectively, and fermentation was carried out; the culture temperature was 25-30° C., the culture time was 72 hours, and the fermentation was completed to obtain three bacterial liquids;

[0087] (4) Collection of bacterial cells: The three bacterial liquids were mixed at a volume ratio of 1:1:1 to obtain a mixed bacterial liquid, and the mixed bacterial liquid was inoculated into a fermentation tank at an inoculum amount of 8%, and fermented at a temperature of 25-30 degrees for 48 hours to obtain a mixed fermentation liquid, which was centrifuged, the supernatant was discarded, and the bacterial cells after centrifugation were collected;

[0088] (5) Preparation of inorganic nutrient solution: MgSO 4 , L-phenylalanine, tartaric acid, KH 2 PO 4 , water in a mass ratio of 4:2:2:2:10000, stir evenly, fully dissolve, stand and cool, to obtain an inorganic nutrient solution;

[0089] (6) Preparation of composite bacterial solution: Add the bacterial cells obtained in step (4) to the inorganic nutrient solution obtained in step (5) to obtain an effective viable bacterial count of (1-3)×10 8 cfu / mL of composite bacterial solution;

[0090] (7) Raw material cooking: Soak the sorghum in water at 70-75°C for 24 hours, first cook it at 98°C for 2 hours, then stew it at 75°C for 8 hours, drain the stewing water, and then spread the steamed sorghum to cool to 35°C;

[0091] (8) Adding the koji: adding crushed Luzhou-flavor Daqu that has passed through a 30-mesh sieve to the cooled sorghum in an amount of 20% of the weight of the sorghum before steaming, and mixing evenly to obtain a koji-sorghum mixture;

[0092] (9) Fermentation: Spray the microbial liquid evenly on the sorghum mixture obtained in step (8), stir and mix evenly, and ferment at 28° C.-35° C. for 32-36 days to obtain fermented grains;

[0093] (10) Distilling the liquor: Distill the fermented mash in a distillation steamer to obtain Luzhou-flavor liquor.

[0094] The composition of the culture medium in the fermentation tank in step (3) is: 10 g glucose, 10 g yeast extract, 10 g calcium carbonate, 1000 mL distilled water, pH 7.5, and sterilized at 121°C for 20 min.

[0095] In step (8), the Luzhou-flavor Daqu is prepared by mixing the medium-high temperature Daqu and the high temperature Daqu in a ratio of 3:1.

[0096] In step (9), the solid-to-liquid ratio of the microbial liquid and the sorghum mixture is 10 mL / kg.

[0097] Comparative Examples 1-9

[0098] The microbial composition was changed for further comparison, that is, in step (4) of the method for brewing Luzhou-flavor liquor with a composite bacterial liquid:

[0099] Bacteria collection: The three bacterial solutions were mixed in a ratio different from that in Example 1 (as shown in Table 1) to obtain a mixed bacterial solution, and the mixed bacterial solution was inoculated into a fermenter at an inoculation rate of 8%, and fermented at a temperature of 25-30 degrees for 48 hours to obtain a mixed fermentation solution, which was centrifuged, the supernatant was discarded, and the bacteria after centrifugation were collected.

[0100] The remaining raw materials and process steps are the same as in Example 1.

[0101] The composition of the microorganisms in the comparative example is shown in Table 1:

[0102] Table 1 Volume ratio of three bacterial solutions

[0103]

[0104] Experimental testing

[0105] Glycoamylase and protease are important enzymes for hydrolyzing grain raw materials. Glycoamylase mainly plays a saccharification role, breaking down starch into monosaccharides that can be used by other microorganisms during the fermentation of liquor. Protease can break down protein into amino acids and alcohols. The presence of these decomposing enzymes can improve the utilization rate of raw materials, provide a material basis for ethanol fermentation and the generation of aroma substances in the liquor brewing process, and have an important impact on the yield and aroma of liquor.

[0106] Enzyme production capacity test of three strains:

[0107] Use an inoculation loop to pick up a loop of Aspergillus Fischerii, Acinetobacter beijerinckii and Saccharomyces cerevisiae and inoculate them into beef extract peptone slant culture medium respectively, place it in a constant temperature incubator at 25-30°C for 24 hours, and obtain three activated strains.

[0108] Glucoamylase:

[0109] Select activated strains respectively, inoculate them on saccharifying enzyme culture medium, and make three parallel cultures, culture at 25-30℃ for 48h, add appropriate amount of iodine solution to the culture dish for staining, let it stand, and use the cross method to determine the diameter of the transparent circle (D) and the diameter of the colony (d), and calculate the hydrolysis circle (HC) value (HC=D / d).

[0110] The composition of the saccharifying enzyme culture medium is: 12 g soluble starch, 8 g yeast extract, 5 g NaCl, 20 g agar, and 1 L water.

[0111] At the same time, use a sterile puncher to punch out several 6mm diameter bacterial cakes at the edge of the colony and inoculate them into the test culture medium. Culture them at 25-30℃ for 72h, and determine the saccharification capacity according to DB34 / T3085-2018 "General Analysis Method for Brewing Daqu".

[0112] The composition of the test medium is: 10 g fresh bran, 8 mL water, sterilized at 121°C for 40 min.

[0113] Table 2 Test results of the ability of strains to produce saccharifying enzymes

[0114]

[0115] Protease:

[0116] Select the activated strains and inoculate them on the protease screening medium for three replicates. Culture them at 25-30℃ for 24h, measure the diameter of the transparent zone and colony, and calculate the hydrolysis zone value. After the strains are activated, configure them to have an effective viable count of 1×10 6 / ml, and inoculated into the re-screening culture medium, cultured at 25-30℃ for 72 h, and determined the acidic protease activity according to DB34 / T3085-2018 "General Analysis Method for Liquor Making".

[0117] Table 3 Test results of protease production ability of strains

[0118]

[0119] From the data in Table 2-3, we can see that the three strains screened in the present invention all have good enzyme production capabilities.

[0120] Strain tolerance test:

[0121] Tolerance of strains to alcohol: Activate the target strain, inoculate 4% seed liquid into liquid LB medium with alcohol content of 1, 2, 3, 4, 5, 6, 7, 8% vol, culture at 25-30 ℃, 180r / min shaking for 24h, and measure OD 600 nm, three parallel experiments were performed in each group, and the average value was taken.

[0122] Temperature tolerance of strains: Activate the target strains, inoculate 4% of the seed solution into liquid LB medium at 30, 35, 40, 45, 50, 55, 60, and 65°C, shake and culture at 180 r / min for 24 h, and measure the OD 600 nm value, each group was tested three times in parallel and the average value was taken.

[0123] Test results such as 图3-8 shown.

[0124] Liquor index determination:

[0125] According to the methods of Examples 1-3 and Comparative Examples 1-9 of the present invention, liquor brewing was performed, and the original liquor yield and volatile aroma components were detected. Calculation of liquor yield: The alcohol content was measured with an alcohol meter, and the weight of the newly distilled original liquor was converted into 65% vol original liquor weight. The ratio of the liquor weight to the feed was the liquor yield.

[0126] Gas chromatography was used to detect the aroma components in the original wine.

[0127] Sensory evaluation of liquor: According to the style characteristics of Luzhou-flavor liquor, the original liquor samples in the study were sensory evaluated from four aspects: aroma, taste, mouthfeel and style in accordance with the provisions of GB / T33404-2016 "Guidelines for Sensory Evaluation of Liquor".

[0128] Table 4 Alcohol content and yield of the original liquor produced by the embodiment group and the comparative example group.

[0129]

[0130] Table 5 Quality and evaluation of wines in the examples and comparative examples

[0131]

[0132] As can be seen from the data in Tables 4-5, the quality of the wine obtained in the embodiment of the present invention is good. Among them, ethyl hexanoate is a landmark substance for measuring the quality of the rich-flavor original wine. The higher its content, the better the quality of the original wine. Too high ethyl acetate content will cause the original wine to have an improper aroma and poor typicality, which seriously affects the quality of the original wine. The alcohol substances in white wine are generally generated by amino acid deamination or sugar decarboxylation reactions, mainly higher alcohols. An appropriate amount of higher alcohols can increase the thickness of the wine and increase the coordination of the wine, which mainly affects the style of the white wine and the comfort of drinking. The organic acid in the white wine is an important aroma and flavor substance, and an appropriate amount of organic acid can make the wine more coordinated. The ester, alcohol, and acid content of the embodiment of the present invention are all higher than those of the comparative example, thereby bringing a rich aroma and a soft taste of the wine. This is because three functional microorganisms are added to ferment together, and the three work synergistically. The content of saccharifying enzymes and proteases in the fermented grains is high and active, and the efficiency of decomposing starch and protein to generate reducing sugars and amino acids is higher, and the fermentation is more sufficient. In comparative example 1-9 where the composition of the strains was changed, the synergistic effect among the three strains was significantly weakened, thus resulting in a weakened fermentation effect, and the quality of the wine was inferior to that of the embodiment.

[0133] It should be noted that the above embodiments are only partial embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Claims

1. A composite bacterial liquid, characterized in that: The composite bacterial liquid includes Aspergillus Fischer ( Aspergillus fishery )、Acinetobacter beijerinckii( Acinetobacter beijerinckii ) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae The strain number of Aspergillus fisheri is CGMCC No.3.15369, the strain number of Acinetobacter beijerinckii is CGMCC No.1.10392, and the strain number of Saccharomyces cerevisiae is CGMCC No.2.3871. All three strains were purchased from the General Microbiology Center of China National Microbiological Culture Collection Administration; the volume ratio of Aspergillus fisheri, Acinetobacter beijerinckii and Saccharomyces cerevisiae is 1:1:

1.

2. A method for brewing Luzhou-flavor liquor using the composite bacterial liquid of any one of claim 1, characterized in that: The following steps are involved: (1) Strain activation: Use an inoculation loop to pick up a loop of Aspergillus Fischerii, Acinetobacter beijerinii, and Saccharomyces cerevisiae, respectively, and inoculate it into beef extract peptone slant culture medium, and place it in a constant temperature incubator at 25-30°C for 20-24 hours to obtain three activated strains; (2) Seed solution preparation: Pick one ring of activated strains of Aspergillus Fischerii, Acinetobacter beijerinii and Saccharomyces cerevisiae, respectively, and inoculate them into 100 mL of beef extract peptone liquid culture medium, and culture them in a shaking incubator at 25-30° C. and 180-200 r / min for 24 h to obtain seed solutions of the three bacteria; (3) Fermentation culture: Inoculate the seed liquid of Aspergillus Fischerii, Acinetobacter beijerinii and Saccharomyces cerevisiae into a fermentation tank at an inoculum rate of 5% respectively, and ferment; culture temperature is 25-30° C., culture for 60-72 hours, and fermentation is completed to obtain three kinds of bacterial liquid; (4) Bacteria collection: The three bacterial solutions were mixed at a volume ratio of 1:1:1 to obtain a mixed bacterial solution, and the mixed bacterial solution was inoculated into a fermentation tank at an inoculum amount of 8%, and fermented at a temperature of 25-30° C. for 48-72 hours to obtain a mixed fermentation solution, which was centrifuged, the supernatant was discarded, and the bacteria after centrifugation were collected; (5) Preparation of inorganic nutrient solution: MgSO4, L-phenylalanine, tartaric acid, KH2PO4, and water are mixed in a mass ratio of (2-4):(1-2):(1-2):(1-2):10000, stirred evenly, fully dissolved, and allowed to stand and cool to obtain an inorganic nutrient solution; (6) Preparation of composite bacterial solution: Add the bacterial cells obtained in step (4) to the inorganic nutrient solution obtained in step (5) to obtain an effective viable bacterial count of (1-3)×10 8 cfu / mL of composite bacterial solution; (7) Steaming of raw materials: Soak the sorghum in water at 70-75°C for 24 hours, steam it at 98°C for 2 hours, and then steam it at 75°C for 8 hours. Drain the steaming water and cool the steamed sorghum to 35°C. (8) Adding the koji: adding crushed Luzhou-flavor Daqu that has passed through a 30-mesh sieve to the cooled sorghum in an amount of 20% of the weight of the sorghum before steaming, and mixing evenly to obtain a koji-sorghum mixture; (9) Fermentation: spraying the composite bacterial solution evenly on the sorghum mixture obtained in step (8), stirring and mixing evenly, and fermenting at 28° C.-35° C. for 32-36 days to obtain fermented grains; (10) Distilling the liquor: The fermented mash is distilled in a distillation steamer to obtain Luzhou-flavor liquor.

3. The method for brewing Luzhou-flavor liquor using the composite bacterial liquid according to claim 2, characterized in that: Step (3) The composition of the culture medium in the fermentation tank is: 10 g glucose, 10 g yeast extract, 10 g calcium carbonate, 1000 mL distilled water, pH 7.5, and sterilized at 121°C for 20 min.

4. The method for brewing Luzhou-flavor liquor using the composite bacterial liquid according to claim 2, characterized in that: In step (8), the Luzhou-flavor Daqu is prepared by mixing the medium-high temperature Daqu and the high temperature Daqu in a ratio of (2-3):

1.

5. The method for brewing Luzhou-flavor liquor with the composite bacterial liquid according to claim 2, characterized in that: In step (9), the solid-to-liquid ratio of the microbial liquid to the sorghum mixture is 5-10 mL / kg.

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