Method for improving quality and liquor yield of bran-free fen-flavor liquor and application of bran-free fen-flavor liquor

Abstract

The invention discloses a method for improving the quality and yield of bran-free fen-flavor liquor and application of the method, two kinds of liquor zymophyte obtained through separation are prepared into a complex microbial inoculant, and therefore the yield of the bran-free fen-flavor liquor is effectively improved. Meanwhile, the combined complex microbial inoculant effectively improves the taste of the bran-free fermented fen-flavor liquor, reduces the pungency, increases the content of flavor substances, improves the drinking safety, and provides a new thought for brewing the bran-free fermented fen-flavor liquor.

Description

Technical Field

[0001] The invention relates to the technical field of winemaking technology, and in particular to a method for improving the quality and yield of bran-free light-fragrant liquor and an application thereof. Background Art

[0002] Chinese liquor has a long history. It not only plays a vital role in China's economic and social development, but is also an important part of China's food industry. According to the flavor characteristics of liquor, it can be divided into twelve major flavors, among which strong-flavored, sauce-flavored and light-flavored types account for the largest proportion. Based on the technological characteristics of light-flavor liquor, it can be further divided into three types: Daqu light-flavored, Xiaoqu light-flavored and Fuqu light-flavored. Among them, Daqu light-flavored liquor and Xiaoqu light-flavored liquor are the most famous in the Chinese liquor industry. The former is represented by Fenjiu, which is popular among consumers for its aromatic, mellow, soft and elegant, refreshing and long-lasting flavor; the latter is produced in Yunnan, Guizhou and Sichuan, with elegant wine quality, few side effects of drinking, low price and wide market.

[0003] In the brewing process of traditional light-fragrance liquor, some fibrous auxiliary materials (such as rice husks, rice husks, sorghum residues, etc.) are added. According to previous studies, rice husks, as an auxiliary material commonly used in liquor production, play a role in reducing the adhesion of raw materials, avoiding steam collapse, and keeping the grain dregs cooked but not greasy when steaming wine and grains. This provides excellent attachment conditions and micro-oxygen environment for the growth and reproduction of microorganisms in the liquor brewing process, and also effectively regulates the starch content, acidity, etc., thereby further promoting microorganisms to better metabolize nutrients in the raw materials. However, the study also found that while providing these conveniences, rice husks also release a furan aldehyde inhibitor (including furfural and methanol), which are common volatile flavor compounds in Xiaoqu liquor flavor substances, which are not conducive to the formation of liquor flavor quality. Among them, furfural can cause the accumulation of yeast reactive oxygen species (ROS) and damage its mitochondrial and vacuole membranes, nuclear chromatin and actin cytoskeleton, thereby seriously affecting the cell activity of yeast and reducing the production of key esters and alcohols in the wine. In addition, furfural itself also has fermentation inhibitory toxicity to yeast. Moreover, yeast will reduce furfural to derivatives such as furan methanol through NAD(P)H-dependent reactions, and this substance is also the main source of bitterness in the wine, so it will also bring abnormal bran taste and bitter taste to the wine. In a sensory study of Xiaoqu wine, it was found that the content of furfural in distilled wine was 2.9-10 mg / L, while the astringency threshold of furfural was relatively low, at 5.201 mg / L, so the wine was prone to astringency. Moreover, furfural and methanol themselves are substances that are harmful to human health, and their production and content should be reduced as much as possible.

[0004] The bran-free fermentation process disclosed in the prior art can effectively reduce the furfural content, but at the cost of losing the support of wood fiber in the fermentation raw materials, which in turn will lead to the destruction of the micro-oxygen environment in the cellar, and environmental factors such as acidity and starch cannot be better regulated, thereby reducing the yield of wine, reducing the diversity of volatile compounds, and significantly reducing the total concentration. Therefore, there is an urgent need for a bran-free fragrant liquor production method that can effectively reduce the furfural content while ensuring the yield of wine and flavor substances. Summary of the invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the purpose of the present invention is to provide a method and application thereof for improving the quality and yield of bran-free light-fragrance liquor. The present invention screened out two strains of liquor fermentation bacteria, and found that the combined use of the two can effectively improve the quality and yield of bran-free light-fragrance liquor, thereby having extremely high economic value and practical value.

[0006] The first aspect of the present invention provides a composite bacterial agent, which comprises Cyberlindnera fabianii and Saccharomyces cerevisiae.

[0007] In the present invention, the above-mentioned Fabianseberlindner yeast and Saccharomyces cerevisiae were both deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on June 19, 2024, with the deposit numbers CGMCC NO.31011 and CGMCC NO.31012, respectively.

[0008] In some embodiments of the present invention, the Fabianseberlindner Saccharomyces cerevisiae and Saccharomyces cerevisiae include living bacteria, cultures, metabolites, extracts or lysates of Fabianseberlindner Saccharomyces cerevisiae and Saccharomyces cerevisiae.

[0009] In the present invention, the term "culture" refers to a liquid or solid culture medium that forms a microbial population under appropriate conditions after inoculation and cultivation. In the present invention, the culture can be a pure culture, that is, a culture medium containing only one microbial population; or it can be a non-pure culture, such as a culture medium that can simultaneously contain other microbial populations, but the presence of other microbial populations does not have an adverse effect on the growth, reproduction, and other activities of the target microorganism.

[0010] In the present invention, the term "metabolite" refers to various metabolites produced by microorganisms during metabolism, including metabolites produced based on anabolism or catabolism of microorganisms.

[0011] In the present invention, the term "extract" refers to a pure substance or mixture obtained by extracting a microorganism or its culture or metabolites using conventional extraction methods in the art.

[0012] In the present invention, the term "lysis product" refers to a substance produced after a microorganism is decomposed or lysed under specific conditions. These substances generally include components such as cell walls, cell membranes, proteins, nucleic acids, etc. of the microorganism. The lysis product can be obtained by physical, chemical or biological methods, such as treating the microorganism with high pressure or ultrasound or using specific enzymes for enzymatic hydrolysis.

[0013] In some embodiments of the present invention, in the composite bacterial agent, the inoculation ratio of Fabianseberlindner yeast and Saccharomyces cerevisiae is 0.5-1:0.5-1.

[0014] In some embodiments of the present invention, in the composite bacterial agent, the inoculation ratio of Fabianseber Lindner yeast and Saccharomyces cerevisiae is 1-1.5:1.

[0015] In some embodiments of the present invention, in the composite bacterial agent, the inoculation ratio of Fabianseberlindnerella yeast and Saccharomyces cerevisiae is 1:1 or 1.1:0.9.

[0016] In some embodiments of the present invention, the activity of Fabianseberlindnerella yeast and Saccharomyces cerevisiae in the composite bacterial agent is 10 5 -10 9 CFU / mL.

[0017] In some embodiments of the present invention, the activity of Fabianseberlindnerella yeast and Saccharomyces cerevisiae in the composite bacterial agent is 10 7 CFU / mL.

[0018] The second aspect of the present invention provides a fermentation agent, which includes the composite bacterial agent described in the above aspect and at least one auxiliary agent.

[0019] In some embodiments of the present invention, the adjuvant includes a lyoprotectant, an inert substance, a humectant, a dispersant, a filler and a carrier.

[0020] The third aspect of the present invention provides use of the composite bacterial agent described in the above aspect or the leavening agent described in the above aspect in food fermentation.

[0021] In some embodiments of the invention, the food comprises wine.

[0022] In some embodiments of the present invention, the wine comprises white wine.

[0023] In some embodiments of the present invention, the liquor is a light-fragrance liquor.

[0024] In some embodiments of the present invention, the liquor is a bran-free and light-fragrance liquor.

[0025] A fourth aspect of the present invention provides a winemaking method, comprising:

[0026] The saccharified grain is fermented using the composite bacterial agent described in the above aspects or the fermentation agent described in the above aspects.

[0027] In some embodiments of the present invention, the mass ratio of the composite bacterial agent or fermentation agent to the saccharified grain is 3-5:95-100.

[0028] In some embodiments of the present invention, the mass ratio of the composite bacterial agent or fermentation agent to the saccharified grain is 1-1.2:20-25.

[0029] In some embodiments of the invention, the fermentation is a bran-free fermentation.

[0030] In the present invention, the term "bran-free fermentation" refers to a fermentation technique that does not use bran husks.

[0031] In some embodiments of the present invention, the food grain comprises sorghum, corn or wheat.

[0032] In some embodiments of the present invention, the winemaking method further comprises: steaming cooked grains, mixing koji, and saccharifying before fermentation; and distilling after fermentation.

[0033] In some embodiments of the present invention, steaming cooked grains, mixing with koji, saccharification and distillation can be performed according to conventional methods in the art, and the present invention is not limited thereto.

[0034] A fifth aspect of the present invention provides a method for improving the quality and yield of bran-free light-fragrant liquor, comprising:

[0035] The saccharified grain is fermented using the composite bacterial agent described in the above aspects or the fermentation agent described in the above aspects.

[0036] In some embodiments of the present invention, the mass ratio of the composite bacterial agent or fermentation agent to the saccharified grain is 3-5:95-100.

[0037] In some embodiments of the present invention, the mass ratio of the composite bacterial agent or fermentation agent to the saccharified grain is 1-1.2:20-25.

[0038] In some embodiments of the present invention, the food grain comprises sorghum, corn or wheat.

[0039] In some embodiments of the present invention, the quality includes: drinking safety, flavor substance content, and volatile compound content.

[0040] In some embodiments of the present invention, the method can improve the taste (reduce the spiciness), drinking safety, increase the content of flavor substances, and adjust the content of volatile compounds (increase the content of beneficial substances and reduce the content of unfavorable substances).

[0041] In some embodiments of the present invention, the flavor substances include ester compounds and ketone compounds.

[0042] In some embodiments of the present invention, the flavoring substance comprises ethyl acetate.

[0043] In some embodiments of the present invention, the method reduces the content of aldehyde compounds in wine and increases the content of alcohol and acid compounds.

[0044] The sixth aspect of the present invention provides the application of the method described in the above aspects in liquor brewing.

[0045] The seventh aspect of the present invention provides a white wine fermentation bacterium, which is Cyberlindnera fabianii. The Cyberlindnera fabianii was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration (CGMCC) on June 19, 2024, with a deposit number of CGMCCNO.31011.

[0046] The eighth aspect of the present invention provides a white wine fermentation bacterium, which is Saccharomyces cerevisiae. The Saccharomyces cerevisiae was deposited in the China General Microbiology Center (CGMCC) on June 19, 2024, with a deposit number of CGMCC NO.31012.

[0047] The ninth aspect of the present invention provides the use of the liquor fermentation bacteria described in the above aspects in liquor brewing.

[0048] The beneficial effects of the present invention are:

[0049] The present invention isolates two strains of liquor fermentation bacteria, and finds that when used alone or in combination, they can effectively improve the quality and yield of bran-free light-flavor liquor. Among them, the combined composite bacterial agent can achieve a liquor yield of 52% to 55%. At the same time, the inventors also found that the combined composite bacterial agent effectively improves the taste of bran-free fermented light-flavor liquor, reduces the spiciness, and also increases the content of flavor substances, improves drinking safety, and provides a new idea for the brewing of bran-free fermented light-flavor liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The color development results of TTC experiments for different strains.

[0051] Figure 2 Shows the colonies and microscopic morphology of representative yeast strains.

[0052] Figure 3 The colony morphology of strains A1 and 7 is combined with microscopic images.

[0053] Figure 4 Phylogenetic tree of representative yeast strains based on 16S rRNA sequences.

[0054] Figure 5 It is a bar graph of ethanol production and total ester content of 10 yeast strains.

[0055] Figure 6 is the content of ethanol and total esters in the mixed fermentation broth with different inoculation ratios (g / L).

[0056] Figure 7 This is a comparison chart of the yield of bran-free, light-fragrant liquor obtained by using a composite bacterial agent, a single strain, and the traditional method of bran-free fermentation.

[0057] Figure 8 This is a sensory comparison chart of the bran-free and fragrant liquor obtained by using a composite bacterial agent, a single strain, and the traditional method of bran-free fermentation.

[0058] Fig. 9 This is a comparison chart of the changes in the concentration of volatile compounds in the mash during the bran-free fermentation process using a composite bacterial agent, a single strain, and the traditional method.

[0059] Fig.10 This is a comparison chart of the test results of the main flavor substances concentration of the bran-free and light-fragrant liquor obtained by using the composite bacterial agent and the traditional method of bran-free fermentation.

[0060] Fig.11 This is a comparison chart of the changes in the concentration of volatile compounds in the mash during the bran-free fermentation process using a composite bacterial agent and the traditional method.

[0061] Fig.12 This is the collinear network diagram of the microbial community of the mash after bran-free fermentation using a composite bacterial agent and the traditional method. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0063] Example 1 Breeding and functional determination of yeast

[0064] In the present embodiment, a famous Luzhou-flavor liquor brewing factory in Sichuan Province collected Luzhou-flavor liquor lees and cellar mud as important sources of yeast screening, and two microorganisms were selected and bred for subsequent brewing of bran-free light-flavor liquor. In order to ensure the uniformity of sampling, 5 points (the sample amount of lees and cellar mud at each point was 50g) were selected from different positions of three parallel cellars (10 years of cellar age) in the production workshop of the brewing factory for sampling. Take 3 parallel samples at each sampling point. Mix samples from all positions. The mixed samples were immediately transported back to the laboratory for microbial separation and purification or stored at 4°C on the day of collection.

[0065] (1) Isolation of yeast:

[0066] The specific steps of yeast separation and purification are as follows:

[0067] Accurately weigh 25g of the collected mash and pit mud samples and place them in 225mL of sterile physiological saline, shake at 30℃, 200r / min for 30min, and let it rest for 15min. After aspirating the supernatant at a 10% (v / v) inoculation rate, enrichment culture was carried out in a 250mL Erlenmeyer flask containing 200mL of yeast enrichment medium (45℃, 150r / min) until obvious mixing occurred to obtain yeast enrichment solution. Add 1mL of yeast enrichment solution to 9mL of sterile sodium chloride solution to prepare a dilution factor of 10. -1 Follow the same procedure to obtain 10 -2 -10 -5 0.1 mL of bacterial suspension was extracted from each of these gradient dilutions and evenly spread on the separation medium plate. The coated plate was placed in a 30°C constant temperature incubator for 48 hours. The cultured colonies were transferred to the YEPD solid culture dish and separated by streaking until they were completely purified.

[0068] Among them, the preparation method of yeast enrichment medium is: malt extract medium (purchased from Senpulai Technology Co., Ltd.) 130.1g / L, sodium propionate 10g / L, olive oil 1% are mixed with water according to the final concentration, after high-temperature sterilization, cooled to room temperature, chloramphenicol 12.5mg / L is added, and then 1% of 0.05g / L vitamin solution is added.

[0069] The preparation method of 0.05g / L vitamin solution is as follows: weigh 0.005g of each vitamin C, B1, B3, B5, B6, and B12, dissolve in 10mL of distilled water to obtain 0.5g / L vitamin solution, then take out 1mL and add it to 9mL of distilled water to obtain 0.05g / L vitamin solution. Filter with a 0.22μm filter membrane for sterilization before use.

[0070] The preparation method of yeast separation medium is as follows: 20g / L glucose, 20g / L peptone, 10g / L yeast extract powder, 10g / L sodium propionate, 2% agar are mixed with water according to the final concentration, and then sterilized at high temperature. When the medium is cooled to about 60°C, 0.01% chloramphenicol is added, and then 0.05g / L vitamin solution is added at a 1% amount.

[0071] The preparation method of YEPD solid culture medium is as follows: 20g / L glucose, 20g / L peptone, 10g / L yeast extract powder, 2% agar and water are mixed and sterilized at high temperature. The pH is 5.6.

[0072] Inoculate the colonies purified in the above steps on the TTC lower culture medium and culture them in a 30°C constant temperature incubator for 48 hours. After a single colony grows, slowly inject the upper culture solution cooled to 27°C into the culture dish in an amount that can completely cover the culture medium. Keep it away from light for 1 to 2 hours, then perform TCC color development to determine the colony's alcohol production capacity based on the degree of color development.

[0073] The TTC lower culture medium is prepared from 10.1 g of glucose, 2.0 g of peptone, 1.5 g of yeast extract, 1.0 g of acid potassium phosphate, 0.4 g of magnesium sulfate, 0.27 g of citric acid, 30.0 g of agar and 200 mL of water.

[0074] The TTC supernatant culture medium is prepared by triphenyltetrazolium chloride (TTC) 0.05 g, glucose 0.5 g, agar 1.5 g, and water 100 mL.

[0075] The color development results of different colonies obtained by screening are as follows Figure 1 shown.

[0076] It can be found that colony 7 has the darkest color and exhibits better metabolic activity, so colony 7 is preliminarily considered to be the yeast with the strongest ethanol production ability.

[0077] (2) Identification of yeast:

[0078] Based on the color development results, colonies 3-5, 7 and A1 were selected for morphological identification. The specific steps were as follows: the yeast of the corresponding colonies was activated and inoculated into YEPD solid culture medium, cultured at 28°C for 48h, and then the colony morphology was observed.

[0079] The results are as follows Figure 2 shown.

[0080] Among them, A1 and 7 were further observed for cell morphology. The specific steps were: put them under a microscope, adjust the eyepiece to 100×, drip pine tar oil on the pick, and observe the cell microscopic morphology.

[0081] The observations of A1 and 7 are as follows Figure 3 shown.

[0082] In order to further clarify the bacterial strain information, the inventors conducted molecular biological identification on the finally selected colonies A1 and 7.

[0083] The specific steps are:

[0084] After colonies A1 and 7 were inoculated on YEPD solid medium for activation, genomic DNA was extracted using a fungal genomic DNA extraction kit (purchased from Thermo Fisher Scientific Inc.) according to the instructions (PCR amplification primers, reaction system and amplification procedures are shown in the instructions). The PCR amplification products obtained were identified by 1% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequence determination. The basic local alignment search tool (BLAST) platform was used for sequencing. The obtained DNA sequences were compared with existing sequences in the GenBank database of the National Center for Biotechnology Information (NCBI) by BLAST. After BLAST homology alignment of the DNA sequences, the sequences with the highest homology were selected to identify the strains, and the phylogenetic tree of the ITS rRNA gene of the strains was constructed by the neighbor-joining method of MEGAX software, and the bootstrap value (Bootstraps) was set to 1000.

[0085] Phylogenetic trees of colonies A1 and 7 Figure 4 shown.

[0086] Combining the above morphological characteristics, physiological and biochemical, and molecular identification results, it can be determined that colony 7 is Cyberlindnera fabianii and A1 is Saccharomyces cerevisiae. The two isolated and purified strains were deposited at the General Microbiological Center of the China Microbiological Culture Collection Administration (deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​June 19, 2024, with the deposit numbers CGMCC NO.31011 (colony 7) and CGMCC NO.31012 (colony A1).

[0087] (3) Yeast fermentation effect test:

[0088] Determination of yeast ester production performance: Use sorghum and wheat hydrolyzate culture medium as yeast fermentation matrix, inoculate the tested yeast into it for single bacterial fermentation, the inoculation concentration of each yeast strain is 2.5% (v / v), the culture temperature is set to 28°C, shake at 150r / min for 8 days, then accurately pipette 50mL of sample into a 250mL Erlenmeyer flask with a lid, add 2 drops of phenolphthalein, neutralize with 0.1mol / LNaOH standard solution, titrate until slightly reddish and does not fade for 30s (do not overdo), record the number of milliliters of sodium hydroxide standard titration solution consumed. Then accurately add 25mL of 0.1mol / LNaOH standard solution (if the total ester content of the sample is high, add 50mL), shake well, put in a few zeolites or glass beads, install a condenser (the cooling water temperature should be lower than 15°C), reflux on a boiling water bath for 30min, remove, and cool. Titrate with 0.1mol / L H2SO4 sulfuric acid standard titration solution until the red color just completely disappears as the endpoint, and record the volume V1 of the sulfuric acid standard titration solution consumed. At the same time, draw 50mL of ethanol (ester-free) solution (40%, volume fraction), and perform a blank test in the same way as above, and record the volume V0 of the sulfuric acid standard titration solution consumed.

[0089] in,

[0090] Among them, the total ester content in the sample is expressed as mass concentration (in terms of ethyl acetate) in grams per liter (g / L);

[0091] The actual molar concentration of the sulfuric acid standard titration solution, in moles per liter (mol / L);

[0092] The volume of sulfuric acid standard titration solution consumed by the blank test sample, in milliliters (mL);

[0093] The volume of sulfuric acid standard titration solution consumed by the sample, in milliliters (mL);

[0094] Where 88 is the molar mass of ethyl acetate, expressed in grams per mole (g / mol) [M(CH:COOC,H;) = 88];

[0095] 50.0 is the volume of the sample aspirated, in milliliters (mL).

[0096] The calculation result is expressed to two decimal places.

[0097] Among them, the preparation method of sorghum wheat hydrolyzate medium is: pulverize sorghum and wheat and mix them evenly (mass ratio of sorghum: wheat = 1:1), then add an appropriate amount of distilled water to make the ratio of the mixture to water 1:4, and then gelatinize in a constant temperature water bath at 90°C for 90 minutes. In order to maintain uniform heating, stir continuously during the gelatinization process. After the gelatinization is completed, α-amylase is added to the gelatinized product according to the amount of 80U / g sorghum-wheat mixture, and then liquefaction is continued in a constant temperature water bath at 90°C for 90 minutes. After cooling, lactic acid is added to make its pH = 4.5. Then, saccharifying enzyme is added to the sorghum-wheat mixture according to the amount of 150U / g, and saccharification is carried out at 60°C for 3-4h. Then the temperature of the constant temperature water bath is raised to 90°C (or boiling) and heated for 5 minutes to inactivate the enzyme. The filtrate is filtered through four layers of gauze to obtain the sorghum wheat hydrolyzate medium. The obtained sorghum-wheat hydrolyzate culture medium was dispensed into 500 mL conical flasks, 200 mL in each conical flask, and then sterilized at 121° C. for 20 min.

[0098] The results are as follows Figure 5 shown.

[0099] It can be found that colonies A1 and 7 have the strongest ester production ability.

[0100] Determination of yeast ethanol production ability: Sorghum wheat hydrolyzate medium was also used as the yeast fermentation matrix for single-bacteria fermentation. The inoculation concentration of each yeast strain was 2.5% (v / v), the culture temperature was set to 28°C, and the shaker was shaken at 150 r / min for 8 days. The ethanol concentration in the fermentation broth was then determined by gas chromatography.

[0101] The chromatographic conditions are as follows: a TG-5MS elastic quartz capillary column (30m×0.25mm×0.25mm) was used, with high-purity He as the carrier gas and a flow rate of 0.8mL / min to analyze the sample. In terms of programmed temperature rise, it was first set at 40°C for 5min, then increased from 5°C / min to 80°C, and then increased to 230°C at a rate of 10°C / min and maintained for 7min. In the vaporization chamber at 250°C, a non-divided flow mode was used. The mass spectrometry conditions are as follows: the interface temperature is 250°C, an electron bombardment ionization source with a temperature of 230°C and an energy of 70eV is used, the scanning range is 30-550amu, and the scanning speed is 0.2s / scan. C6-C30 normal alkane solutions were analyzed on the same chromatographic column, and the linear retention indices (LRIs) were calculated using the retention time. Before injection, the sample was pretreated as follows: the fermentation broth sample was centrifuged at 10000 r / min for 30 min, 9 mL of supernatant was extracted into a 10 mL volumetric flask, 0.10 mL of isopropanol was added, the volume was fixed and mixed, and the solution was filtered through a 0.22 μm needle filter membrane before use.

[0102] The results are as follows Figure 6 shown.

[0103] The results showed that the ethanol content in the fermentation broth of colony 7 was the highest, at 108.78 g / L, with a volume fraction (percentage) of 10.8%. Under the same fermentation conditions, the ethanol content in the fermentation broth of A1 accounted for 3.79% of the fermentation broth, which was lower than that of colony 7, but significantly higher than that of other colonies.

[0104] Therefore, considering the ethanol production and ester production effects, it can be considered that colonies 7 and A1 have good application prospects and can be used as fermentation strains for subsequent fermentations.

[0105] Example 2 Optimization of composite fermentation agent

[0106] In this example, S.cerevisiae A1 and C.fabianii 7 obtained in the above examples were mixed as a composite bacterial agent for fermentation, and the effect of the mixing ratio of the two on the fermentation was investigated.

[0107] The specific experimental steps are:

[0108] The sorghum and wheat hydrolyzate culture medium in the above embodiment was sterilized in a high pressure steam sterilizer, and then inoculated with different ratios of S.cerevisiae A1 and C.fabianii 7, and the inoculation ratios were 1:1, 1:5 and 1:10, respectively. The culture was carried out in a constant temperature shaking incubator at 28°C and 150r / min for 8 days. After the culture was completed, the total ester and ethanol content in each group of fermentation broth was determined according to the above method.

[0109] The results are as follows Figure 6 shown.

[0110] Depend on Figure 6 It can be seen that when the inoculation ratio of S.cerevisiae A1 and C.fabianii 7 was 1:1 for mixed fermentation, the total ester content produced was the richest, which was 46.59±0.96g / L. When the inoculation ratio was 1:10, the ethanol production capacity was the strongest (132.78±2.39g / L), and when the two yeasts were mixed and fermented at a ratio of 1:1, the ethanol production capacity was slightly weaker than that of the 1:10 group, but it could still reach an ethanol content of 128.15±5.42g / L. Therefore, the mixed fermentation ratio of 1:1 was selected as the optimal ratio.

[0111] Example 3 Actual wine production effect of the composite fermentation agent

[0112] (1) Preparation of bacterial stock solution:

[0113] The preparation method of the mother solution of S.cerevisiae A1 (A1 bacterial solution) is as follows: S.cerevisiae A1 is inoculated at a 10% inoculum into a 250 mL Erlenmeyer flask filled with 100 mL of yeast extract-peptone-dextrose (YEPD) liquid medium, and cultured at 30°C and 150 r / min for 36 h until the number of viable bacteria is controlled at about 10 7 CFU / mL, prepare a total of 60L for use.

[0114] The preparation method of the mother solution of C. fabianii 7 (7 bacterial solution) is as follows: C. fabianii 7 is inoculated into a 250 mL Erlenmeyer flask containing 100 mL yeast proliferation medium at a 10% inoculation rate, and cultured at 30°C and 150 r / min for 36 h until the number of viable bacteria is controlled at about 10 7 CFU / mL, prepare a total of 60L for use.

[0115] Preparation of composite fermentation agent: Take the mother liquor of S.cerevisiae A1 and the mother liquor of C.fabianii 7 respectively, collect the bacteria by centrifugation, add equal volumes of sterile physiological saline to resuspend them into bacterial suspensions, and then mix the two bacterial suspensions in a volume ratio of 1:1 to obtain a composite fermentation agent. The number of live bacteria of both bacteria in the composite fermentation agent is controlled at about 10 7 CFU / mL, prepare a total of 60L for use.

[0116] Among them, YEPD liquid culture medium: glucose 20g / L, peptone 20g / L, yeast extract powder 10g / L, and the rest is water.

[0117] (2) Bran-free fermentation:

[0118] A. Raw material processing: soak the shelled sorghum in water, then steam, suffocate and re-steam to obtain cooked grain;

[0119] B. Spread the cooked grains to cool and then sprinkle koji three times (once each when the raw material temperature cools to 56°C, 48°C and 40°C), and stir evenly after each spreading to obtain cooked grains with koji;

[0120] C. placing the cooked grain mixed with koji in a container for bacterial culture and saccharification to obtain saccharified cooked grain mixed with koji;

[0121] D. Fermentation in the pit: The volume of the pit is set to 3.6m×3.6m×1.4m. The A1 bacterial solution, 7 bacterial solution and composite fermentation bacterial agent are mixed with saccharified and koji-mixed cooked grains (spread to cool) respectively (the mass ratio of bacterial solution / agent to raw material is about 1-1.2:25-30). Take 15L and spray it evenly on the upper layer of the pit for anaerobic fermentation at room temperature. The fermentation cycle is 28 days.

[0122] E. After fermentation is completed, it is taken out of the cellar and distilled to obtain the bran-free and fragrant raw liquor.

[0123] At the same time, the bran-free light-fragrant original liquor produced by the traditional method (reference patent CN 116656443) was used as the control group (CK), and the fermentation period was 28 days.

[0124] The wine yield, various flavor components and sensory organs of each group were tested and compared.

[0125] The calculation formula for the wine yield is:

[0126] Volatile compounds are detected by gas chromatography-mass spectrometry equipment, the specific method is as follows:

[0127] 1 mL of liquor sample, 4 mL of distilled water and 1.5 g of sodium chloride were added to a 20 mL headspace bottle, and then 20 μL of 0.822 mg / L 2-octanol was added for gas chromatography mass spectrometry analysis. The chromatographic conditions were: TG-5MS elastic quartz capillary column (30 m × 0.25 mm × 0.25 mm), high purity He as carrier gas, flow rate of 0.8 mL / min. 40 ° C, 5 min; 5 ° C / min increased to 80 ° C; 10 ° C / min increased to 230 ° C, maintained for 7 min. In the 250 ° C vaporization chamber, non-divided mode was used. The mass spectrometry conditions were: interface temperature 250 ° C, using an electron bombardment ionization ion source with a temperature of 230 ° C and an energy of 70 eV, a scanning range of 30-550 amu, and a scanning speed of 0.2 s / scan. A solution of C6-C30 n-alkanes was analyzed on the same chromatographic column and the retention times were used to calculate the linear retention indices (LRIs) by comparing the mass spectra of the compounds with those of the standards.

[0128] The sensory analysis method is as follows: 10 assessors (4 males and 6 females, with an average age of 35 years) were selected for the analysis. All assessors received at least one year of nasal perception training and performed a positive nasal perception test, followed by a sensory evaluation. All tests were conducted in a sensory evaluation laboratory at 20±1℃. The sensory descriptive analysis technology for liquor flavor (GB / T12313) was used as the source of the qualitative and quantitative description analysis method for liquor flavor sensory, and the flavor characteristics of the product were qualitatively analyzed according to the liquor flavor wheel (GB / T 33405-2016). Among them, according to GB / T 33405-2016, a numerical scale (as shown in Table 1 below) was used to quantify the intensity or retention of the characteristics. Panelists were asked to rate the intensity of each attribute on a six-point scale of 0-5, with 0 indicating no odor, increasing in order, and 5 indicating very strong. Each sample (20mL) was then transferred to a glass tube and coded. The assessors provided the results after sniffing for 90 seconds, and each round of sniffing lasted about 30 minutes. This process was repeated three times for each sample. Between each evaluation step, a 10-min interval was maintained and the results obtained by the 10 evaluators were averaged.

[0129] Table 1 Sensory evaluation scale of liquor

[0130]

[0131] The results are as follows Figure 7 And as shown in Table 2.

[0132] Table 2 Physical and chemical test results of fermented grains of each group

[0133]

[0134] from Figure 7 and Figure 8 It can be found that the CK group has the lowest wine yield. Compared with the CK (42%) group, the wine yields of the mixed fermentation group (A1&7), A1 alone group and 7 alone group have improved, among which the A1&7 group has the highest wine yield, reaching 52% to 55%. In addition, through sensory evaluation, it can be found that the mixed fermentation group (A1&7), A1 alone group and 7 alone group all showed good sensory results, with high scores in fruity aroma, floral aroma and sweetness. Among them, the A1&7 group had a significant improvement in sweetness and floral aroma, and the spicy stimulation was reduced. The overall sensory score was significantly higher than that of single strain fermentation.

[0135] In addition, from Fig. 9It can be found that in the late fermentation period, the content of ester compounds and ketone compounds in the fermented grains in the A1&7 group was significantly increased compared with other groups, which may be one of the reasons for the stronger floral and fruity aroma. In addition, the content of aldehyde compounds in the A1&7 group was significantly reduced, which reduced its spicy and irritating feeling. It can be seen that the mixed bacteria fermentation in the present invention can better improve the quality of bran-free liquor.

[0136] It can be further observed from Table 2 that, compared with a single strain, the consumption of starch and reducing sugars by groups A1&7 was significantly higher, indicating that multi-strain fermentation is more conducive to improving the utilization of raw materials by microorganisms.

[0137] In order to further explore the effect of the composite bacterial agent on the fermentation of bran-free light-fragrance liquor, the inventors adjusted the mixing volume ratio of S.cerevisiae A1 and C.fabianii 7 in the composite fermentation bacterial agent from 1:1 to 1.1:0.9 based on the above-mentioned bran-free fermentation method.

[0138] Then, various flavor components were detected according to the above method, and the group without adding any bacterial agent was used as the control.

[0139] The results are as follows Fig.10 and Fig.11 shown.

[0140] It can be found that compared with the group without any bacterial agent added, the ester, alcohol, acid and ketone compounds in the original liquor distilled from A1&7 increased by 1349.43 mg / L, 146.02 mg / L, 35.75 mg / L and 5.65 mg / L respectively. Among them, the amount of ethyl acetate, which is a characteristic flavor substance of light-fragrance liquor, increased by 911.96 mg / L. This shows that the composite bacterial agent in the present invention can effectively increase the various volatile compounds in bran-free light-fragrance liquor under different ratios, thereby improving its quality. Further, Fig.11 It can be seen that the increase in the concentration of volatile compounds in the CK group mainly occurred on the 15th to 28th day of fermentation. The A1&7 group was able to significantly increase the content of volatile compounds starting from the 7th day of fermentation, which shows that the composite bacterial agent in the present invention can accelerate the production and accumulation of volatile compounds in the fermented grains. In addition, the inventors also found that more terpenoid compounds were detected during the fermentation process of the A1&7 group, and the increase in the content of terpenoid compounds reflects the improvement of the drinking safety of bran-free light-fragrant liquor.

[0141] The inventors further performed high-throughput sequencing on the microbial communities of the mash after fermentation in the experimental group and the control group, and further conducted co-occurrence network analysis.

[0142] The results are as follows Fig.12 shown.

[0143] It can be found that the composite bacterial agent in the present invention strengthens the connection between the bacterial and fungal communities in the mash, making the microbial community in the mash more stable, thereby reducing the quality differences between batches of liquor and being more conducive to the sustainability of brewing.

[0144] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A composite bacterial agent, characterized in that: The composite bacterial agent includes Cyberlindnerafabianii and Saccharomyces cerevisiae; Among them, the Fabianseberlindner yeast and Saccharomyces cerevisiae were both deposited in the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC) on June 19, 2024, with the deposit numbers CGMCC NO.31011 and CGMCC NO.31012 respectively; Preferably, the Fabianseberlindner yeast and Saccharomyces cerevisiae include live bacteria, cultures, metabolites, extracts or lysates of Fabianseberlindner yeast and Saccharomyces cerevisiae.

2. The composite bacterial agent according to claim 1, characterized in that In the composite bacterial agent, the inoculation ratio of Fabianseber Lindner yeast and Saccharomyces cerevisiae is 0.5-1:0.5-1.

3. A leavening agent, characterized in that The fermentation agent comprises the composite bacterial agent according to any one of claims 1 to 2, and at least one auxiliary agent; Preferably, the auxiliary agent includes a lyoprotectant, an inert substance, a humectant, a dispersant, a filler and a carrier.

4. Use of the composite bacterial agent according to any one of claims 1 to 2 or the starter according to claim 3 in food fermentation; Preferably, the food comprises wine.

5. A winemaking method comprising: Fermenting the saccharified grain using the composite bacterial agent according to any one of claims 1 to 2 or the fermentation agent according to claim 3; Preferably, the mass ratio of the composite bacterial agent or fermentation agent to the saccharified grain is 3-5:95-100; Preferably, the fermentation is a bran-free fermentation.

6. A method for improving the quality and yield of bran-free light-fragrant liquor, comprising: Fermenting the saccharified grain using the composite bacterial agent according to any one of claims 1 to 2 or the fermentation agent according to claim 3; Preferably, the mass ratio of the composite bacterial agent or fermentation agent to the saccharified grain is 3-5:95-100.

7. The method according to claim 6, characterized in that The qualities include: taste, drinking safety, flavor content, and volatile compound content.

8. Application of the method according to claim 6 or 7 in liquor brewing.

9. A liquor fermentation bacterium, characterized in that: The liquor fermentation bacteria is Cyberlindnerafabianii or Saccharomyces cerevisiae; Among them, the Fabianseberlindner yeast and brewer's yeast were both deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC) on June 19, 2024, with the deposit numbers CGMCC NO.31011 and CGMCCNO.31012 respectively.

10. Use of the liquor fermentation bacteria according to claim 9 in liquor brewing.

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