Method for improving liquor quality and antioxidant activity through semi-solid brewing of Gujing Luzhou-flavor Daqu using a collaborative bacterial strain combination

Abstract

The invention discloses a method for improving the quality and antioxidant activity of liquor by using a strain collaborative combination of Gujing Luzhou-flavor Daqu semi-solid brewing. The present invention utilizes the strain isolated from Daqu and uses sorghum as raw material for semi-solid brewing of liquor. The results show that a certain concentration of Escherichia coli, Bacillus subtilis and Bacillus velez form collaborative strains and Gujing Daqu semi-solid brewing of liquor with good quality and antioxidant activity. Compared with Daqu brewing liquor alone, the total ester content of the collaborative strain combination Gujing Luzhou-flavor Daqu brewing liquor is increased by 84.09-120.45%, the DPPH scavenging free radical scavenging rate is increased by 49.87-66.63%, and the content of guaiacols (4-vinyl guaiacol, guaiacol) is increased by 1176.55-2873.79%. The strain collaborative combination Gujing Daqu fermentation has the effect of improving the total ester content and antioxidant activity of liquor simultaneously.

Description

Technical Field

[0001] The present invention belongs to the field of liquor brewing, and specifically relates to a method for improving the quality and antioxidant activity of liquor by semi-solid brewing of Gujing Luzhou-flavor Daqu using a cooperative combination of bacterial strains. More specifically, it relates to a method for brewing high-quality antioxidant Luzhou-flavor liquor by semi-solid brewing of Luzhou-flavor Daqu using a combination of different bacterial strains separated from Daqu and fermented grains. Background Art

[0002] Free radicals are highly reactive intermediates produced during metabolism. They all share a common characteristic: they contain unpaired electrons, which can cause instability in reactive oxygen species (ROS). Under normal physiological conditions, moderate amounts of free radicals participate in intracellular signaling cascades, defending against pathogen invasion. However, excessive free radicals can not only be toxic but also cause cellular oxidative stress, increasing the risk of diabetes, cardiovascular disease, and cancer.

[0003] Baijiu (Chinese liquor) is a traditional fermented food in my country. 2,020 compounds have been detected in it, including 138 functionally active ingredients. These compounds include volatile compounds such as phenols, organic acids, esters, aldehydes, pyrazines, sulfides, terpenes, and furans, as well as non-volatile compounds such as amino acids, bioactive peptides, polyols, vitamins, and minerals. Guaifenesin compounds, such as guaiacol, 4-methylguaiacol, and 4-ethylguaiacol, exhibit strong antioxidant activity. 4-Methylguaiacol and 4-ethylguaiacol can enhance the expression of antioxidant enzymes by activating the Keap1-Nrf2 signaling pathway, alleviating or reversing intracellular oxidative stress induced by azobisisobutyramidine hydrochloride (AAPH) in a dose-dependent manner. Phenol, 4-methylphenol, 4-ethylphenol, and vanillin also possess excellent free radical scavenging abilities and are considered health-promoting components of baijiu.

[0004] Liquor brewing is an open, complex, and collaborative fermentation process of functional microbial communities. Microbial interactions are key to regulating the structure and function of microorganisms in the liquor microecosystem. Microbial interactions are mainly divided into competition, cooperation, symbiosis, and parasitism based on ecological types. They can be regulated by changing metabolic pathways, intercellular communication, and spatial structure to achieve specific functions. In the liquor brewing microecosystem, microbial interactions mainly involve synergistic (cooperative) and antagonistic (competitive) effects. At present, researchers have increased the abundance of hexanoic acid bacteria and methanogens and strengthened the hydrogen transfer interaction between the two bacteria in Daqu fermentation, thereby increasing the content of hexanoic acid and ethyl hexanoate in the Luzhou-flavor liquor microecosystem. In the liquor fermentation process, it is common to inhibit growth but promote flavor metabolic activity in the microbial co-culture system. For example, when Zygosaccharomyces Bailii and Bacillus licheniformis coexisted, the growth of Bacillus licheniformis was significantly inhibited, but the genes GAPDH, PGM1, ENO1, PDC1, COX1 and MEP2 involved in the glycolysis, Ehrlich and oxidative phosphorylation pathways of Zygosaccharomyces Bailii were upregulated, thereby producing more alcohols, acids, esters and aldehydes; the addition of Pichia membranifaciens and Bacillus amyloliquefaciens alleviated the competition between Saccharomyces cerevisiae, Issatchenkiaorientalis and Bacillus licheniformis, changed their growth rates, and promoted the production of baijiu aroma. Microbial interactions are conducive to the rational construction of synthetic bacterial communities, thereby directional regulation of the production of volatile substances in liquor. However, at present, they are mostly used to achieve directional regulation of flavor quality during liquor fermentation, while there are few applications in functional regulation such as antioxidant activity of liquor. Summary of the Invention

[0005] The present invention aims to improve the quality and antioxidant activity of liquor and provides a method for semi-solid brewing of Luzhou-flavor liquor by isolating and screening different strains from Daqu and fermented grains to form a composite Luzhou-flavor Daqu. The method comprises combining the isolated different strains with Luzhou-flavor Daqu, inoculating the mixture into a fermentation system using glutinous sorghum as a raw material, and fermenting to obtain Luzhou-flavor liquor. During the preparation process, different strain combinations are combined with Luzhou-flavor Daqu for brewing to improve the functional properties of the liquor and enhance its antioxidant activity. The present invention creatively uses different strains isolated from Luzhou-flavor Daqu for fermentation to improve the quality and antioxidant activity of the liquor.

[0006] The method for brewing high-quality antioxidant Luzhou-flavor liquor using a strain-cooperative combination of Gujing Luzhou-flavor Daqu provided by the present invention comprises the following steps:

[0007] (1) Cultivation of fermentation strains

[0008] ①Single strain liquid seed culture

[0009] Escherichia coli, Bacillus subtilis and Bacillus velezensis were inoculated into PCA liquid culture medium respectively, and cultured at 37℃ with shaking for 20-36h to obtain seed solution. The concentration of seed solution was adjusted to OD 600nm The value is 0.36-0.37;

[0010] ② Preparation of cooperative strengthening bacteria seed solution

[0011] The cooperative strengthening bacteria seed liquid is prepared by taking the dry weight of glutinous sorghum as 100%, the Escherichia coli seed liquid accounts for 3-4%, the Bacillus subtilis seed liquid accounts for 3-4%, and the Bacillus Velez seed liquid accounts for 1-2%.

[0012] (2) Fermentation system preparation:

[0013] ① Weigh glutinous sorghum and crush it into 6-8 petals, with a crushing particle size of about 70% of the portion above a 20-mesh sieve;

[0014] ② Moistening: Add tap water to the chopped sorghum at a ratio of 60% (based on dry sorghum) and moisten the grain for 10-12 hours;

[0015] ③ Steaming: Steam the moistened sorghum crumbs for 0.3-2 hours until the material becomes cooked and changes color to obtain cooked sorghum crumbs;

[0016] ④ Mixing: Take 2.6-3.4 times the mass of tap water or distilled water of the dry material, add cooled cooked sorghum and 30% Luzhou-flavor Daqu (based on dry material sorghum), add 7%-10% (based on dry material sorghum) cooperative strengthening bacteria seed liquid, and seal for fermentation;

[0017] Wherein, the Daqu is the Luzhou-flavor Daqu produced by Anhui Gujing Gongjiu Co., Ltd.;

[0018] (3) Fermentation and distillation (liquor brewing):

[0019] The fermentation conditions are: fermentation at 25-37°C for 15-20 days;

[0020] Specifically: 28°C on the first day, 33-35°C on the second day, 30-33°C on the third day, 28°C on the fourth to the 18th day, fermentation for 18 days to obtain fermented mash;

[0021] The method adopts a semi-solid state method for direct distillation, intercepts 40° and above distilled liquors and mixes them to obtain 45° liquor, wherein the total ester is greater than 1.60 g / L, the DPPH free radical scavenging rate is greater than 34%, and the content of guaiacol substances (4-vinylguaiacol, guaiacol) is greater than 18 mg / L.

[0022] In the above method, the Escherichia coli is classified as Escherichia coli and is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 30682.

[0023] The taxonomic name of the Bacillus subtilis is Bacillus subtilis, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 28796.

[0024] The taxonomic name of the Bacillus velezensis is Bacillus velezensis, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 28797.

[0025] The Luzhou-flavor liquor prepared by the above method also falls within the protection scope of the present invention.

[0026] The Luzhou-flavor liquor has good aroma and good antioxidant activity.

[0027] Another object of the present invention is to provide a synergistically enhanced strain that can work together with Daqu to produce high-quality liquor with high antioxidant activity.

[0028] The collaborative strengthening bacteria are mainly composed of Escherichia coli, Bacillus subtilis and Bacillus velez;

[0029] The Escherichia coli is classified as Escherichia coli and is deposited in the General Microbiology Center of China Culture Collection Administration under the deposit number CGMCC No. 30682.

[0030] The taxonomic name of the Bacillus subtilis is Bacillus subtilis, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 28796.

[0031] The taxonomic name of the Bacillus velezensis is Bacillus velezensis, which is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration with a deposit number of CGMCC No. 28797.

[0032] The application of the above-mentioned cooperatively strengthened strains in the semi-solid brewing of Luzhou-flavor liquor and improving the antioxidant activity of the semi-solid brewed Luzhou-flavor liquor also falls within the scope of protection of the present invention.

[0033] The advantages and positive effects achieved by the present invention are:

[0034] 1. The present invention utilizes composite Daqu with different strains isolated from Daqu to carry out semi-solid brewing of liquor with sorghum as raw material. The results show that the semi-solid brewing liquor of Gujing Daqu with the collaborative combination of strains has good quality, and the total ester content is increased by 84.09-120.45% compared with the liquor brewed with Daqu alone.

[0035] 2. The present invention utilizes the three selected strains to brew a composite Luzhou-flavor Daqu, and the prepared liquor has good antioxidant activity. Compared with the liquor brewed with Daqu alone, the DPPH free radical scavenging rate is increased by 49.87-66.63%, and the content of guaiacols (4-vinylguaiacol, guaiacol) is increased by 1176.55-2873.79%.

[0036] Preservation Instructions

[0037] Species name: Escherichia coli

[0038] Latin name: Escherichia coli

[0039] Strain number: EC

[0040] Depository: General Microbiology Center of China Culture Collection Administration

[0041] Abbreviation of depository institution: CGMCC

[0042] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0043] Deposit date: May 17, 2024

[0044] CGMCC registration number: CGMCC No.30682

[0045] Bacteria name: Bacillus subtilis

[0046] Latin name: Bacillus subtilis

[0047] Strain ID: BS

[0048] Depository: General Microbiology Center of China Culture Collection Administration

[0049] Abbreviation of depository institution: CGMCC

[0050] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0051] Deposit date: October 27, 2023

[0052] CGMCC registration number: CGMCC No.28796

[0053] Bacteria name: Bacillus velezensis

[0054] Latin name: Bacillus velezensis

[0055] Strain ID: BV

[0056] Depository: General Microbiology Center of China Culture Collection Administration

[0057] Abbreviation of depository institution: CGMCC

[0058] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0059] Deposit date: October 27, 2023

[0060] CGMCC registration number: CGMCC No.28797 BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is the total ion current chromatogram of volatile substances in the liquor prepared in Example 1 of the present invention.

[0062] Figure 2 This is the total ion current chromatogram of volatile substances in the liquor prepared in Example 2 of the present invention.

[0063] Figure 3 This is the total ion current chromatogram of volatile substances in the liquor prepared in Example 3 of the present invention.

[0064] Figure 4 This is the total ion current chromatogram of volatile substances in the liquor prepared in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0065] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0066] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0067] Example 1: Semi-solid brewing of antioxidant liquor using composite Luzhou-flavor Daqu with different strains at specific concentrations

[0068] (1) Screening and determination of enhanced strains in Daqu

[0069] Accurately weigh 10g of the mixed Daqu and add it to a conical flask containing 90mL of sterile saline. Oscillate it in a shaker at 28℃ and 150r / min for 30min. -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 For each dilution, 100 μL of the dilution was evenly spread onto YPD, MRS, and PCA media, with triplicate replicates for each dilution. For the cultivation of molds and yeasts on YPD, 0.01% chloramphenicol was added to inhibit bacterial growth. For the cultivation of lactic acid bacteria on MRS, 0.2% natamycin was added to inhibit fungal growth. For the cultivation of Bacillus on PCA, the Daqu mixture was first incubated at 80°C in a water bath for 10 minutes before serial dilutions. After spreading, the YPD, MRS, and PCA media were incubated inverted at 28°C, 35°C, and 37°C incubators, respectively, for 2-3 days. Morphologically distinct colonies were selected for 16S rRNA sequencing, confirming that Escherichia coli, Bacillus subtilis, and Bacillus velezensis were fortified strains. These strains have been registered and deposited at the General Microbiology Center of the China Culture Collection Administration.

[0070] 16S rRNA sequencing results of Escherichia coli (SEQ.1):

[0071] AAGCTACCTACTTCTTTTGCACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTGGTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGGACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCACGGTTCCCGAAGGCACATTCTCATCTCTGAAAACTTCCGTGGATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTCGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACGAGACTCAAGCTTGCCAGTATCAGATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCTGACTTAACAAACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAACGTCAATGAGCAAAGGTATTAACTTTACTCCCTTCCTCCCCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCGTTACCCCACCTACTAGCTAATCCCATCTGGGCACATCCGATGGCAAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCACTCGTCAGCAAA

[0072] 16S rRNA sequencing results of Bacillus subtilis (SEQ.2):

[0073] CGGCGGCTGGCTCCTAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTGCA

[0074] 16S rRNA sequencing results of Bacillus velezensis (SEQ.3):

[0075] GCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCT ACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCC

[0076] (2) Cultivation of fermentation strains

[0077] ①Single strain liquid seed culture

[0078] Escherichia coli (EC), Bacillus subtilis (BS) and Bacillus velezensis (BV) were inoculated into PCA liquid culture medium respectively, and cultured at 37℃ for 24h to obtain seed solution. The concentration of seed solution was adjusted to OD 600nm value 0.36;

[0079] ② Preparation of cooperative strengthening bacteria seed solution

[0080] Based on the dry weight of glutinous sorghum as 100%, 3% of EC seed liquid, 4% of BS seed liquid and 2% of BV seed liquid were prepared to prepare the cooperative strengthening bacteria seed liquid;

[0081] (3) Fermentation system preparation:

[0082] ① Weigh glutinous sorghum and crush it into 6-8 petals, with the particle size of the sorghum above a 20-mesh sieve accounting for about 70%;

[0083] ② Moistening: Add tap water to the chopped sorghum at a ratio of 60% (based on dry sorghum) and moisten the grain for 12 hours;

[0084] ③ Steaming: Steam the sorghum crumbs in atmospheric air for 1 hour until the material becomes cooked and changes color to obtain cooked sorghum crumbs;

[0085] ④ Mixing: Take 3.0 times the mass of tap water of dry material, add cooled cooked sorghum and 30% (based on dry material sorghum) of Luzhou-flavor Daqu, add the above-mentioned cooperative strengthening bacteria seed liquid, and seal and ferment;

[0086] (4) Fermentation and distillation (liquor brewing):

[0087] The fermentation conditions are: 28°C on the first day, 33-35°C on the second day, 30-33°C on the third day, and 28°C on the fourth to eighteenth days. The fermentation takes 18 days to obtain fermented mash.

[0088] For semi-solid distilled liquor, the total ester content of liquor cut at 45° and above was measured to be 1.94±0.13 g / L, which is higher than the 0.60 g / L required by GB / T 20822-2007 "Solid-Liquid Distilled Liquor"; the DPPH free radical scavenging rate of the brewed liquor was 38.09±0.13%, and the test results of ester and phenolic volatile substances are shown in Table 2-3. The content of guaiacol substances (4-vinylguaiacol, guaiacol) was 43.12±11.14 mg / L.

[0089] Example 2: Semi-solid brewing of antioxidant liquor using composite Luzhou-flavor Daqu with different strains at specific concentrations

[0090] (1) Screening and determination of enhanced strains in Daqu

[0091] Same as Example 1.

[0092] (2) Cultivation of fermentation strains

[0093] ①Single strain liquid seed culture

[0094] Same as Example 1.

[0095] ② Preparation of cooperative strengthening bacteria seed solution

[0096] Based on the dry weight of glutinous sorghum as 100%, 3% EC seed liquid, 3% BS seed liquid and 1% BV seed liquid were prepared to prepare the cooperative strengthening bacteria seed liquid.

[0097] (3) Fermentation system preparation:

[0098] ① Weigh glutinous sorghum and crush it into 6-8 petals, with the particle size of the sorghum above a 20-mesh sieve accounting for about 70%;

[0099] ② Moistening: Add tap water to the chopped sorghum at a ratio of 60% (based on dry sorghum) and moisten the grain for 12 hours;

[0100] ③ Steaming: Steam the sorghum crumbs in atmospheric air for 1 hour until the material becomes cooked and changes color to obtain cooked sorghum crumbs;

[0101] ④ Mixing: Take 3.0 times the mass of tap water of dry material, add cooled ripe sorghum and 30% (based on dry material sorghum) of Luzhou-flavor Daqu, add the above-mentioned cooperative strengthening bacteria seed liquid, and seal and ferment;

[0102] (4) Fermentation and distillation (liquor brewing):

[0103] Same as Example 1.

[0104] For semi-solid distilled liquor, the total ester content of liquor cut at 45° and above was measured to be 1.62±0.12 g / L, which is higher than the 0.60 g / L required by GB / T 20822-2007 "Solid-Liquid Distilled Liquor"; the DPPH free radical scavenging rate of the brewed liquor was 37.21±1.18%, and the test results of ester and phenolic volatile substances are shown in Table 2-3. The total content of guaiacols (4-vinylguaiacol, guaiacol) was 28.46±1.44 mg / L.

[0105] Example 3: Semi-solid brewing of antioxidant liquor using composite Luzhou-flavor Daqu with different strains at specific concentrations

[0106] (1) Screening and determination of enhanced strains in Daqu

[0107] Same as Example 1.

[0108] (2) Cultivation of fermentation strains

[0109] ①Single strain liquid seed culture

[0110] Same as Example 1.

[0111] ② Preparation of cooperative strengthening bacteria seed solution

[0112] Based on 100% dry weight of glutinous sorghum, 3% EC seed liquid, 4% BS seed liquid and 1% BV seed liquid were prepared to prepare the cooperative strengthening bacteria seed liquid.

[0113] (3) Fermentation system preparation:

[0114] ① Weigh glutinous sorghum and crush it into 6-8 petals, with the particle size of the sorghum above a 20-mesh sieve accounting for about 70%;

[0115] ② Moistening: Add tap water to the chopped sorghum at a ratio of 60% (based on dry sorghum) and moisten the grain for 12 hours;

[0116] ③ Steaming: Steam the sorghum crumbs in atmospheric air for 1 hour until the material becomes cooked and changes color to obtain cooked sorghum crumbs;

[0117] ④ Mixing: Take 3.0 times the mass of tap water of dry material, add cooled cooked sorghum and 30% (based on dry material sorghum) of Luzhou-flavor Daqu, add the above-mentioned composite cooperative bacteria seed liquid, and seal and ferment;

[0118] (4) Fermentation and distillation (liquor brewing):

[0119] Same as Example 1.

[0120] For semi-solid distilled liquor, the total ester content of liquor cut at 45° and above was measured to be 1.83±0.16g / L, which is higher than the 0.60g / L required by GB / T 20822-2007 "Solid-Liquid Distilled Liquor"; the DPPH free radical scavenging rate of the brewed liquor was 34.26±0.53%, and the test results of ester and phenolic volatile substances are shown in Table 2-3. The total content of guaiacols (4-vinylguaiacol, guaiacol) was 18.51±0.24mg / L.

[0121] Comparative Example 1: Semi-solid-state brewing of Luzhou-flavor Daqu liquor

[0122] (1) Fermentation system preparation:

[0123] ① Weigh glutinous sorghum and crush it into 6-8 petals, with the particle size of the sorghum above a 20-mesh sieve accounting for about 70%;

[0124] ② Moistening: Add tap water to the chopped sorghum at a ratio of 60% (based on dry sorghum) and moisten the grain for 12 hours;

[0125] ③ Steaming: Steam the sorghum crumbs in atmospheric air for 1 hour until the material becomes cooked and changes color to obtain cooked sorghum crumbs;

[0126] ④ Mixing: Take 3.0 times the mass of tap water of dry material, add cooled cooked sorghum and 30% Luzhou-flavor Daqu (calculated based on dry material sorghum), do not add fortified bacteria, and seal for fermentation.

[0127] (2) Fermentation and distillation (liquor brewing):

[0128] Same as Example 1.

[0129] For semi-solid distilled liquor, the total ester content of liquor cut at 45° and above was measured to be 0.88±0.02g / L, which is higher than the 0.60g / L required by GB / T 20822-2007 "Solid-Liquid Distilled Liquor"; the DPPH free radical scavenging rate of the brewed liquor was 22.86±0.43%, and the test results of ester and phenolic volatile substances are shown in Table 2-3. The content of guaiacol substances (4-vinylguaiacol, guaiacol) was 1.45±0.21mg / L.

[0130] Comparative Example 2: Semi-solid brewing of Luzhou-flavor Daqu with Escherichia coli slightly improves total esters and antioxidant activity of liquor

[0131] (1) Screening and determination of enhanced strains in Daqu

[0132] Same as Example 1.

[0133] (2) Cultivation of fermentation strains

[0134] ①Single strain liquid seed culture

[0135] Same as Example 1.

[0136] ② Preparation of enhanced bacterial seed solution

[0137] No BS or BV was added, and only 4% of EC seed solution based on the weight percentage of sorghum was added.

[0138] (3) Fermentation system preparation:

[0139] ① Weigh glutinous sorghum and crush it into 6-8 petals, with the particle size of the sorghum above a 20-mesh sieve accounting for about 70%;

[0140] ② Moistening: Add tap water to the chopped sorghum at a ratio of 60% (based on dry sorghum) and moisten the grain for 12 hours;

[0141] ③ Steaming: Steam the sorghum crumbs in atmospheric air for 1 hour until the material becomes cooked and changes color to obtain cooked sorghum crumbs;

[0142] ④ Mixing: Take 3.0 times the mass of tap water of dry material, add cooled cooked sorghum and 30% Luzhou-flavor Daqu (calculated based on dry material sorghum), add 4% (calculated based on dry material sorghum) fortified fungus seed liquid, and seal for fermentation.

[0143] (3) Fermentation and distillation (liquor brewing):

[0144] Same as Example 1.

[0145] For semi-solid distilled liquor, the total ester content of liquor cut at 45° and above was measured to be 1.32±0.04g / L, which is higher than the 0.60g / L required by GB / T 20822-2007 "Solid-Liquid Distilled Liquor"; the DPPH free radical scavenging rate of the brewed liquor was 28.69±0.68%, and the test results of ester and phenolic volatile substances are shown in Table 2-3. The content of guaiacol substances (4-vinylguaiacol, guaiacol) was 13.22±0.22mg / L.

[0146] Comparative Example 3: Semi-solid brewing of Bacillus subtilis composite Luzhou-flavor Daqu slightly improves total esters and antioxidant activity of liquor

[0147] (1) Screening and determination of enhanced strains in Daqu

[0148] Same as Example 1.

[0149] (2) Cultivation of fermentation strains

[0150] ①Single strain liquid seed culture

[0151] Same as Example 1.

[0152] ② Preparation of enhanced bacterial seed solution

[0153] No EC or BV was added, and only 3% of BS seed solution was added based on the weight percentage of sorghum.

[0154] (3) Fermentation system preparation:

[0155] ① Weigh glutinous sorghum and crush it into 6-8 petals, with the particle size of the sorghum above a 20-mesh sieve accounting for about 70%;

[0156] ② Moistening: Add tap water to the chopped sorghum at a ratio of 60% (based on dry sorghum) and moisten the grain for 12 hours;

[0157] ③ Steaming: Steam the sorghum crumbs in atmospheric air for 1 hour until the material becomes cooked and changes color to obtain cooked sorghum crumbs;

[0158] ④ Mixing: Take 3.0 times the mass of tap water of dry material, add cooled ripe sorghum and 30% (based on dry material sorghum) Luzhou-flavor Daqu, add 3% (based on dry material sorghum) fortified fungus seed liquid, and seal for fermentation.

[0159] (4) Fermentation and distillation (liquor brewing):

[0160] Same as Example 1.

[0161] For semi-solid distilled liquor, the total ester content of liquor cut at 45° and above was measured to be 1.35±0.09 g / L, which is higher than the 0.60 g / L required by GB / T 20822-2007 "Solid-Liquid Distilled Liquor"; the DPPH free radical scavenging rate of the brewed liquor was 26.89±0.23%, and the test results of ester and phenolic volatile substances are shown in Table 2-3. The content of guaiacol substances (4-vinylguaiacol, guaiacol) was 7.45±0.11 mg / L.

[0162] Comparative Example 4: Semi-solid brewing of Luzhou-flavor Daqu with Bacillus Velezii significantly improves the antioxidant activity of liquor and reduces total esters

[0163] (1) Screening and determination of enhanced strains in Daqu

[0164] Same as Example 1.

[0165] (2) Cultivation of fermentation strains

[0166] ①Single strain liquid seed culture

[0167] Same as Example 1.

[0168] ② Preparation of enhanced bacterial seed solution

[0169] No EC or BS was added, and only 1% of BV seed solution was added based on the weight percentage of sorghum.

[0170] (3) Fermentation system preparation:

[0171] ① Weigh glutinous sorghum and crush it into 6-8 petals, with the particle size of the sorghum above a 20-mesh sieve accounting for about 70%;

[0172] ② Moistening: Add tap water to the chopped sorghum at a ratio of 60% (based on dry sorghum) and moisten the grain for 12 hours;

[0173] ③ Steaming: Steam the sorghum crumbs in atmospheric air for 1 hour until the material becomes cooked and changes color to obtain cooked sorghum crumbs;

[0174] ④ Mixing: Take 3.0 times the mass of tap water of dry material, add cooled cooked sorghum and 30% (based on dry material sorghum) Luzhou-flavor Daqu, add 1% (based on dry material sorghum) fortified fungus seed liquid, seal and ferment.

[0175] (4) Fermentation and distillation (liquor brewing):

[0176] Same as Example 1.

[0177] For semi-solid distilled liquor, the total ester content of liquor cut at 45° and above was measured to be 0.44±0.03g / L, which is lower than the 0.60g / L required by GB / T 20822-2007 "Solid-Liquid Distilled Liquor"; the DPPH free radical scavenging rate of the brewed liquor was 36.07±1.43%, and the test results of ester and phenolic volatile substances are shown in Table 2-3. The content of guaiacol substances (4-vinylguaiacol, guaiacol) was 24.43±5.17mg / L.

[0178] Comparative Example 5: Semi-solid brewing of Luzhou-flavor Daqu with Escherichia coli and Bacillus subtilis slightly improves total esters and antioxidant activity of liquor

[0179] (1) Screening and determination of enhanced strains in Daqu

[0180] Same as Example 1.

[0181] (2) Cultivation of fermentation strains

[0182] ①Single strain liquid seed culture

[0183] Same as Example 1.

[0184] ② Preparation of cooperative strengthening bacteria seed solution

[0185] No BV was added, and only 3% EC seed solution and 3.5% BS seed solution were added based on the weight percentage of sorghum.

[0186] (3) Fermentation system preparation:

[0187] ① Weigh glutinous sorghum and crush it into 6-8 petals, with the particle size of the sorghum above a 20-mesh sieve accounting for about 70%;

[0188] ② Moistening: Add tap water to the chopped sorghum at a ratio of 60% (based on dry sorghum) and moisten the grain for 12 hours;

[0189] ③ Steaming: Steam the sorghum crumbs in atmospheric air for 1 hour until the material becomes cooked and changes color to obtain cooked sorghum crumbs;

[0190] ④ Mixing: Take 3.0 times the mass of tap water of dry material, add cooled cooked sorghum and 30% (based on dry material sorghum) Luzhou-flavor Daqu, add 6.5% (based on dry material sorghum) fortified fungus seed liquid, seal and ferment.

[0191] (4) Fermentation and distillation (liquor brewing):

[0192] Same as Example 1.

[0193] For semi-solid distilled liquor, the total ester content of liquor cut at 45° and above was measured to be 1.14±0.03g / L, which is higher than the 0.60g / L required by GB / T 20822-2007 "Solid-Liquid Distilled Liquor"; the DPPH free radical scavenging rate of the brewed liquor was 29.93±0.53%, and the test results of ester and phenolic volatile substances are shown in Table 2-3. The content of guaiacol substances (4-vinylguaiacol, guaiacol) was 13.34±1.52mg / L.

[0194] Comparative Example 6: Semi-solid brewing of Luzhou-flavor Daqu with Escherichia coli and Bacillus Velezii slightly improves the antioxidant activity of liquor

[0195] (1) Screening and determination of enhanced strains in Daqu

[0196] Same as Example 1.

[0197] (2) Cultivation of fermentation strains

[0198] ①Single strain liquid seed culture

[0199] Same as Example 1.

[0200] ② Preparation of cooperative strengthening bacteria seed solution

[0201] No BS was added, and only 4% of EC seed solution and 1.5% of BV seed solution were added based on the weight percentage of sorghum.

[0202] (3) Fermentation system preparation:

[0203] ① Weigh glutinous sorghum and crush it into 6-8 petals, with the particle size of the sorghum above a 20-mesh sieve accounting for about 70%;

[0204] ② Moistening: Add tap water to the chopped sorghum at a ratio of 60% (based on dry sorghum) and moisten the grain for 12 hours;

[0205] ③ Steaming: Steam the sorghum crumbs in atmospheric air for 1 hour until the material becomes cooked and changes color to obtain cooked sorghum crumbs;

[0206] ④ Mixing: Take 3.0 times the mass of tap water of dry material, add cooled cooked sorghum and 30% (based on dry material sorghum) Luzhou-flavor Daqu, add 5.5% (based on dry material sorghum) fortified fungus seed liquid, seal and ferment.

[0207] (4) Fermentation and distillation (liquor brewing):

[0208] Same as Example 1.

[0209] For semi-solid distilled liquor, the total ester content of liquor cut at 45° and above was measured to be 0.88±0.05g / L, which is higher than the 0.60g / L required by GB / T 20822-2007 "Solid-Liquid Distilled Liquor"; the DPPH free radical scavenging rate of the brewed liquor was 32.59±1.01%, and the test results of ester and phenolic volatile substances are shown in Table 2-3. The content of guaiacol substances (4-vinylguaiacol, guaiacol) was 16.76±1.17mg / L.

[0210] Comparative Example 7: Semi-solid-state brewing of Luzhou-flavor liquor using low-concentration collaborative strains and composite Luzhou-flavor Daqu

[0211] (1) Screening and determination of enhanced strains in Daqu

[0212] Same as Example 1.

[0213] (2) Cultivation of fermentation strains

[0214] ①Single strain liquid seed culture

[0215] Same as Example 1.

[0216] ② Preparation of cooperative strengthening bacteria seed solution

[0217] The cooperative strengthening bacteria seed solution was prepared by mixing 2% EC seed solution, 2% BS seed solution and 1% BV seed solution by weight percentage of sorghum.

[0218] (3) Fermentation system preparation:

[0219] ① Weigh glutinous sorghum and crush it into 6-8 petals, with the particle size of the sorghum above a 20-mesh sieve accounting for about 70%;

[0220] ② Moistening: Add tap water to the chopped sorghum at a ratio of 60% (based on dry sorghum) and moisten the grain for 12 hours;

[0221] ③ Steaming: Steam the sorghum crumbs in atmospheric air for 1 hour until the material becomes cooked and changes color to obtain cooked sorghum crumbs;

[0222] ④ Mixing: Take 3.0 times the mass of tap water of dry material, add cooled cooked sorghum and 30% (based on dry material sorghum) Luzhou-flavor Daqu, add 5% (based on dry material sorghum) cooperative strengthening bacteria seed liquid, and seal for fermentation.

[0223] (4) Fermentation and distillation (liquor brewing):

[0224] Same as Example 1.

[0225] For semi-solid distilled liquor, the total ester content of liquor cut at 45° and above was measured to be 0.88±0.08g / L, which is higher than the 0.60g / L required by GB / T 20822-2007 "Solid-Liquid Distilled Liquor"; the DPPH free radical scavenging rate of the brewed liquor was 35.83±0.43%, and the test results of ester and phenolic volatile substances are shown in Table 2-3. The content of guaiacol substances (4-vinylguaiacol, guaiacol) was 24.71±2.95mg / L.

[0226] Comparative Example 8: Semi-solid-state brewing of Luzhou-flavor liquor using high-concentration collaborative strains and compound Luzhou-flavor Daqu

[0227] (1) Screening and determination of enhanced strains in Daqu

[0228] Same as Example 1.

[0229] (2) Cultivation of fermentation strains

[0230] ①Single strain liquid seed culture

[0231] Same as Example 1.

[0232] ② Preparation of cooperative strengthening bacteria seed solution

[0233] The cooperative strengthening bacteria seed solution was prepared by mixing 5% EC seed solution, 5% BS seed solution and 5% BV seed solution by weight percentage of sorghum.

[0234] (3) Fermentation system preparation:

[0235] ① Weigh glutinous sorghum and crush it into 6-8 petals, with the particle size of the sorghum above a 20-mesh sieve accounting for about 70%;

[0236] ② Moistening: Add tap water to the chopped sorghum at a ratio of 60% (based on dry sorghum) and moisten the grain for 12 hours;

[0237] ③ Steaming: Steam the sorghum crumbs in atmospheric air for 1 hour until the material becomes cooked and changes color to obtain cooked sorghum crumbs;

[0238] ④ Mixing: Take 3.0 times the mass of tap water of dry material, add cooled cooked sorghum and 30% (based on dry material sorghum) Luzhou-flavor Daqu, add 15% (based on dry material sorghum) cooperative strengthening fungus seed liquid, and seal for fermentation.

[0239] (4) Fermentation and distillation (liquor brewing):

[0240] Same as Example 1.

[0241] For semi-solid distilled liquor, the total ester content of liquor cut at 45° and above was measured to be 0.90±0.05g / L, which is higher than the 0.60g / L required by GB / T 20822-2007 "Solid-Liquid Distilled Liquor"; the DPPH free radical scavenging rate of the brewed liquor was 23.66±0.35%, and the test results of ester and phenolic volatile substances are shown in Table 2-3. The content of guaiacol substances (4-vinylguaiacol, guaiacol) was 6.47±1.99mg / L.

[0242] Comparative Example 9: Semi-solid-state brewing of Luzhou-flavor Daqu using different proportions of cooperative strains

[0243] (1) Screening and determination of enhanced strains in Daqu

[0244] Same as Example 1.

[0245] (2) Cultivation of fermentation strains

[0246] ①Single strain liquid seed culture

[0247] Same as Example 1.

[0248] ② Preparation of cooperative strengthening bacteria seed solution

[0249] The cooperative strengthening bacteria seed solution was prepared by mixing 5% EC seed solution, 1% BS seed solution and 3% BV seed solution by weight percentage of sorghum.

[0250] (3) Fermentation system preparation:

[0251] ① Weigh glutinous sorghum and crush it into 6-8 petals, with the particle size of the sorghum above a 20-mesh sieve accounting for about 70%;

[0252] ② Moistening: Add tap water to the chopped sorghum at a ratio of 60% (based on dry sorghum) and moisten the grain for 12 hours;

[0253] ③ Steaming: Steam the sorghum crumbs in atmospheric air for 1 hour until the material becomes cooked and changes color to obtain cooked sorghum crumbs;

[0254] ④ Mixing: Take 3.0 times the mass of tap water of dry material, add cooled cooked sorghum and 30% (based on dry material sorghum) Luzhou-flavor Daqu, add 15% (based on dry material sorghum) cooperative strengthening fungus seed liquid, and seal for fermentation.

[0255] (4) Fermentation and distillation (liquor brewing):

[0256] Same as Example 1.

[0257] For the semi-solid distilled liquor, the total ester content of liquor cut at 45° and above was measured to be 0.94±0.07g / L, which is higher than the 0.60g / L required by GB / T 20822-2007 "Solid-Liquid Distilled Liquor"; the DPPH free radical scavenging rate of the brewed liquor was 32.85±0.97%, and the test results of ester and phenolic volatile substances are shown in Table 2-3. The content of guaiacol substances (4-vinylguaiacol, guaiacol) was 20.11±2.21mg / L.

[0258] Related test results of the present invention:

[0259] (1) Determination of alcohol content of liquor: Use an alcohol refractometer to determine the alcohol content of different distilled liquors.

[0260] (2) Determination of total ester content: Refer to the complete manual of liquor production technology. The specific method is as follows: Pipette 10.0 mL of distilled liquor sample into a 100 mL conical flask, add 2 drops of phenolphthalein indicator, and titrate with 0.1 mol / L NaOH standard solution until it turns pink. Record the volume of NaOH standard solution consumed. Then accurately add 5.00 mL of 0.1 mol / L NaOH, shake well, add a few zeolites or glass beads, install a condenser, condense and reflux in a boiling water bath for 30 minutes, remove and cool, and titrate with 0.1 mol / L HCl. The endpoint is when the slightly red color just disappears. Record the volume of HCl standard solution consumed. At the same time, pipette 10.00 mL of ethanol solution for a blank test and record the volume of HCl consumed.

[0261] (3) Sensory evaluation

[0262] The evaluation was conducted in accordance with the national standard GB / T 33405-2016, "Terminology for Sensory Evaluation of Liquor." A sensory evaluation panel of six experienced evaluators was selected. After systematic training, they discussed and screened the sensory characteristics of Luzhou-flavor liquors. They ultimately settled on eight sensory descriptors: "cereal aroma," "fruity aroma," "floral aroma," "mellow flavor," "sweetness," "sourness," "bitterness," and "exotic flavors." Each attribute was represented on a scale of 0-5, with "0" representing the weakest intensity and increasing in intensity to "5" representing the strongest. The final score for each attribute was the average of all panelists' scores.

[0263] (4) Determination of antioxidant activity:

[0264] ①Determination of DPPH free radical scavenging rate

[0265] Dilute the sample solution to 50%, take 2 mL of the sample and add 2 mL of DPPH anhydrous ethanol solution, vortex, and place in the dark for 30 minutes. Use distilled water as the blank solution (adjust to 0) and measure B1 at 517 nm. Replace the DPPH ethanol solution with anhydrous ethanol and conduct the experiment to measure the background absorbance of the sample, B2; replace the sample solution with distilled water and measure the absorbance of the blank control solution, B0. Calculation formula:

[0266] DPPH free radical scavenging rate (%) = [1-(B1-B2) / B0] × 100%

[0267] (5) Determination of Volatile Components: A sample of the fermented liquor of the present invention was diluted with deionized water to an alcohol content of approximately 10%. 2 g of NaCl was weighed and added to a 20 mL headspace sample bottle. 5 mL of the diluted liquor sample was then accurately pipetted into the bottle. 10 μL of a 1 mg / mL internal standard solution (4-methyl-2-pentanol) was then accurately pipetted into the bottle. The sample was shaken at 50°C (shaking speed 450 rpm) for 15 min, then extracted at 50°C for 45 min, and then subjected to GC-MS analysis by thermal desorption at the GC injection port for 5 min. Chromatographic conditions: Chromatographic column: (DB-WAX UI, 60 m×0.25 mm, 0.25 μm), injection port temperature: 240°C, temperature program: 40°C, hold for 3 min, 3°C / min temperature program: 40°C, hold for 3 min, then increase to 240°C at 3°C / min, hold for 10 min. Mass spectrometry conditions were as follows: electron energy 70 eV, ion source temperature 230 °C, quadrupole temperature 150 °C, scan range 20–550 m / z, and threshold value 100.

[0268] As can be seen from the sensory evaluation results (Table 1), the aroma characteristics of the liquor brewed in the examples are mainly fruity, floral, and mellow, with a prominent sweetness and insignificant sourness, bitterness, and foreign flavors, which is consistent with the characterization of the corresponding total ester content determined by the indicator method. Compared with the liquor brewed using only Luzhou-flavor Daqu (Comparative Example 1), the fruity, floral, mellow, and sweet flavors of the liquors in Examples 1, 2, and 3 were all improved. Although the flavor quality (fruity and floral) of Comparative Examples 2, 3, and 5 was slightly improved, their antioxidant activity (DPPH free radical scavenging rate) was lower than that of the examples. Brewing using this method can improve the sensory quality of liquor.

[0269] Esters are the most abundant and diverse of all flavor compounds in baijiu, determining its flavor, aroma, grade, and style. As shown in Table 2, the total ester content of the Examples, as measured by GC-MS and by the indicator method, was higher than that of the Comparative Examples. Compared with Comparative Example 1, the total ester content of Examples 1, 2, and 3 increased by 138.92%, 102.82%, and 125.27%, respectively, indicating that the addition of Escherichia coli, Bacillus subtilis, and Bacillus velezine in the brewing of baijiu increased ester content. Adding Bacillus subtilis alone (Comparative Example 5) slightly increased ester content, by 27.35% compared to Daqu fermentation alone. However, blending with other strains significantly increased the ester content in the fermented mash, likely due to the synergistic effect of Bacillus subtilis with Escherichia coli and Bacillus velezine on ester production. The total ester content of Comparative Examples 2, 3, and 5, as measured by the indicator method, was significantly higher than that of Comparative Example 1. Although comparative examples 2 and 3 have higher volatile ester content, their antioxidant activity is still lower than that of the embodiment. Ethyl acetate, ethyl caprate, ethyl caprate and ethyl palmitate are higher in the liquor, and have fruity, coconut and creamy flavors. Ethyl palmitate increases the body of the liquor, reduces the dryness of the liquor, enhances the aftertaste, and plays a vital role in the stability and mouthfeel of the liquor. In addition, isoamyl acetate (strong fruity and floral aroma), ethyl butyrate (pineapple and banana smell), ethyl benzoate (fruity aroma), phenylethyl acetate (honey aroma) and ethyl laurate (peanut aroma) also constitute the special aroma of the liquor. Adopting this method to brew liquor can increase the ester content and improve the liquor flavor while ensuring the local flavor.

[0270] Phenolic compounds are key aromatic compounds in liquor, and guaiacols have antioxidant activity. The antioxidant activity index DPPH free radical scavenging rate of the embodiment is all higher than that of Comparative Example 1. As shown in Table 3, guaiacols (4-vinylguaiacol, guaiacol) and the total content of phenols in the embodiment liquor are significantly higher than those in the comparative example, which is consistent with the antioxidant activity index. Although the liquors brewed by Comparative Examples 4 and 7 show good antioxidant activity, their total ester content is reduced or not improved compared with (Comparative Example 1) without adding fortified strains, and the liquor quality is not improved. Compared with Comparative Example 1, the guaiacols content (4-vinylguaiacol, guaiacol) of Examples 1, 2, and 3 is respectively 29.74 times, 19.63 times, and 12.77 times that of Comparative Example 1, indicating that the present method for brewing liquor can increase the phenolic content in the wine body and improve antioxidant activity.

[0271] Table 1 Sensory evaluation results of brewed liquors of Examples 1-3 and Comparative Examples 1-9 of the present invention

[0272]

[0273] Table 2 Analysis of ester content in the liquor brewed in Examples 1-3 and Comparative Examples 1-9

[0274]

[0275]

[0276]

[0277]

[0278] Table 3 Analysis of guaiacol components in the liquors brewed in Examples 1-3 and Comparative Examples 1-9 of the present invention

[0279]

[0280] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A method for brewing high-quality antioxidant Luzhou-flavor liquor by using a combination of bacterial strains and Gujing Luzhou-flavor Daqu, comprising the following steps: (1) Cultivation of fermentation strains ①Single strain liquid seed culture Escherichia coli, Bacillus subtilis and Bacillus velezensis were inoculated into PCA liquid culture medium respectively, and cultured at 37℃ for 20-36 h to obtain seed solution. The concentration of seed solution was adjusted to OD 600nm The value is 0.36-0.37; The classification name of the Escherichia coli is Escherichia coli; The gene is deposited in the General Microbiology Center of China Culture Collection Administration under the accession number: CGMCC No.30682; The classification name of the Bacillus subtilis is Bacillus subtilis; The gene is deposited in the General Microbiology Center of China Culture Collection Administration under the accession number: CGMCC No. 28796; The taxonomic name of the Bacillus velez is Bacillus velezensis; The gene is deposited in the General Microbiology Center of China Culture Collection Administration under the accession number: CGMCC No.28797; ② Preparation of cooperative strengthening bacteria seed solution The cooperative strengthening bacteria seed liquid is prepared by taking the dry weight of glutinous sorghum as 100%, the Escherichia coli seed liquid accounts for 3-4%, the Bacillus subtilis seed liquid accounts for 3-4%, and the Bacillus velezensis seed liquid accounts for 1-2%. (2) Fermentation system preparation: ① Weigh glutinous sorghum and crush it into 6-8 petals, with the particle size of the sorghum above a 20-mesh sieve accounting for 70%; ② Moistening: Add tap water to the chopped sorghum at a ratio of 60% based on the dry material sorghum, and moisten the grain for 10-12 hours; ③ Steaming: Steam the moistened sorghum crumbs in atmospheric air for 0.3-2 hours until the material becomes cooked and changes color to obtain cooked sorghum crumbs; ④ Mixing: Take 2.6-3.4 times the mass of tap water or distilled water of the dry material, add cooled cooked sorghum and 30% of Luzhou-flavor Daqu based on the dry material sorghum, add 7%-10% of cooperative strengthening bacteria seed liquid based on the dry material sorghum, and seal for fermentation; in, The Daqu is a Luzhou-flavor Daqu produced by Anhui Gujing Gongjiu Co., Ltd. (3) Fermentation and distillation: The fermentation conditions are: 28° C. on the first day, 33-35° C. on the second day, 30-33° C. on the third day, and 28° C. on the fourth to the 18th day, and fermentation for 18 days to obtain fermented mash; The semi-solid state method is used for direct distillation, and distilled liquors with a temperature of 40° or above are intercepted and mixed to obtain 45° liquor, wherein the total ester is greater than 1.60 g / L, the DPPH free radical scavenging rate is greater than 34%, and the content of guaiacols is greater than 18 mg / L.

2. Luzhou-flavor liquor prepared by the method according to claim 1.

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