Bacillus licheniformis and method for preparing strong Fengxiang-type Daqu

Abstract

The application discloses a bacillus licheniformis and a method for preparing a phoenix-scented intensified Daqu. Bacillus licheniformis ), which is preserved in the China Center for Type Culture Collection (CCTCC) and has a preservation number of CCTCC NO: M 2026955; the bacillus licheniformis has the activity of producing enzymes under high-temperature conditions of 65 DEG C and can resist 9% vol alcohol concentration, and the maximum protease production is 513 U / mL. The strain is used to prepare the phoenix-scented intensified Daqu at an optimal inoculation concentration of 10 7 CFU / mL, and the saccharifying power and liquefying power of the obtained intensified Daqu are significantly improved compared with common Daqu; the application integrates metagenomic sequencing, HS-SPME / GC-MS technology and a multivariate statistical analysis system to establish a comprehensive evaluation method for the quality of the intensified Daqu, which is a trinity of'microbial community structure-physicochemical properties-flavor metabolism'.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a Bacillus licheniformis and a method for preparing a type of fortified Daqu (a type of starter culture). Background Technology

[0002] Daqu, or koji, is the core saccharification and fermentation agent in Chinese Baijiu brewing. Its microbial community structure and enzyme system composition directly determine the quality and style of Baijiu. Fengxiang-style Baijiu, one of the four basic aroma types in China, possesses a unique style characterized by "mellow and elegant aroma, sweet and robust taste." The quality stability of its daqu has always been a key issue restricting the industry's development. Traditional Fengxiang-style daqu uses an open natural fermentation process, which is greatly affected by environmental factors, resulting in significant quality fluctuations, insufficient functional enzyme activity, and poor controllability of flavor compounds.

[0003] Bacillus licheniformis ( Bacillus licheniformis Due to its high-temperature resistance, abundant enzyme production, and strong aroma-producing ability, *Bacillus licheniformis* has attracted widespread attention in the fortification of Daqu (a type of starter culture). However, the *Bacillus licheniformis* strains used for Daqu fortification in existing technologies are mainly derived from other aroma types of Daqu or environmental samples, lacking homologous functional strains specific to the *feng* aroma type of Daqu. Current methods for preparing fortified Daqu often employ compound microbial agents or combinations of multiple strains, resulting in complex processes and unclear mechanisms. Quality evaluation of fortified Daqu largely remains at the level of conventional physicochemical indicators, lacking systematic analysis from the perspectives of microbial community structure and flavor metabolites. Summary of the Invention

[0004] The problem solved by this invention is to provide a Bacillus licheniformis strain and a method for preparing a pungent-flavored fortified koji, which is derived from a pungent-flavored koji and a high-protease-producing Bacillus licheniformis strain with excellent high-temperature and alcohol tolerance, and to establish a method for preparing a pungent-flavored fortified koji with this strain as the core.

[0005] This invention is achieved through the following technical solution: The first objective of this invention is to provide a high-protein-producing Bacillus licheniformis strain isolated from a type of Daqu (a type of Chinese liquor). This strain exhibits excellent high-temperature tolerance (it can still produce enzymes at 65°C) and alcohol tolerance (9% vol), with a maximum protease yield of 513 U / mL.

[0006] The second objective of this invention is to provide a method for preparing a type of enhanced Daqu (a type of starter culture) using the aforementioned Bacillus licheniformis.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A type of Bacillus licheniformis, this bacterium is Bacillus licheniformis ( Bacillus licheniformis The ), is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 2026955.

[0008] Furthermore, the *Bacillus licheniformis* isolated from the Daqu (a type of starter culture) of Fenghuang-flavored Baijiu (Chinese liquor) possesses the following biological characteristics: Morphological characteristics: Forms round, milky white, dry colonies on LB solid medium with irregular edges, Gram-positive, and rod-shaped cells; Enzyme production characteristics: The maximum protease yield is 513 U / mL; Tolerance characteristics: It can maintain enzyme activity even at a high temperature of 65℃ and can grow and metabolize normally in an environment with a 9% vol alcohol concentration.

[0009] Furthermore, its 16S rDNA sequence is shown in SEQ.ID.NO.1.

[0010] This invention provides a method for preparing a type of enhanced Daqu (a type of starter culture) using Bacillus licheniformis, comprising the following steps: (1) Inoculate Bacillus licheniformis CCTCC NO: M 2026955 into LB solid slant medium and activate it at 35-40℃ for 18-24 hours; (2) Seed culture preparation: The activated strain is inoculated into LB liquid medium and cultured in a shaker at 35-40℃ and 150-200rpm for 16-20 hours to obtain the seed culture; (3) Preparation of enhanced inoculum: Expand the seed culture to 1×10⁻⁶ according to the specified ratio. 8 CFU / mL was used to obtain the enhanced inoculum; (4) Strengthening the preparation of Daqu: Using the Fengxiang-type Daqu preparation process, in the wheat soaking or mixing process, the inoculation concentration is 10 5 -10 9 CFU / mL, the enhanced inoculum solution is evenly sprayed onto the koji-making raw materials; (5) Fermentation management: Fermentation management is carried out in accordance with the process of fermentation of Fengxiang type Daqu, including the stages of mold growth, mold drying, fire period, high fire, and post-fire. The temperature and humidity of the fermentation room are controlled and the fermentation cycle is 25-35 days. (6) Processing of the starter culture: After the starter culture is completed, it is dried on a drying rack and aged in the starter culture warehouse to obtain the Phoenix Fragrance type enhanced starter culture.

[0011] Furthermore, the concentration of the inoculation is 10. 7 CFU / mL.

[0012] This invention also provides a method for evaluating the quality of phoenix-flavored enhanced koji, comprising the following steps: (1) Test the conventional physicochemical properties of enhanced Daqu (a type of Chinese liquor); (2) Metagenomic sequencing technology was used to analyze the microbial community structure of enhanced Daqu, a sequencing library was constructed, and Venn analysis of α diversity and OTU distribution and LEfSe differential species analysis were performed to identify the core dominant bacterial genera and characteristic microbial biomarkers. (3) The volatile flavor compounds of the enhanced Daqu were analyzed using HS-SPME / GC-MS technology, and potential marker metabolites were screened by multivariate statistical analysis. (4) Correlation analysis was conducted between the microbial community structure and physicochemical indicators and flavor substances.

[0013] Furthermore, the conventional physicochemical index tests include indicators such as moisture, acidity, starch content, saccharification power, liquefaction power, and protease activity.

[0014] Furthermore, the core dominant bacterial genus identified in the microbial community structure analysis should be *Saccharopolysporum* (Saccharopolysporum). Saccharopolyspora ), Straight-spined polysporum ( Saccharopolyspora rectivirgula ); Strengthening the Daqu (a type of starter culture) with Polysaccharide Polyporus spp. ( Saccharopolyspora The relative abundance of ) should reach 59-61%; Straight-spined polysporum ( Saccharopolyspora rectivirgula The relative abundance of ) should reach 70-71%.

[0015] Furthermore, the potential marker metabolites should include alcohols such as teratol, 1-nonanol, (S)-2-methylbutanol, linalool, and phenylethanol; Esters including γ-nonanolide; Ketones, including 2-heptanone; Flavor compounds including pyrazines such as 2-vinyl-6-methylpyrazine and 2,3,5-trimethylpyrazine, and caryophyllene.

[0016] This invention also provides the application of Bacillus licheniformis CCTCC NO: M 2026955 as a saccharification and fermentation agent, enzyme preparation and / or flavor enhancer in the brewing of Baijiu (Chinese liquor).

[0017] Compared with the prior art, the present invention has the following significant advantages: (1) Strain innovation: The Bacillus licheniformis LB-6 provided by this invention was isolated from the Fengxiang type of Daqu and has the characteristics of strong homology adaptability. It can still maintain enzyme production activity under high temperature conditions of 65℃ and tolerate 9% vol alcohol concentration. It has both high temperature tolerance and alcohol tolerance characteristics. The maximum protease production reaches 513 U / mL. Its comprehensive performance is better than most of the existing Bacillus licheniformis strains.

[0018] (2) Technological innovation: This invention has identified 10 7CFU / mL is the optimal inoculum concentration for fortification. At this concentration, the saccharification and liquefaction power of the fortified Daqu are significantly improved compared with ordinary Daqu (saccharification power: 250±3U, liquefaction power: 0.79±0.02U). At the same time, the basic indicators such as moisture, acidity, and starch content meet the requirements for storage and fermentation, achieving synergistic optimization of fortification effect and basic quality.

[0019] (3) Innovative Mechanism Analysis: This invention is the first to systematically reveal the regulatory effect of Bacillus licheniformis on the microbial community of Fengxiang-type Daqu—enhancing the presence of Saccharomyces polysporus in Daqu ( Saccharopolyspora ) and Streptomyces ( Saccharopolyspora rectivirgula The relative abundance of *Bacillus licheniformis* was significantly higher than that of ordinary Daqu, while ordinary Daqu had higher microbial species richness and evenness. This finding confirms that exogenous inoculation of *Bacillus licheniformis* can indirectly enhance the dominant position of endogenous functional flora through community remodeling, providing a new perspective for understanding the intrinsic mechanism of enhanced processes.

[0020] (4) Flavor and Quality Enhancement: This invention identified 151 volatile compounds in the enhanced Daqu (a type of starter culture) and screened out 10 potential marker metabolites. The enhanced Daqu showed significant upregulation of key flavor compounds such as linalool, terpineol, phenethyl alcohol, and various pyrazines, while ordinary Daqu was characterized by significant enrichment of γ-nonanolide. Correlation analysis further revealed a significant positive correlation between core bacterial groups such as *Saccharomyces* and *Aspergillus* and physicochemical indicators such as saccharification power and liquefaction power, as well as alcohol flavor compounds, providing a scientific basis for the targeted regulation of the flavor and quality of enhanced Daqu.

[0021] (5) System evaluation system: This invention integrates metagenomic sequencing, HS-SPME / GC-MS technology and multivariate statistical analysis system to establish a three-in-one comprehensive evaluation method for the quality of enhanced Daqu, which can be extended to the quality evaluation and process optimization of enhanced Daqu for other types of Baijiu.

[0022] Preservation Instructions Bacillus licheniformis ( Bacillus licheniformis The sample is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026955 and deposit date of May 14, 2026. Attached Figure Description

[0023] Figure 1 This is a phylogenetic tree diagram of the LB-6 strain constructed based on the 16S rRNA gene sequence.

[0024] Figure 2 The graph shows the enzyme activity of Bacillus licheniformis LB-6 at different temperatures.

[0025] Figure 3This is a growth diagram of Bacillus licheniformis LB-6 at different alcohol concentrations.

[0026] Figure 4 For different inoculation concentrations, strengthen the use of Daqu (10) 5 10 6 10 7 Comparison of saccharification power and liquefaction power between koji and ordinary koji.

[0027] Figure 5 To enhance the comparison of α-diversity analysis of microbial communities between Daqu and ordinary Daqu (Shannon index).

[0028] Figure 6 To enhance the Venn analysis of OTU distribution between Daqu and ordinary Daqu.

[0029] Figure 7 This is a diagram showing the differential species analysis between enhanced Daqu and ordinary Daqu based on LEfSe analysis.

[0030] Figure 8 A pie chart showing the volatile compounds of Daqu and ordinary Daqu.

[0031] Figure 9 To enhance the PCA analysis charts of Daqu and ordinary Daqu.

[0032] Figure 10 Heat map to highlight the differences in flavor compounds between Daqu and ordinary Daqu.

[0033] Figure 11 This is a graph showing the correlation between physicochemical indicators and microbial communities.

[0034] Figure 12 This is a graph showing the correlation between microbial communities and potential marker metabolites. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments. These embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0036] Example 1: Isolation and Identification of Bacillus licheniformis LB-6 (1) Source of strain: High-quality Daqu samples were collected from a certain Fengxiang-style winery in Shaanxi Province, and samples of the core part of the Daqu were taken using aseptic operation.

[0037] (2) Isolation method: Take 10 g of sample and add it to 90 mL of sterile physiological saline. After shaking and homogenization, dilute serially and spread it on casein selection medium plates to screen for protease-producing strains. Incubate at 37℃ for 48 hours. Select typical single colonies that produce clear hydrolysis zones, streak them for purification, and number them as LB-6.

[0038] (3) Morphological identification: LB-6 strain forms round, milky white, dry colonies with irregular edges on LB solid medium; Gram staining positive, the bacteria are rod-shaped and produce spores.

[0039] (4) Molecular biological identification: Genomic DNA of LB-6 strain was extracted, and the 16S rDNA gene sequence was amplified using universal primers 27F and 1492R. The sequencing results (as shown in SEQ.ID.NO.1) were compared with the NCBI database using BLAST. The results showed that it had more than 99% homology with the type strain of Bacillus licheniformis. The phylogenetic tree of LB-6 strain constructed based on the 16S rRNA gene sequence is shown in Figure 1. Figure 1 As shown; the results indicate that LB-6 is Bacillus licheniformis ( Bacillus licheniformis (), is a new Bacillus licheniformis strain.

[0040] (5) Enzyme production capacity determination: The Folin-phenol method was used to determine the protease activity. Under optimized fermentation conditions, the maximum protease production of LB-6 strain reached 513 U / mL.

[0041] Example 2: Temperature tolerance test of Bacillus licheniformis LB-6 The activated LB-6 strain was inoculated into LB liquid medium and cultured at 22℃, 25℃, 28℃, 30℃, 34℃, 37℃, 40℃, 43℃, 46℃, 48℃, 51℃, 55℃, 58℃, 60℃, 63℃ and 65℃ respectively. Samples were taken at regular intervals to determine the protease activity.

[0042] The growth temperature range of *Bacillus licheniformis* is 15-55℃; however, the enzyme production activity of *Bacillus licheniformis* LB-6 varies at different temperatures, such as... Figure 2 As shown, the results indicate that LB-6 strain still exhibits significant protease activity (124 U / mL) at 65℃, demonstrating excellent high-temperature tolerance.

[0043] Example 3: Alcohol tolerance test of Bacillus licheniformis LB-6 LB liquid medium was supplemented with 0%, 3%, 6%, 9%, and 12% (v / v) anhydrous ethanol, respectively. After inoculation with LB-6 strain, the medium was incubated at 37°C, and the OD values ​​at different time points were measured. 600 Values ​​and protease activities. Common Bacillus cannot grow normally in medium with a 6% vol alcohol concentration; while Bacillus licheniformis LB-6 grows as expected under different alcohol concentrations. Figure 3 As shown, the results indicate that strain LB-6 can still grow normally in a medium with a 9% vol alcohol concentration (OD). 600 It reached more than 65% of the control (and maintained enzyme activity), showing good alcohol tolerance.

[0044] In summary, Bacillus licheniformis CCTCC NO: M 2026955 possesses the following biological characteristics: Morphological characteristics: Forms round, milky white, dry colonies on LB solid medium with irregular edges, Gram-positive, and rod-shaped cells; Enzyme production characteristics: The maximum protease yield is 513 U / mL; Tolerance characteristics: It can maintain enzyme activity even at a high temperature of 65℃ and can grow and metabolize normally in an environment with a 9% vol alcohol concentration. Its 16S rDNA sequence is shown in SEQ.ID.NO.1.

[0045] This invention also provides a method for preparing a type of aromatic-flavored fortified koji based on Bacillus licheniformis CCTCC NO: M 2026955, comprising the following steps: (1) Inoculate Bacillus licheniformis CCTCC NO: M 2026955 into LB solid slant medium and activate it at 35-40℃ for 18-24 hours; (2) Seed culture preparation: The activated strain is inoculated into LB liquid medium and cultured in a shaker at 35-40℃ and 150-200rpm for 16-20 hours to obtain the seed culture; (3) Preparation of enhanced inoculum: Expand the seed culture to 1×10⁻⁶ according to the specified ratio. 8 CFU / mL was used to obtain the enhanced inoculum; (4) Strengthening the preparation of Daqu: Using the Fengxiang-type Daqu preparation process, in the wheat soaking or mixing process, the inoculation concentration is 10 5 -10 9 CFU / mL, the enhanced inoculum solution is evenly sprayed onto the koji-making raw materials; (5) Fermentation management: Fermentation management is carried out in accordance with the process of fermentation of Fengxiang type Daqu, including the stages of mold growth, mold drying, fire period, high fire, and post-fire. The temperature and humidity of the fermentation room are controlled and the fermentation cycle is 25-35 days. (6) Processing of the starter culture: After the starter culture is completed, it is dried on a drying rack and aged in the starter culture warehouse to obtain the Phoenix Fragrance type enhanced starter culture.

[0046] Example 4: Determination of the optimal booster inoculation concentration Set 5 inoculation concentration gradients: 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL, prepare the Phoenix-flavored fortified Daqu according to the following steps: Raw material ratio: Barley, wheat, and peas are ground and sieved in a mass ratio of 35:50:15; Lubrication: Add water to the crushed raw materials at 40% of the total mass of the raw materials and mix well; Inoculation: Different concentrations of LB-6 enhanced inoculum were evenly sprayed onto the raw materials; Blank making: pressing and shaping into curved blanks; Incubation in the fermentation room: The koji blanks are moved into the fermentation room and go through the following stages in sequence: mold growth period (25-30℃, 3-5 days), mold drying period (35-40℃, 2-3 days), high-temperature fermentation period (45-55℃, 7-10 days), and low-temperature fermentation period (40-45℃, 7-10 days). After the fermentation process is completed, the koji is dried on a drying rack and stored in a koji warehouse for more than 30 days.

[0047] The obtained fortified Daqu and ordinary Daqu (uninoculated control) were tested for seven physicochemical indicators, including moisture, acidity, starch content, saccharification power, liquefaction power, fermentation power and protease activity.

[0048] Comparison of saccharification and liquefaction power of enhanced Daqu and ordinary Daqu at different inoculation concentrations, for example Figure 4 As shown; the results indicated that the moisture content (≤14%), acidity (0.8-1.2 mmol / 10g), and starch content (55-60%) of the fortified Daqu at various inoculation concentrations all met the basic requirements for storage and fermentation; at 10 7 The optimal inoculum concentration of CFU / mL showed the best results, with the saccharification power of the enhanced Daqu (a type of starter culture) increasing by 42.6% (P<0.01) and the liquefaction power increasing by 38.2% (P<0.01) compared to ordinary Daqu.

[0049] Example 5: Analysis of the microbial community structure of enhanced Daqu and ordinary Daqu (1) Sample collection: 10 samples were collected in Example 4. 7 Three biological replicates were taken from both enhanced and ordinary Daqu samples at an inoculation concentration of CFU / mL.

[0050] (2) Metagenomic sequencing: Total DNA was extracted from the samples, sequencing libraries were constructed, and metagenomic sequencing was performed using the Illumina platform. Each sample obtained no less than 10 Gb of high-quality data.

[0051] (3) Bioinformatics analysis: After quality control, assembly, gene prediction and species annotation, sequencing data are used for community structure analysis.

[0052] The results showed that 15 bacterial phyla and 4 fungal phyla were identified in both the enhanced and ordinary Daqu (a type of starter culture), with the core dominant bacterial genus being *Saccharopolysporum*. Saccharopolyspora However, the relative abundance of this genus in enhanced Daqu (60.3%) was significantly higher than that in ordinary Daqu (49%), among which *Polyspora linearis* (… Saccharopolyspora rectivirgula The relative abundance of ) increased particularly significantly.

[0053] (4) α diversity analysis: Shannon index and Simpson index were used to assess species diversity.

[0054] Enhanced analysis of microbial community α-diversity between Daqu and ordinary Daqu, for example Figure 5 As shown; The results showed that the Shannon index of ordinary Daqu (2.25) was higher than that of enhanced Daqu (1.83), indicating that ordinary Daqu had a higher microbial species richness and evenness; while the dominant position of core functional bacteria (Saccharomyces spp. and Saccharomyces linearis) was more prominent in enhanced Daqu.

[0055] (5) Venn analysis and LEfSe analysis of OTU distribution: Venn diagrams of enhanced Daqu and ordinary Daqu are shown below. Figure 6 As shown, a total of 1960 OTUs were detected in both enhanced and ordinary Daqu. Among them, 1706 core OTUs were common to both types of Daqu, accounting for 87.04% of the total OTUs; 60 OTUs were unique to enhanced Daqu, and 124 OTUs were unique to ordinary Daqu.

[0056] Differential species analysis between enhanced and ordinary Daqu based on LEfSe analysis, such as... Figure 7 As shown; LEfSe analysis (LDA>4) successfully screened two types of characteristic microbial biomarkers for Daqu (a type of starter culture). The characteristic biomarkers for enhanced Daqu included *Saccharomyces* and *Saccharomyces*, while the characteristic biomarkers for ordinary Daqu included *Streptomyces*, *Rhizopus*, and *Rhizopus deltaeni*.

[0057] Example 6: Analysis of volatile flavor compounds in enhanced Daqu and ordinary Daqu (1) Sample pretreatment: The enhanced Daqu and ordinary Daqu samples were crushed and sieved. 2.0 g of the sample was accurately weighed and placed in a 20 mL headspace vial. 5 mL of saturated NaCl solution and 10 μL of internal standard (2-octanol, 10 μg / mL) were added.

[0058] (2) HS-SPME extraction: 50 / 30 μm DVB / CAR / PDMS extraction fiber was used, and the extraction was carried out at 60℃ for 40 minutes, followed by desorption at 250℃ injection port for 5 minutes.

[0059] (3) GC-MS analysis: An HP-5MS column (30 m × 0.25 mm × 0.25 μm) was used. The temperature program was as follows: 40℃ for 3 min, then increased to 120℃ at 5℃ / min, and then increased to 250℃ at 10℃ / min, and held for 5 min. The mass spectrometer used an EI ion source, 70 eV, in full scan mode (33-450 amu).

[0060] (4) Data analysis: A total of 151 volatile compounds were identified, including 36 alcohols, 13 esters, 11 aldehydes, 27 ketones, 20 aromatic compounds, 7 acids, 10 pyrazines, 2 furans, and 25 other compounds. Differentially expressed metabolites with VIP>1 and P<0.05 were screened using PCA analysis, resulting in 10 potential marker metabolites: Alcohols including terpineol, 1-nonanol, (S)-2-methylbutanol, linalool, and phenylethanol; Esters including γ-nonanolide; Ketones, including 2-heptanone; Flavor compounds including pyrazines such as 2-vinyl-6-methylpyrazine and 2,3,5-trimethylpyrazine, and caryophyllene.

[0061] The pie chart of volatile compounds in enhanced Daqu and ordinary Daqu is shown below. Figure 8 As shown; PCA analysis of enhanced Daqu and ordinary Daqu is as follows. Figure 9 As shown; the heat map of flavor compounds that differentiate Daqu from ordinary Daqu is as follows. Figure 10 As shown; The results showed that key flavor compounds such as terpineol, 1-nonanol, (S)-2-methylbutanol, linalool, phenethyl alcohol, γ-nonanolide, 2-heptanone, pyrazines including 2-vinyl-6-methylpyrazine and 2,3,5-trimethylpyrazine, and caryophyllene were significantly upregulated in fortified Daqu, while γ-nonanolide was significantly enriched in ordinary Daqu.

[0062] Therefore, this invention proposes a quality evaluation method for a type of enhanced Daqu (a type of starter culture) with a phoenix-like aroma, characterized by the following steps: (1) Test the conventional physicochemical properties of enhanced Daqu (a type of Chinese liquor); (2) Metagenomic sequencing technology was used to analyze the microbial community structure of enhanced Daqu, a sequencing library was constructed, and α diversity, OUT distribution Venn analysis and LEfSe differential species analysis were performed to identify the core dominant bacterial genera and characteristic microbial biomarkers. (3) The volatile flavor compounds of the enhanced Daqu were analyzed using HS-SPME / GC-MS technology, and potential marker metabolites were screened by multivariate statistical analysis. (4) Correlation analysis was conducted between the microbial community structure and physicochemical indicators and flavor substances.

[0063] The core dominant bacterial genus identified in the microbial community structure analysis should be *Saccharopolysporum* (Saccharopolysporum). Saccharopolyspora ), Straight-spined polysporum ( Saccharopolyspora rectivirgula ); Strengthening the Daqu (a type of starter culture) with Polysaccharide Polyporus spp. ( Saccharopolyspora The relative abundance of ) should reach 59-61%; Straight-spined polysporum ( Saccharopolyspora rectivirgula 70-71% of ).

[0064] Example 7: Correlation Analysis of Microorganism-Physicochemical-Flavor Spearman correlation analysis was performed on the relative abundance of the core microbial community in Example 5, the physicochemical indicators (saccharification power, liquefaction power, etc.) in Example 4, and the content of differential flavor compounds in Example 6. The results showed that the core microbial communities, such as *Thermophilic Actinobacterium* and *Streptomyces*, were significantly positively correlated with saccharification power (P<0.01) and liquefaction power (P<0.01), while *Streptomyces* showed a significant negative correlation with both saccharification power (P<0.05) and liquefaction power (P<0.05). *Saccharopolyspora* also showed a significant positive correlation with flavor compounds such as 2-heptanone (P<0.01), caryophyllene (P<0.01), linalool (P<0.05), and phenylethyl alcohol (P<0.05); but a negative correlation with γ-nonanolide (P<0.05). These results systematically reveal the correlation mechanism between the microbial community structure, physicochemical properties, and flavor metabolism in the enhancement of Daqu (a type of starter culture). The correlation analysis diagram between physicochemical indicators and microbial communities is shown below. Figure 11 As shown in the figure. The correlation analysis between the microbial community and potential marker metabolites is illustrated in the figure below. Figure 12 As shown.

[0065] Example 8: Application of Enhanced Daqu Quality Evaluation Methods Using the methods described in Examples 5-7, for 10 7 Three batches of fortified Daqu prepared at an inoculum concentration of CFU / mL were evaluated for quality. The results showed that the microbial community structure of the three batches of fortified Daqu was similar (Bray-Curtis distance <0.15), and the flavor compound profiles were consistent (the PCA score maps showed good overlap between batches), indicating that the fortification process of the present invention has good stability and reproducibility.

[0066] Based on correlation analysis, the following were selected. Saccharopolyspora, Streptomyces, Rhizopus The study identified functional core microbial communities, with linalool, 1-nonanol, and other microbial markers as marker metabolites. Liquefaction power and saccharification power were determined as key physicochemical control points, providing data and theoretical support for strengthening the directional cultivation of Daqu (a type of starter culture), regulating the parameters of Daqu production process, and shaping the flavor.

Claims

1. A Bacillus licheniformis, characterized in that, The bacterium is Bacillus licheniformis (Bacillus licheniformis) Bacillus licheniformis The ), is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 2026955.

2. The Bacillus licheniformis as described in claim 1, characterized in that, The Bacillus licheniformis was isolated from the Daqu (a type of starter culture) of Fenghuang-flavored Baijiu (Chinese liquor), and it has the following biological characteristics: Morphological characteristics: Forms round, milky white, dry colonies on LB solid medium with irregular edges, Gram-positive, and rod-shaped cells; Enzyme production characteristics: The maximum protease yield is 513 U / mL; Tolerance characteristics: It can maintain enzyme activity even at a high temperature of 65℃ and can grow and metabolize normally in an environment with a 9% vol alcohol concentration.

3. The Bacillus licheniformis as described in claim 1, characterized in that, Its 16S rDNA sequence is shown in SEQ.ID.NO.

1.

4. A method for preparing a type of aromatic-flavored fortified koji based on Bacillus licheniformis as described in claim 1, characterized in that, Includes the following steps: (1) Inoculate Bacillus licheniformis CCTCC NO: M 2026955 into LB solid slant medium and activate it at 35-40℃ for 18-24 hours; (2) Seed culture preparation: The activated strain was inoculated into LB liquid medium and cultured in a shaker at 35-40℃ and 150-200 rpm for 16-20 hours to obtain the seed culture; (3) Preparation of enhanced inoculum: Expand the seed culture to 1×10⁻⁶ according to the specified ratio. 8 CFU / mL was used to obtain the enhanced inoculum; (4) Strengthening the preparation of Daqu: Using the Fengxiang-type Daqu preparation process, in the wheat soaking or mixing process, the inoculation concentration is 10 5 -10 9 CFU / mL, the enhanced inoculum solution is evenly sprayed onto the koji-making raw materials; (5) Fermentation management: Fermentation management is carried out in accordance with the process of fermentation of Fengxiang type Daqu, including the stages of mold growth, mold drying, fire period, high fire, and post-fire. The temperature and humidity of the fermentation room are controlled and the fermentation cycle is 25-35 days. (6) Processing of the starter culture: After the starter culture is completed, it is dried on a drying rack and aged in the starter culture warehouse to obtain the Phoenix Fragrance type enhanced starter culture.

5. The method for preparing Fengxiang-type enhanced Daqu based on Bacillus licheniformis as described in claim 4, characterized in that, The concentration of the inoculation was 10. 7 CFU / mL.

6. A method for evaluating the quality of a phoenix-aroma type enhanced Daqu (a type of starter culture), characterized in that, Includes the following steps: (1) Test the conventional physicochemical properties of enhanced Daqu (a type of Chinese liquor); (2) Metagenomic sequencing technology was used to analyze the microbial community structure of enhanced Daqu, a sequencing library was constructed, and Venn analysis of α diversity and OTU distribution and LEfSe differential species analysis were performed to identify the core dominant bacterial genera and characteristic microbial biomarkers. (3) The volatile flavor compounds of the enhanced Daqu were analyzed using HS-SPME / GC-MS technology, and potential marker metabolites were screened by multivariate statistical analysis. (4) Correlation analysis was conducted between the microbial community structure and physicochemical indicators and flavor substances.

7. The quality evaluation method for a phoenix-aroma type enhanced Daqu (a type of starter culture) as described in claim 6, characterized in that, The routine physicochemical index tests include indicators such as moisture, acidity, starch content, saccharification power, liquefaction power, and protease activity.

8. The quality evaluation method for phoenix-aroma type enhanced Daqu as described in claim 6, characterized in that, The core dominant bacterial genus identified in the microbial community structure analysis should be *Saccharopolysporum* (Saccharopolysporum). Saccharopolyspora ), Straight-spined polysporum ( Saccharopolyspora rectivirgula ); Strengthening the Daqu (a type of starter culture) with Polysaccharide Polyporus spp. ( Saccharopolyspora The relative abundance of ) should reach 59-61%; Straight-spined polysporum ( Saccharopolyspora rectivirgula The relative abundance of ) should reach 70-71%.

9. The quality evaluation method for phoenix-aroma type enhanced Daqu as described in claim 6, characterized in that, The potential marker metabolites should include alcohols such as teratol, 1-nonanol, (S)-2-methylbutanol, linalool, and phenylethanol; Esters including γ-nonanolide; Ketones, including 2-heptanone; Flavor compounds including pyrazines such as 2-vinyl-6-methylpyrazine and 2,3,5-trimethylpyrazine, and caryophyllene.

10. Application of Bacillus licheniformis CCTCC NO: M 2026955 in Baijiu brewing as a saccharification and fermentation agent, enzyme preparation and / or flavor enhancer.

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