A kind of fortified Daqu and its preparation method and application

Abstract

The scheme discloses a kind of reinforced Daqu in Daqu preparation technical field, preparation includes the following steps: (1) respectively Bacillus stearothermophilus, Bacillus licheniformis and Saccharomyces cerevisiae are activated and expanded culture, obtain the expanded culture fluid of each bacterial species;(2) the expanded culture fluid of Bacillus stearothermophilus and Bacillus licheniformis obtained in step (1) is mixed with Daqu material powder and water, is uniformly stirred, is loaded into Daqu mould and is treading into Daqu blank;(3) the Daqu blank is inoculated and fermented using staged temperature fermentation process;(4) in the late stage of inoculated fermentation, when temperature drops to 35~38 ℃, the expanded culture fluid of Saccharomyces cerevisiae obtained in step (1) is evenly sprayed to the surface of Daqu blank for secondary inoculation, and continues to culture;(5) the matured Daqu block is dried and stored, and the reinforced Daqu is obtained.The reinforced Daqu of the application can realize metabolic complementation, efficiently synthesize acetoin and further convert into tetramethylpyrazine.

Description

Technical Field

[0001] This invention belongs to the field of Daqu preparation technology, and specifically relates to a fortified Daqu, its preparation method and application. Background Technology

[0002] Maotai-flavor liquor is one of the typical aroma types of traditional Chinese liquor. It is beloved by consumers for its prominent Maotai aroma, elegant and delicate flavor, mellow body, and long-lasting aftertaste. High-temperature Daqu (a type of starter culture) is the saccharification and fermentation agent and aroma-generating agent in the production of Maotai-flavor liquor; its quality directly affects the quality and flavor of the liquor.

[0003] Traditional high-temperature koji preparation employs natural inoculation and open solid-state fermentation, with cultivation temperatures reaching 60–65℃ during the process. Under these high-temperature conditions, most heat-sensitive microorganisms (such as yeasts and molds) are eliminated, resulting in a microbial community dominated by thermophilic Bacillus in the high-temperature koji. Studies have shown that the mechanism of action of thermophilic Bacillus and its produced enzymes in the high-temperature koji during the koji-making process is closely related to the composition of the characteristic aroma substances of soy sauce-flavored baijiu, and the aroma substances in the finished koji are one of the main sources of the soy sauce aroma in soy sauce-flavored baijiu.

[0004] Tetramethylpyrazine (TTMP), also known as tetramethylpyrazine, is an important health-promoting component in soy sauce-flavored baijiu (Chinese liquor). It possesses various physiological activities, including improving cardiovascular circulation, anti-oxidation, and liver protection. The biosynthetic pathway of tetramethylpyrazine is as follows: Microorganisms first convert carbon sources such as glucose and fructose into pyruvate through glycolysis. Pyruvate then generates acetoin under the action of α-acetolactate synthase (ALS) and α-acetolactate decarboxylase (ALDC). Acetonitrile then reacts with ammonia produced from the decomposition of amino acids through a non-enzymatic catalytic reaction to synthesize tetramethylpyrazine.

[0005] However, the koji-making temperature of high-temperature koji is as high as 60-65℃. Under this extreme environment, the microbial community naturally evolves, and the main dominant bacteria are heat-resistant Bacillus and some heat-resistant yeasts. The existing naturally inoculated koji has the following problems: (1) It is greatly affected by the season, region, raw materials and operating environment, and the number of functional strains that produce high-methylenepyrazine and acetoin fluctuates greatly, resulting in significant differences in the quality of base liquor between batches. (2) Although natural koji contains potential strains that produce acetoin and tetramethylpyrazine, the conversion efficiency of pyruvate to acetoin and tetramethylpyrazine is often low due to the imbalance of the bacterial community ratio or insufficient metabolic synergy. (3) The existing koji strengthening technology mostly uses single strains or room-temperature strains (such as lactic acid bacteria and common yeast). For example, although the introduction of mesophilic bacteria such as Lactobacillus bulgaricus can produce acetoin, they cannot tolerate the high temperature stage of koji making and die in large numbers in the middle of koji making, making it difficult to continuously participate in the Maillard reaction and enzymatic reaction to generate tetramethylpyrazine in the later stacking fermentation. However, using Bacillus alone often lacks the alcohol precursors provided by yeast and the regulation of anaerobic / microaerobic environments, which limits the upper limit of pyrazine synthesis.

[0006] Therefore, identifying a complex bacterial strain that can efficiently synthesize acetoin through metabolic complementarity and further convert it into tetramethylpyrazine, and applying it to the preparation of fortified Daqu (a type of starter culture), is a technical problem that urgently needs to be solved in the current upgrading of the Maotai-flavor liquor industry. Summary of the Invention

[0007] The present invention aims to provide a method for preparing enhanced Daqu containing a complex bacterial strain, so as to achieve metabolic complementarity for efficient synthesis of acetoin and further conversion into tetramethylpyrazine.

[0008] One method for preparing enhanced Daqu (a type of starter culture) in this scheme includes the following steps: (1) Bacillus stearothermophilus ( Geobacillus stearothermophilus ), Bacillus licheniformis ( Bacillus licheniformis ) and brewer's yeast ( Saccharomyces cerevisiae The bacteria were activated and cultured on a large scale to obtain the culture broth for each strain. (2) Mix the expanded culture medium of thermophilic Bacillus stearothermophilus and Bacillus licheniformis obtained in step (1) with water and mix well. Then, put the mixture into a mold and press it to make koji blanks. (3) A staged variable temperature fermentation process is used to cultivate bacteria for fermentation of the koji blanks; (4) In the later stage of fermentation, when the temperature drops to 35-38℃, the expanded culture of brewing yeast obtained in step (1) is evenly sprayed onto the surface of the koji for a second inoculation and continued to be cultured. (5) After the fermented koji blocks are dried and stored, the fortified koji is obtained.

[0009] Furthermore, the koji powder is at least one of wheat flour and pea flour.

[0010] Furthermore, in step (1), the viable count of each of the thermophilic Bacillus steatophilus, Bacillus licheniformis, and Saccharomyces cerevisiae in the expanded culture medium is ≥10. 9 CFU / mL.

[0011] Furthermore, in step (2), the ratio of viable bacteria of *Bacillus stearothermophilus* and *Bacillus licheniformis* is 3:5 to 5:3, the amount of expanded culture medium added to *Bacillus stearothermophilus* and *Bacillus licheniformis* is 0.5% to 2% of the weight of the koji powder, and the amount of water added is 35% to 45% of the weight of the koji powder. Here, "the amount of water added" does not include the water in the expanded culture medium.

[0012] Furthermore, in step (3), the bacterial fermentation includes the following five stages: Initial stage: Temperature controlled at 35-45℃, cultured for 3-5 days; Intermediate stage: Temperature controlled at 55-65℃, cultured for 5-7 days; Later stage: The temperature is controlled at 45-50℃, and the culture is carried out for 2-3 days; Post-ripening stage: Gradually lower the temperature to 35-38℃ and cultivate for 1-2 days; Low-temperature culture stage: After the second inoculation, continue to culture at 28-32℃ for 2-3 days.

[0013] Furthermore, in step (4), the time point for the second inoculation is when the temperature drops to 35-38℃ during the post-fermentation ripening stage, and the amount of expanded culture medium of brewing yeast added is 0.5%-1% of the weight of the koji powder.

[0014] Furthermore, in step (5), the drying temperature is 35-40℃, and the curd is dried until the moisture content of the curd block is ≤12%.

[0015] This application also seeks protection for the enhanced Daqu prepared by the above method.

[0016] The fortified koji prepared according to this application can efficiently synthesize acetoin through metabolic complementarity and further convert it into tetramethylpyrazine; therefore, this application also requests protection for the application of the above-mentioned fortified koji in the production of sauce-flavored baijiu.

[0017] Furthermore, the application involves mixing the enhanced Daqu with traditional Daqu at a weight ratio of 1:5 to 1:3, and then using the mixture for brewing sauce-flavored Baijiu.

[0018] The beneficial effects of this invention are: 1. This invention employs a combination of three bacteria: *Bacillus stearothermophilus*, *Bacillus licheniformis*, and *Saccharomyces cerevisiae*, which are responsible for acetoin synthesis, nitrogen source supply, and aroma synergy, respectively, forming a complete metabolic pathway and significantly increasing the yield of tetramethylpyrazine. Specifically: *Bacillus stearothermophilus* is heat-resistant and adapted to the high-temperature core stage of Daqu (a type of Chinese liquor); it produces high-temperature amylase and protease, efficiently degrading starch and protein, providing sufficient precursors (pyruvate and α-acetolactate) for acetoin synthesis; and *Bacillus stearothermophilus* possesses a complete 2,3-butanediol pathway, highly expressing α-acetolactate decarboxylase (ALDC) to directly synthesize acetoin; simultaneously, it produces ammonia, providing a nitrogen source for the conversion of acetoin to tetramethylpyrazine (TTMP). Bacillus licheniformis is resistant to medium to high temperatures, acids, and ethanol, making it a dominant functional bacterium in soy sauce-flavored koji (a type of starter culture). It produces high levels of acetoin and tetramethylpyrazine, and synergistically with Bacillus stearothermophilus to further amplify acetoin synthesis. It efficiently utilizes ammonia and acetoin to generate tetramethylpyrazine through a non-enzymatic reaction, enhancing the characteristic flavor of soy sauce-flavored koji. Brewing yeast rapidly proliferates in the medium and low-temperature stages of koji, converting fermentable sugars into ethanol, thus enhancing the koji and providing a microaerobic environment and metabolic substrates for Bacillus licheniformis. Its metabolic byproducts (such as pyruvate) can serve as precursors to acetoin.

[0019] 2. In this invention, the inoculation time of brewing yeast is delayed until the temperature drops to 35-38°C in the later stage of koji making, and a low-temperature culture period of 28-32°C is set after inoculation. This allows the brewing yeast to grow and metabolize at the optimal temperature, avoiding direct exposure of the brewing yeast during the high-temperature period (55-65°C) or the medium-temperature period (45-50°C), which would result in inactivation or metabolic inhibition. This ensures that the yeast can produce aroma substances during the koji making stage.

[0020] 3. The variable temperature fermentation process of the present invention (35-45℃, 55-65℃, 45-50℃, 35-38℃, 28-32℃) is perfectly matched with the temperature adaptation range of the three strains. Each strain plays a dominant role during its optimal temperature window, avoiding the problem of single strains becoming inactive in environmental changes.

[0021] 4. Through initial cultivation at 35-45℃, Bacillus licheniformis is allowed to grow fully at the optimal temperature and produce highly active proteases, which efficiently decompose the proteins in the raw materials into amino acids, providing sufficient nitrogen source precursors for the synthesis of tetramethylpyrazine.

[0022] 5. The invented enhanced Daqu can be used in combination with traditional Daqu without changing the original brewing equipment and process, making it easy to promote and apply in existing Baijiu production enterprises. Attached Figure Description

[0023] Figure 1 This is a gas chromatogram of the enhanced Daqu prepared in Example 2 of the present invention.

[0024] Figure 2This is a gas chromatogram of the enhanced Daqu prepared in Example 3 of the present invention.

[0025] Figure 3 This is a gas chromatogram of the enhanced Daqu prepared in Example 4 of the present invention.

[0026] Figure 4 This is a gas chromatogram of the enhanced Daqu prepared in Example 5 of the present invention. Detailed Implementation

[0027] The following detailed description illustrates the specific implementation method: Example 1: Activation and scale-up of bacterial strains 1. Source of microbial strains Thermophilic Bacillus stearothermophilus (CICC: 10267), Bacillus licheniformis (CICC: 10092), and Saccharomyces cerevisiae (CICC: 1308).

[0028] 2. Culture medium formulation Nutrient gravy agar slant medium (Bacillus activated): 5g peptone, 30g beef extract, 5g NaCl, 15g agar, 1L distilled water, pH 7.0; YPD medium (yeast activation): 10g yeast extract, 20g peptone, 20g glucose, 15g agar, 1L distilled water, natural pH; Liquid seed culture medium: 20g glucose, 15g peptone, 5g sodium chloride, 0.5g beef extract, 1L distilled water, pH 7.0.

[0029] 3. Activation and scale-up culture Bacillus thermophilus: Bacillus thermophilus was inoculated onto nutrient gravy agar slant medium and cultured at 55℃ for 20 h to prepare primary seed culture. The seed culture was scraped off the slant with sterile water to prepare a bacterial suspension. After heating in an 80℃ water bath for 10 min, it was inoculated at a 5% (v / v) inoculation rate into a 1000 mL Erlenmeyer flask containing 300 mL of liquid seed culture medium and cultured at 55℃ and 200 rpm for 24 h to prepare secondary seed culture (expansion culture medium).

[0030] Bacillus licheniformis: Bacillus licheniformis was inoculated onto nutrient gravy agar slant medium and cultured at 37°C for 20 h to prepare primary seed culture. This was then inoculated into liquid seed culture medium at a 5% (v / v) inoculation rate and cultured at 37°C and 200 rpm for 24 h to prepare secondary seed culture (expansion culture medium).

[0031] Saccharomyces cerevisiae: Saccharomyces cerevisiae was inoculated onto YPD slant medium and cultured at 30℃ for 24 h to prepare primary seed culture. Then, 5% (v / v) of the inoculum was added to YPD liquid medium and cultured at 30℃ and 180 r / min for 24 h to prepare secondary seed culture (expansion culture medium).

[0032] Example 2: Preparation of enhanced Daqu (a type of starter culture) 1. Pretreatment of koji materials Take 200 kg of wheat and grind it to obtain wheat flour (grinding degree: ≥80% passing through a 20-mesh sieve). Sterilize it with steam (121℃, 30 min) and cool it to below 50℃ to obtain koji powder.

[0033] 2. Single inoculation and preparation The secondary seed cultures of *Bacillus stearothermophilus* and *Bacillus licheniformis* prepared in Example 1 were mixed at a viable cell ratio of 1:1 to obtain a composite bacterial agent solution, and the total viable cell count was adjusted to 5 × 10⁻⁶. 9 CFU / mL. Take 2.4L of the compound microbial agent solution and dilute it with 80L of water (this helps the compound microbial agent solution to mix more evenly with the koji powder later). Mix the diluted compound microbial agent solution with 200kg of koji powder. Put the mixed koji powder into koji molds and press them to form koji blanks. After unmolding, let it stand for 1.5 hours and then transfer it to the koji room.

[0034] 3. Microbial culture and fermentation A staged, variable-temperature fermentation process is adopted: Preliminary stage (medium-temperature incubation period): Close the doors and windows of the fermentation room to allow the fermented koji to heat up naturally. Control the temperature at 35-45℃ and incubate for 4 days. During this stage, Bacillus licheniformis grows and metabolizes fully. Bacillus licheniformis efficiently produces proteases, breaking down the proteins in the raw materials into amino acids.

[0035] Intermediate stage (high-temperature incubation period): Gradually increase the temperature to 55-65℃ and incubate for 6 days. During this stage, *Bacillus stearothermophilus* grows vigorously, efficiently metabolizing the amino acids and sugars accumulated in the previous stage to produce acetoin. *Bacillus stearothermophilus* then forms spores and enters a dormant state.

[0036] Later stage (mesothermal recovery period): Gradually ventilate and cool down to 45-50℃, and incubate for 2 days. During this stage, Bacillus licheniformis recovers from its spore state and continues to exert its metabolic functions.

[0037] Post-ripening stage: Continue to cool down, gradually reducing the temperature to 35-38℃, and culture for 1.5 days to prepare for the second inoculation.

[0038] 4. Second vaccination When the temperature of the starter culture drops to 35-38℃, take the secondary seed culture of brewer's yeast prepared in Example 1 and adjust the total viable count to 3×10⁻⁶. 9CFU / mL; spray evenly onto the surface of the koji blank at 0.8% (i.e., 1.6L) of the koji powder weight.

[0039] 5. Low-temperature culture stage After the second inoculation, the yeast was cultured at 28–32°C for another 2.5 days. During this stage, the brewer's yeast grew and multiplied at the optimal temperature, producing a variety of aroma precursors, which, in synergy with acetoin and amino acids, provided favorable conditions for the formation of tetramethylpyrazine.

[0040] 6. Drying and Storage After fermentation, the koji blocks are removed and air-dried at 35-40℃ until the moisture content is ≤12%. They are then stored in a koji warehouse for 30 days to obtain the finished fortified koji product.

[0041] The only difference between Example 3 and Example 2 is that Saccharomyces cerevisiae is not inoculated.

[0042] The only difference between Example 4 and Example 2 is that Bacillus licheniformis and Saccharomyces cerevisiae are not inoculated.

[0043] The only difference between Example 5 and Example 2 is that Bacillus licheniformis and Bacillus thermophilus are not inoculated.

[0044] In Examples 3-5, in the examples where no bacterial solution was inoculated, an equal amount of sterile water was used instead to maintain a consistent total water volume.

[0045] Detection of acetoin and tetramethylpyrazine content: Preparation of fermentation broth for testing: Glucose, fructose, and water (obtained by boiling and cooling purified water) were added to the fortified koji (the fortified koji product prepared in Examples 2-5) to obtain a mixed solution. The weight ratio of glucose to fructose was 1:1, and the total sugar content in the mixed solution was 200 g / kg. The weight ratio of koji, glucose, fructose, and sterile water in the mixed solution was 3:2:2:13. The mixed solution was fermented at 30-35℃ for 30 days (open fermentation for the first 10 days and closed fermentation for the last 20 days) to obtain the koji fermentation broth. The koji fermentation broth was centrifuged, and the supernatant was collected and filtered through qualitative filter paper to obtain the fermentation broth for testing.

[0046] The contents of acetoin and tetramethylpyrazine in the fermentation broth were detected using a gas chromatograph (Shimadzu GC-2010PLUS). For detection, 10 ml of the fermentation broth was taken, 0.2 ml of 2% amyl acetate internal standard was added, and the mixture was shaken well. Chromatographic analysis was performed using a known quantitative method. The chromatogram was read, and the content of the target peak was recorded. The results are detailed in the appendix. Figures 1-4 As can be seen from the attached figures, the contents of acetoin and tetramethylpyrazine in the fermentation broth of the enhanced Daqu prepared in Examples 2-5 are shown in the table below:

[0047] The data in the table above show that the combined use of thermophilic Bacillus stearothermophilus, Bacillus licheniformis, and Saccharomyces cerevisiae can significantly improve the detection of acetoin and tetramethylpyrazine content in fermentation broth.

[0048] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing enhanced Daqu (a type of starter culture), characterized in that: Includes the following steps: (1) The thermophilic Bacillus stearothermophilus, Bacillus licheniformis and Saccharomyces cerevisiae were activated and cultured on a large scale to obtain the culture medium of each strain; (2) Mix the expanded culture medium of thermophilic Bacillus stearothermophilus and Bacillus licheniformis obtained in step (1) with water and mix well. Then, put the mixture into a mold and press it to make koji blanks. (3) A staged variable temperature fermentation process is used to cultivate bacteria for fermentation of the koji blanks; (4) In the later stage of fermentation, when the temperature drops to 35-38℃, the expanded culture of brewing yeast obtained in step (1) is evenly sprayed onto the surface of the koji for a second inoculation and continued to be cultured. (5) After the fermented koji blocks are dried and stored, the fortified koji is obtained.

2. The method for preparing a strengthened Daqu (a type of starter culture) according to claim 1, characterized in that: The koji powder is at least one of wheat flour and pea flour.

3. The method for preparing a strengthened Daqu (a type of starter culture) according to claim 2, characterized in that: In step (1), the viable count of each of the thermophilic Bacillus steatophilus, Bacillus licheniformis, and Saccharomyces cerevisiae in the expanded culture medium is ≥10. 9 CFU / mL.

4. The method for preparing a strengthened Daqu (a type of starter culture) according to claim 3, characterized in that: In step (2), the ratio of viable bacteria of Bacillus stearothermophilus and Bacillus licheniformis is 3:5 to 5:3, the amount of expanded culture medium of Bacillus stearothermophilus and Bacillus licheniformis added is 0.5% to 2% of the weight of koji powder, and the amount of water added is 35% to 45% of the weight of koji powder.

5. The method for preparing a strengthened Daqu (a type of starter culture) according to claim 4, characterized in that: In step (3), the bacterial culture and fermentation process includes the following five stages: Initial stage: Temperature controlled at 35-45℃, cultured for 3-5 days; Intermediate stage: Temperature controlled at 55-65℃, cultured for 5-7 days; Later stage: The temperature is controlled at 45-50℃, and the culture is carried out for 2-3 days; Post-ripening stage: Gradually lower the temperature to 35-38℃ and cultivate for 1-2 days; Low-temperature culture stage: After the second inoculation, continue to culture at 28-32℃ for 2-3 days.

6. The method for preparing a strengthened Daqu (a type of starter culture) according to claim 5, characterized in that: In step (4), the second inoculation is performed when the temperature drops to 35-38℃ during the post-fermentation ripening stage, and the amount of expanded culture medium of brewing yeast added is 0.5%-1% of the weight of the koji powder.

7. The method for preparing a strengthened Daqu (a type of starter culture) according to claim 6, characterized in that: In step (5), the drying temperature is 35-40℃, and the curd blocks are dried until the moisture content is ≤12%.

8. A type of enhanced Daqu (a type of Chinese liquor), characterized in that: The enhanced Daqu is prepared by the method described in any one of claims 1 to 7.

9. The application of the enhanced Daqu (a type of starter culture) as described in claim 8 in the production of Maotai-flavor liquor.

10. The application according to claim 9, characterized in that: The enhanced Daqu and traditional Daqu are mixed at a weight ratio of 1:5 to 1:3 and then used for brewing sauce-flavored Baijiu.

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