Method for strengthening pyrazine substances in high-temperature yeast based on mixed fermentation

Abstract

The invention relates to a method for strengthening pyrazine substances in high-temperature yeast based on mixed fermentation, and belongs to the technical field of wine brewing and yeast making. According to the method, mixed bacteria of kluyveromyces marxianus, high-temperature-resistant lactobacillus plantarum, bacillus licheniformis and pichia pastoris are combined, and organic / inorganic nitrogen source compounding and precursor addition are combined, so that flora metabolism synergy and efficient synthesis of pyrazine substances are realized. The fermentation process is further optimized through segmented fermentation, dynamic temperature and humidity control and a CO2-assisted regulation and control strategy. After fermentation, the pyrazine yield is increased by 54% through detection. After optimization, the total amount of pyrazine substances in the yeast for making hard liquor reaches 643.58 mg / kg, and the content of tetramethylpyrazine reaches 440.38 mg / kg. According to the method, the content and diversity of pyrazine substances are remarkably improved, and the quality of the yeast for making hard liquor is improved. In addition, by introducing a precursor slow release and energy field coupling mass transfer technology, the fermentation efficiency and the product quality are further enhanced, and a new way is provided for wine quality optimization.

Description

Technical Field

[0001] The present invention relates to the technical field of wine making and koji making, in particular to a method for enhancing pyrazine substances in high-temperature koji based on mixed bacteria fermentation. Background Art

[0002] High-temperature Daqu is the core fermentation agent for sauce-flavored liquor. The pyrazine substances (such as tetramethylpyrazine, trimethylpyrazine, etc.) produced by its metabolism are the key components that give liquor flavors such as burnt aroma and nutty aroma.

[0003] The current traditional koji making process relies on the microbial community in the natural environment and has the following defects:

[0004] (1) Low pyrazine content: The proportion of pyrazine-producing functional bacteria in the natural fermentation microbiome is insufficient, resulting in low efficiency in synthesizing the target substance. Extensive process control: Single-temperature fermentation inhibits the synergistic metabolism of thermotolerant and mesophilic bacteria, resulting in insufficient utilization of precursor substances. (2) Single flavor: The existing process is difficult to achieve the diversity of pyrazine substances (such as alkylpyrazine, methoxypyrazine, etc.). (3) Failure to solve the problems of multi-species synergistic metabolism and process adaptability.

[0005] Therefore, there is an urgent need for a method for enhancing the ability of high-temperature Daqu to produce multiple pyrazines through mixed fermentation. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a method for enhancing the production of pyrazines in high-temperature Daqu based on mixed-bacteria fermentation. The present invention is based on a mixed-bacteria directional enhancement koji-making process with cross-feeding and segmented fermentation. Through the sequential co-activation and segmented fermentation of Kluyveromyces marxianus, thermostable Lactobacillus plantarum, Bacillus licheniformis and aroma-producing Pichia pastoris, combined with the organic / inorganic nitrogen source compound + precursor addition for directional regulation, the synergistic metabolism of the bacterial community and the efficient synthesis of pyrazines are achieved. The method of the present invention can improve the quality of Baijiu Daqu and is suitable for the quality optimization of Baijiu and other flavored wines.

[0007] The present invention is achieved through the following technical solutions:

[0008] The purpose of the present invention is to provide a method for enhancing the content of high-temperature pyrazine substances by mixed bacteria fermentation, comprising the following steps:

[0009] (1) activating Kluyveromyces marxianus, thermostable Lactobacillus plantarum, Bacillus licheniformis and Pichia pastoris respectively, mixing them, and further activating them in a complex nitrogen source medium to obtain activated strains;

[0010] (2) After the crushed wheat is moistened, it is mixed with the activated bacteria obtained in step (1). The inoculation amount is 3×10 5 CFU / g~5×10 5CFU / g, fermented dynamically in temperature and humidity in stages under the conditions of low-frequency ultrasound and intermittent high-frequency microwave, and pyrazine synthesis precursors were added in batches during the fermentation process.

[0011] Furthermore, in step (1), the amount of Kluyveromyces marxianus used is 5×10 5 CFU / mL-1.0×10 6 CFU / mL; the usage of the thermostable Lactobacillus plantarum is 7.5×10 6 CFU / mL-8×10 6 CFU / mL; the usage of the Bacillus licheniformis is 1.5×10 7 CFU / mL-2×10 7 CFU / mL; the amount of the aroma-producing Pichia yeast used was 1.0×10 5 CFU / m-1.2×10 5 CFU / mL.

[0012] Furthermore, in step (1), the activation conditions in the complex nitrogen source culture medium are: culturing at 30°C-32°C, 100rpm-110rpm for 24h-26h; and the pH value is 6.0-6.5.

[0013] Furthermore, in step (1), the composite nitrogen source culture medium includes an organic nitrogen source, an inorganic nitrogen source and a pyrazine precursor substance.

[0014] Furthermore, the organic nitrogen source includes yeast extract and / or soybean peptide;

[0015] The added amount of the yeast extract powder is 5wt%-10wt%; the added amount of the soybean peptide is 3wt%-5wt%;

[0016] and / or, the inorganic nitrogen source comprises ammonium sulfate and / or sodium nitrate;

[0017] The amount of ammonium sulfate added is 10wt%-15wt%; the amount of sodium nitrate added is 2wt%-4wt%.

[0018] Furthermore, the pyrazine precursor is obtained by compounding glycine and alanine;

[0019] And / or, the added amount of the pyrazine precursor is 0.1 wt%-0.3 wt%.

[0020] Furthermore, in step (2), the conditions of the low-frequency ultrasound are: frequency of 20kHz-25kHz; power of 90W-100W; ultrasound for 10min-15min every 3h-5h.

[0021] Furthermore, in step (2), the conditions of the intermittent high-frequency microwave are: frequency of 2.25GHz-2.45GHz; power of 200W-300W; microwave for 3min-5min every 10h-12h.

[0022] Furthermore, in step (2), the staged temperature and humidity dynamic fermentation includes the following steps: in the first stage, the temperature is increased and maintained at 45°C-50°C and the humidity is 70%-75% for 0-48 hours; in the second stage, the temperature is increased and maintained at 55°C-60°C for 49 hours-120 hours, and the humidity is controlled at 60%-65%; in the third stage, the temperature is lowered to 40°C-45°C and the humidity is 50%-55% for 121 hours-168 hours.

[0023] Furthermore, in step (2), the pyrazine synthesis precursor is one or more of α-ketoglutarate-alanine microcapsules, 1,2-dicarbonyl microcapsules and 1,2-diamine microcapsules.

[0024] The above technical solution of the present invention has the following advantages over the prior art:

[0025] (1) The present invention provides a method for enhancing the production of pyrazines in high-temperature Daqu based on mixed-bacteria fermentation. The present invention introduces Bacillus licheniformis to assist in enzyme production, and simultaneously activates the strain using a combination of organic / inorganic nitrogen sources and the addition of precursors to the culture medium. Compared with the traditional method, the pyrazine production detected at the end of fermentation increased by 54%. After optimization, the total amount of pyrazines in the Daqu reached 643.58 mg / kg, and the content of tetramethylpyrazine reached 440.38 mg / kg.

[0026] (2) The present invention dynamically couples the temperature and humidity of the fermentation environment and adds CO2 to assist in regulation. Compared with traditional static temperature and humidity control (temperature fluctuation > ±3°C), the bacteria rate is found to decrease by 70%, and the fermentation cycle is shortened and the total fermentation time is shortened by 20%. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0028] Figure 1 Comparison of the total amount of pyrazines in the examples of the present invention and the comparative example Daqu;

[0029] Figure 2 The figure is a comparison of the tetramethylpyrazine content in the Daqu of the embodiment of the present invention and the comparative example. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0032] The Kluyveromyces marxianus CICC 1276, thermostable Lactobacillus plantarum CICC 24833, Bacillus licheniformis CICC 10291, Pichia pastoris CICC 33194, and Wickhamella anomala CICC 31460 used in the following examples were purchased from the China Industrial Microbial Culture Collection Center.

[0033] The culture medium used in the following examples is as follows:

[0034] (1) Yeast universal medium (YPD-X): glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, KH2PO4 3 g / L (pH 6.0, sterilized at 121°C for 15 min).

[0035] (2) Modified MRS medium (MRS-T): peptone 10 g / L, beef extract 8 g / L, yeast extract 4 g / L, glucose 15 g / L, Tween 80 1 mL / L, sodium acetate 5 g / L, MgSO4 0.2 g / L, MnSO4 0.05 g / L (pH 6.5, anaerobic sterilization).

[0036] (3) LB-G medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L, glycerol 5 g / L (pH 7.0, sterilized at 121°C for 15 min).

[0037] (4) Complex nitrogen source final medium (CNM): 10 g / L soy peptone, 5 g / L corn steep liquor, 15 g / L glucose, 2 g / L KH2PO4, and 0.5 g / L MgSO4. The medium was prepared using 8% yeast extract and 4% soybean peptide as organic nitrogen sources, 12% ammonium sulfate and 3% sodium nitrate as inorganic nitrogen sources, and 0.1% glycine-alanine complex (molar ratio 1:1) as a pyrazine synthesis precursor.

[0038] Example 1: Stepwise activation and optimized combination of organic nitrogen sources

[0039] This embodiment provides a method for enhancing pyrazines in high-temperature Daqu based on mixed bacteria fermentation, and the specific steps are as follows:

[0040] (1) Strain activation treatment: Kluyveromyces marxianus CICC 1276 (1.0×10 6 CFU / mL), thermostable Lactobacillus plantarum CICC 24833 (8×10 6 CFU / mL), Bacillus licheniformis CICC10291 (2×10 7 CFU / mL) and Pichia pastoris CICC33194 (1.0×10 5 CFU / mL) were activated separately and then compounded as follows:

[0041] 1) Kluyveromyces marxianus: inoculate into YPD-X medium and culture with shaking at 30°C and 180 rpm for 24 h.

[0042] Pichia pastoris: inoculate into YPD-X medium and culture at 28°C for 36 hours.

[0043] Thermoresistant Lactobacillus plantarum: inoculated into MRS-T medium and cultured anaerobically at 37°C (anaerobic jar + gas-producing bag) for 48 hours.

[0044] Bacillus licheniformis: Inoculate into LB-G medium and culture at 37°C and 200 rpm with shaking for 24 h.

[0045] 2) Mix Kluyveromyces marxianus and Pichia pastoris cultures at a volume ratio of 1:1, transfer to fresh YPD-X medium, and culture at 28°C for 12 h to obtain a yeast complex solution.

[0046] After centrifugation and washing, the thermostable Lactobacillus plantarum and Bacillus subtilis were inoculated into CNM culture medium at a ratio of 2:1, and cultured at 37°C microaerophilically (stationary) for 18 hours to obtain a bacterial complex solution.

[0047] 3) Composite activation

[0048] The yeast complex solution and the bacterial complex solution were added to the CNM culture medium at a ratio of 3:1, and cultured at 30°C and 100 rpm with shaking for 24 h, with the pH value maintained at 6.0-6.5.

[0049] (2) Daqu fermentation process: Wheat is used as raw material, cleaned and impurities removed, then crushed, passed through a 40-mesh sieve, and the coarse-fine ratio is 6:4. After reaching the appropriate particle size, the crushed material is placed in a clean container. Then, according to the characteristics of the raw material, 40% of clean water is slowly added, and at the same time, the activated strain obtained in step (1) is inoculated into the Daqu raw material and fully mixed. Based on the mass of the koji block, the inoculation amount is 4×10 5CFU / g; Subsequently, the mixed wet material was filled into a mold and pressed into a specific brick-shaped koji for fermentation. The specific fermentation conditions are as follows: three-stage temperature and humidity dynamic fermentation was carried out under the conditions of low-frequency ultrasound (25kHz, 100W, 10 minutes every 3 hours) and intermittent high-frequency microwave (2.45GHz, 300W, 3 minutes every 12 hours). The fermentation process was carried out by exogenously providing pyrazine synthesis precursors. Based on the key metabolic window period of pyrazine synthesis (73h, 96h, and 120h), α-ketoglutaric acid-alanine microcapsules (1g α-ketoglutaric acid-alanine microcapsules per kilogram of Daqu) were added in three times, with a segmented addition ratio of 3:4:3 to match the bacterial flora metabolic window period (73h, 96h, and 120h).

[0050] The sustained-release properties of α-ketoglutarate-alanine microcapsules match the metabolic rhythm of the bacterial flora, ensuring that the precursor is continuously released and efficiently utilized in the high-temperature stage (60°C), avoiding substrate waste or toxic inhibition caused by one-time addition.

[0051] Among them, the three-stage temperature and humidity dynamic control: 0-48 hours: when fermentation starts, slowly rise from room temperature to 48°C within 12 hours, and then maintain the temperature at 48°C and the humidity at 75% to promote rapid proliferation of bacteria; 49h-73h slowly increase the temperature to 60°C, 74h-120h maintain 60°C and control the humidity at 65%, triggering the Maillard reaction and microbial pyrazine synthesis pathway; 121h-168h slowly cool down to 45°C and humidity 55%, stabilize flavor substances and inhibit miscellaneous bacteria.

[0052] This embodiment utilizes the ultrasonic cavitation effect to promote the rupture of microcapsule wall materials and the diffusion of intracellular metabolites. At the same time, the microwave thermal effect enhances the permeability of bacterial membranes and activates the activity of high-temperature resistant enzymes, thereby achieving a dual enhancement of mass transfer and reaction rate by the energy field.

[0053] After testing, the total amount of pyrazines in the Daqu obtained in Example 1 reached 643.58 mg / kg, and the content of tetramethylpyrazine was 440.38 mg / kg, which was 54% higher than that of the traditional process; the fermentation efficiency was increased by 19%; the survival rate of the key functional bacteria Bacillus licheniformis was >90%, confirming that its high temperature tolerance and metabolic activity were significantly optimized. Among them, the traditional process is that the traditional Daqu fermentation process is a solid-state fermentation process of natural inoculation; first, raw materials such as wheat and barley are crushed and mixed with water, and then manually pressed into brick-shaped koji blanks, and then placed in the koji room for natural fermentation. During fermentation, it relies on the natural growth of microorganisms (molds, yeasts, bacteria) in the environment, and adjusts the temperature and humidity by "turning the koji", going through three stages of "molding", "high temperature" and "post-fire" (about 30-40 days), and finally forming a mature koji block covered with mycelium and emitting koji fragrance. The whole process does not add artificial strains, and relies entirely on the raw materials, environment and the experience of the master craftsman.

[0054] Comparative Example 1

[0055] This comparative example provides a method for enhancing the pyrazines in high-temperature Daqu based on mixed fermentation, which is similar to Example 1 except that: the aroma-producing Pichia pastoris is replaced by abnormal Wickham yeast (10 5 CFU / mL), and the rest of the operations were consistent with those in Example 1.

[0056] Because the abnormal Wickham yeast has a temperature tolerance limit of only 50°C, which is lower than the process's high-temperature stage of 55°C, and lacks the pyruvate decarboxylase gene, it cannot efficiently convert α-ketoglutarate. Testing in this comparative example revealed a total pyrazine content of 341.26 mg / kg, with a tetramethylpyrazine content of 263.13 mg / kg. This significantly reduced yield demonstrates the irreplaceable nature of Pichia pastoris.

[0057] Comparative Example 2

[0058] This comparative example provides a method for enhancing pyrazines in high-temperature Daqu based on mixed-bacteria fermentation, which is similar to Example 1, except that only strains of Kluyveromyces marxianus, thermostable Lactobacillus plantarum, and Bacillus licheniformis are used, and the other operations remain the same.

[0059] The absence of aroma-producing Pichia yeast impaired its core functions—ester synthesis and pyrazine precursor delivery. This resulted in the inability of α-ketoglutaric acid to be effectively converted into pyrazine intermediates after release from the microcapsules, significantly reducing precursor utilization. Furthermore, the reduced ester content reduced the flavor diversity of the bacterial metabolites, and the lack of antagonistic activity against other bacteria increased the contamination rate to 1.2%. Testing revealed that the total amount of pyrazines produced in this comparative example reached 251.94 mg / kg, with a tetramethylpyrazine content of 164.73 mg / kg.

[0060] Comparative Example 3

[0061] This comparative example provides a method for enhancing the production of pyrazines in high-temperature Daqu based on mixed-bacteria fermentation. The method is similar to Example 1, except that the coupled mass transfer conditions are changed, i.e., intermittent high-frequency microwaves are not used during the fermentation process, and the other operations remain the same.

[0062] Based on Example 1, the microwave-assisted effect was eliminated, and only low-frequency ultrasound was retained. The remaining conditions (bacteria ratio, precursor addition method, temperature and humidity, etc.) were consistent with Example 1. Due to the reduced energy field intensity, the ultrasonic cavitation effect was insufficient to efficiently rupture the microcapsule wall material, resulting in a misalignment between the precursor release rate and the bacterial metabolic window period, and a decrease in the α-ketoglutaric acid conversion efficiency. At the same time, the lack of microwave thermal effect reduced the permeability of the bacterial membrane, limiting the activity of the thermostable enzyme of Bacillus licheniformis, further slowing down the reaction kinetics. Ultimately, the total amount of pyrazine was 426.25 mg / kg, an increase of 26% compared to Example 1, the tetramethylpyrazine content was 209.61 mg / kg, and the energy consumption was reduced by only 5%, proving the irreplaceable nature of high-intensity energy fields (ultrasound + microwave coupling) for the targeted release of microcapsule precursors and the stimulation of bacterial metabolic activity.

[0063] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for enhancing the content of high-temperature pyrazine substances by mixed bacterial fermentation, characterized in that: The following steps are involved: (1) activating Kluyveromyces marxianus, thermostable Lactobacillus plantarum, Bacillus licheniformis and Pichia pastoris respectively, mixing them, and further activating them in a complex nitrogen source medium to obtain activated strains; (2) After the crushed wheat is moistened, it is mixed with the activated bacteria obtained in step (1). The inoculation amount is 3×10 5 CFU / g~5×10 5 CFU / g, fermented dynamically in temperature and humidity in stages under the conditions of low-frequency ultrasound and intermittent high-frequency microwave, and pyrazine synthesis precursors were added in batches during the fermentation process.

2. The method according to claim 1, characterized in that In step (1), the amount of Kluyveromyces marxianus used is 5×10 5 CFU / mL-1.0×10 6 CFU / mL; the usage of the thermostable Lactobacillus plantarum is 7.5×10 6 CFU / mL-8×10 6 CFU / mL; the usage of the Bacillus licheniformis is 1.5×10 7 CFU / mL-2×10 7 CFU / mL; the amount of the aroma-producing Pichia yeast used was 1.0×10 5 CFU / m-1.2×10 5 CFU / mL.

3. The method according to claim 1, characterized in that In step (1), the activation conditions in the complex nitrogen source culture medium are: culturing at 30°C-32°C, 100rpm-110rpm for 24h-26h; and pH value of 6.0-6.

5.

4. The method according to claim 1, wherein In step (1), the composite nitrogen source culture medium comprises an organic nitrogen source, an inorganic nitrogen source and a pyrazine precursor substance.

5. The method according to claim 4, characterized in that The organic nitrogen source includes yeast extract and / or soybean peptide; And / or, the inorganic nitrogen source includes ammonium sulfate and / or sodium nitrate.

6. The method according to claim 4, characterized in that The pyrazine precursor is obtained by compounding glycine and alanine; And / or, the added amount of the pyrazine precursor is 0.1 wt%-0.3 wt%.

7. The method according to claim 1, characterized in that In step (2), the conditions of the low-frequency ultrasound are: frequency of 20kHz-25kHz; power of 90W-100W; ultrasound for 10min-15min every 3h-5h.

8. The method according to claim 1, characterized in that In step (2), the conditions of the intermittent high-frequency microwave are: frequency of 2.25 GHz to 2.45 GHz; power of 200 W to 300 W; microwave for 3 min to 5 min every 10 h to 12 h.

9. The method according to claim 1, characterized in that In step (2), the staged temperature and humidity dynamic fermentation includes the following steps: in the first stage, the temperature is increased and maintained at 45°C-50°C and the humidity is 70%-75% for 0-48 hours; in the second stage, the temperature is increased and maintained at 55°C-60°C for 49 hours-120 hours, and the humidity is controlled at 60%-65%; in the third stage, the temperature is lowered to 40°C-45°C and the humidity is 50%-55% for 121 hours-168 hours.

10. The method according to claim 1, characterized in that In step (2), the pyrazine synthesis precursor is one or more of α-ketoglutaric acid-alanine microcapsules, 1,2-dicarbonyl microcapsules and 1,2-diamine microcapsules.

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