Fermentation control process for Maotai-flavor liquor based on brewing environmental microorganisms

Abstract

The application discloses a kind of based on brewing environment microorganism's Maotai-flavor liquor fermentation regulation process, belongs to biological fermentation technical field, including bacillus licheniformis and pichia pastoris are enlarged culture by two stages, centrifugal resuspension and proportion mixing, composite control agent is made, simultaneously, add koji and stack and arrange temperature monitoring network when the window period temperature of grain stack reaches and out of fragrance, start to turn over heap equipment linkage even spray control agent, then, after two times, stack insulation development, after temperature stabilization, into cellar and carry out anaerobic fermentation, the application is by capturing grain stack core temperature window period directional supplement local dominant bacteria, utilize bacillus licheniformis and pichia pastoris synergistic metabolism, promote ester production, balance ethyl lactate, both ensure that the smooth progress of saccharification and aroma production in stacking stage, effectively prevent abnormal acid production in later period of dazao, realize the improvement of core flavor factor content of raw liquor.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, and in particular relates to a fermentation control process for Maotai-flavor liquor based on microorganisms in the brewing environment. Background Technology

[0002] As a typical representative of traditional Chinese distilled spirits, Maotai-flavor baijiu is known for its brewing process, which is characterized by high-temperature koji making, high-temperature stacking, high-temperature distillation, and long-term storage. In the unique open-air stacking fermentation process, the mash after steaming is cooled and mixed with high-temperature koji, and then piled into grain piles. It relies heavily on the natural collection and enrichment of microorganisms in the brewing workshop environment. These collected brewing microorganisms are rich in variety, mainly represented by bacterial communities such as Bacillus licheniformis. Through Maillard reaction and amino acid metabolism, they produce a large amount of important Maotai-flavor precursor substances such as tetramethylpyrazine and pyrazines. At the same time, it also includes aroma-producing yeast communities represented by Pichia kudria, which produce core ester flavor substances such as ethyl acetate through alcohol-ester conversion metabolism.

[0003] In traditional Maotai-flavor liquor fermentation, the excessive proliferation of lactic acid-producing bacteria during the pile fermentation process often leads to a high ethyl lactate content after the liquor is placed in the fermentation pit. This results in a dull liquor with high fusel oil content, which can easily cause adverse effects such as headache and dry mouth after drinking. Although Bacillus licheniformis is widely present in the brewing environment, it is easy for it to miss the optimal window for aerobic heat production and precursor accumulation in open grain piles. This results in a low tetramethylpyrazine content in the raw liquor of traditional Maotai-flavor liquor fermentation. The traditional Maotai-flavor liquor fermentation process involves directly sprinkling a single high-yielding strain of external bacteria into the mash. This strain is not only easily rejected and inactivated by the complex local microbial ecology of the fermentation pit, but also causes abnormal acid production in the later stages of fermentation due to the sudden surge of a single strain in the grain pile, thus disrupting the traditional flavor balance of Maotai-flavor liquor. Summary of the Invention

[0004] The purpose of this invention is to address the problems of large quality fluctuations, low yield of core flavor factors, and imbalance of ester ratios caused by the random microbial gathering during the open-piling fermentation of traditional Maotai-flavor liquor. Therefore, this invention proposes a fermentation control process for Maotai-flavor liquor based on microorganisms in the brewing environment.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A fermentation control process for Maotai-flavor liquor based on microorganisms in the brewing environment includes sterilizing modified LB liquid culture medium and modified YPD liquid culture medium and then inoculating Bacillus licheniformis and Pichia pastoris kudricae for primary seed culture. The obtained primary seed culture was transferred to the corresponding fresh culture medium at the set inoculation amount for secondary seed culture propagation. The expanded secondary bacterial solution was subjected to freeze centrifugation, washing and sterilization, and bacterial resuspension. It was then aseptically mixed according to a set volume ratio to obtain a microbial composite regulator, which was then packaged into a stainless steel spray tank. After the sorghum lees are distilled, they are cooled, water is poured on them, and high-temperature Daqu powder is mixed in. They are then piled into a trapezoidal grain pile, and probes are inserted into different spatial locations of the grain pile to set up a multi-channel temperature monitoring network. When the average temperature of the grain pile reaches the window period temperature, and specific mycelia grow on the surface of the grain pile and produce a fruity aroma, start the turning and spraying linkage equipment to evenly spray the microbial compound regulator into the mash. After spraying, the mash is piled up again for secondary heat preservation and development. When the temperature stabilizes, the mash is moved into the pit, compacted, and covered with sealing mud, and then enters the anaerobic fermentation stage.

[0006] As a further description of the above technical solution: The modified LB liquid culture medium has the following formulation: 10.0 g / L tryptone, 5.0 g / L yeast extract, and 10.0 g / L sodium chloride, with the pH adjusted to 7.3 using 1 mol / L NaOH solution. The modified YPD liquid culture medium has the following formula: yeast extract 10.0 g / L, peptone 20.0 g / L, and glucose 20.0 g / L.

[0007] As a further description of the above technical solution: The specific steps for preparing the modified YPD liquid culture medium are as follows: dissolve yeast extract and peptone in deionized water to prepare bottle A as the base material, and dissolve glucose in deionized water to prepare bottle B as the sugar material; The base material in bottle A and the sugar material in bottle B are placed separately in a high-pressure steam sterilizer for sterilization. After sterilization, under the flame protection of an ultra-clean workbench, the liquid in bottle B is poured into the liquid in bottle A and mixed and shaken well.

[0008] As a further description of the above technical solution: The specific parameters for sterilization and primary seed culture are as follows: high-pressure steam sterilization is performed at 121°C and 0.12MPa for 20 minutes, and the sample is transferred to a clean bench when the temperature drops below 60°C. The Bacillus licheniformis BL-06 was cultured with continuous shaking at 37°C and 180 rpm for 24 h. The Pichia pastoris PK-02 was cultured with continuous shaking at 30°C and 150 rpm for 24 h.

[0009] As a further description of the above technical solution: The specific parameters for the secondary seed culture propagation are as follows: pipette the primary seed culture and transfer it at an inoculation rate of 5%, and continue to culture for 18 hours according to the temperature and rotation speed parameters of the primary seed culture. The propagation endpoint was defined as follows: Bacillus licheniformis was in the form of regular long rods, and the concentration of cells counted under a microscope was ≥1.5×10⁹ CFU / mL. Pichia kudria exhibits an oval budding state, with a microscopic cell count concentration ≥2.0×10⁸ CFU / mL.

[0010] As a further description of the above technical solution: The formulation of the microbial compound regulator includes: placing the secondary bacterial culture in a benchtop refrigerated centrifuge and centrifuging at 5000 rpm and 4°C for 15 min, discarding the supernatant, adding 0.9% sterile physiological saline to resuspend and wash, and repeating the refrigerated centrifugation once; The centrifuged and washed Bacillus licheniformis resuspension and Pichia pastoris resuspension were aseptically stirred in a 5L stainless steel spray tank at a volume ratio of 2:1 for 3 minutes.

[0011] As a further description of the above technical solution: The multi-channel temperature monitoring network is arranged as follows: sorghum lees are gathered into a trapezoidal pile with a height of 1.6m, and the PT100 long rod probe of the 16-channel temperature recorder is driven into the grain pile. The points are arranged including one at the geometric center of the grain pile, and one each at the core heat-generating layer 80cm away from the surface on the east, west, south, and north sides. The parameters are set to automatically record the temperature every 30 minutes.

[0012] As a further description of the above technical solution: The criteria for determining the precise intervention window period are: the average temperature at the five sites (east, west, south, north, and center) rises to 46.8°C, white disc-shaped mycelia are observed when the grain pile is pulled apart 5cm from the surface, and an apple aroma and an amino acid-soy sauce precursor aroma can be smelled.

[0013] As a further description of the above technical solution: The specific parameters for spraying the microbial compound regulator and fermenting in the cellar are as follows: adjust the pressure of the sprayer pressure regulating valve to 0.2MPa, and set the nozzle orifice diameter to 0.5mm to present a fan-shaped atomization surface; The dedicated tracked fully automatic compost turner for distiller's grains has a travel speed of 2m / min and a drum rotation speed of 220rpm; the spray flow rate is controlled to allow each ton of distiller's grains to adsorb 3000mL of microbial compound regulator. After turning and spraying, the pile is reassembled and kept warm for 24 hours. When the surface temperature of the pile stabilizes at 52.3°C, a loader is moved into the pit and compacted. The surface is covered with 10cm of sealing mud and transferred to anaerobic fermentation pit for 30 days.

[0014] As a further description of the above technical solution: When the obtained raw wine was diluted to an alcohol content of 53% vol, solid-phase microextraction and gas chromatography-mass spectrometry analysis showed that the content of tetramethylpyrazine was 8.92 mg / L, ethyl acetate was 2.42 g / L, ethyl lactate was 1.72 g / L, and the total ester content was 4.65 g / L. The ratio of ethyl acetate to ethyl lactate was 1.41. The wine exhibited a prominent soy sauce aroma and a long-lasting fragrance in the empty glass.

[0015] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: This invention captures the window of temperature change in the core of the grain pile and selectively introduces highly active native Bacillus licheniformis to drive the Maillard reaction and precursor metabolism during the pile fermentation stage. It also introduces aroma-producing Pichia pastoris for synergistic metabolism, promoting ethyl acetate production and balancing the excessive accumulation of ethyl lactate. By scientifically adjusting the strain ratio, a synergistic evolutionary balance between Bacillus licheniformis and Pichia pastoris in the solid-state mash is established. This ensures smooth saccharification and aroma production during the pile fermentation stage while effectively preventing abnormal acid production in the mash later due to the overgrowth of a single strain, thereby increasing the content of core flavor factors in the raw liquor. Attached Figure Description

[0016] Figure 1 This is a flowchart of a fermentation control process for Maotai-flavor liquor based on microorganisms in the brewing environment, as proposed in this invention. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Please see Figure 1 The present invention provides a technical solution: a fermentation control process for soy sauce-flavored liquor based on microorganisms in the brewing environment, which includes sterilizing modified LB liquid culture medium and modified YPD liquid culture medium and then inoculating Bacillus licheniformis and Pichia pastoris kudricae for primary seed culture. The obtained primary seed culture was transferred to the corresponding fresh culture medium at the set inoculation amount for secondary seed culture propagation. The expanded secondary bacterial solution was subjected to freeze centrifugation, washing and sterilization, and bacterial resuspension. It was then aseptically mixed according to a set volume ratio to obtain a microbial composite regulator, which was then packaged into a stainless steel spray tank. After the sorghum lees are distilled, they are cooled, water is poured on them, and high-temperature Daqu powder is mixed in. They are then piled into a trapezoidal grain pile, and probes are inserted into different spatial locations of the grain pile to set up a multi-channel temperature monitoring network. When the average temperature of the grain pile reaches the window period temperature, and specific mycelia grow on the surface of the grain pile and produce a fruity aroma, start the turning and spraying linkage equipment to evenly spray the microbial compound regulator into the mash. After spraying, the mash is piled up again for secondary heat preservation and development. When the temperature stabilizes, the mash is moved into the pit, compacted, and covered with sealing mud, and then enters the anaerobic fermentation stage.

[0019] The modified LB liquid medium formula is: 10.0 g / L tryptone, 5.0 g / L yeast extract, and 10.0 g / L sodium chloride. The pH value is adjusted to 7.3 using 1 mol / L NaOH solution. The formula for the modified YPD liquid culture medium is: yeast extract 10.0 g / L, peptone 20.0 g / L, and glucose 20.0 g / L.

[0020] The specific steps for preparing the modified YPD liquid culture medium are as follows: dissolve yeast extract and peptone in deionized water to prepare bottle A as the base material, and dissolve glucose in deionized water to prepare bottle B as the sugar material; The base material in bottle A and the sugar material in bottle B are placed separately in a high-pressure steam sterilizer for sterilization. After sterilization, under the flame protection of an ultra-clean workbench, the liquid in bottle B is poured into the liquid in bottle A and mixed and shaken well.

[0021] The specific parameters for sterilization and primary seed culture are as follows: high-pressure steam sterilization is performed at 121°C and 0.12MPa for 20 minutes, and the sample is transferred to a clean bench when the temperature drops below 60°C. Bacillus licheniformis BL-06 was cultured with continuous shaking at 37°C and 180 rpm for 24 h. Pichia kudria PK-02 was cultured with continuous shaking at 30°C and 150 rpm for 24 h.

[0022] The specific parameters for secondary seed culture propagation are as follows: pipette the primary seed culture at an inoculation rate of 5%, and continue to culture for 18 hours at the same temperature and rotation speed parameters as the primary seed culture. The propagation endpoint was defined as follows: Bacillus licheniformis was in the form of regular long rods, and the concentration of cells counted under a microscope was ≥1.5×10⁹ CFU / mL. Pichia kudria exhibits an oval budding state, with a microscopic cell count concentration ≥2.0×10⁸ CFU / mL.

[0023] The formulation of the microbial compound regulator includes: placing the secondary bacterial culture in a benchtop refrigerated centrifuge and centrifuging at 5000 rpm and 4°C for 15 min, discarding the supernatant, adding 0.9% sterile physiological saline to resuspend and wash, and repeating the refrigerated centrifugation once; The centrifuged and washed Bacillus licheniformis resuspension and Pichia pastoris resuspension were aseptically stirred in a 5L stainless steel spray tank at a volume ratio of 2:1 for 3 minutes.

[0024] The multi-channel temperature monitoring network is set up as follows: sorghum lees are gathered into a trapezoidal pile with a height of 1.6m. The PT100 long rod probe of the 16-channel temperature recorder is driven into the grain pile. The points are set up as follows: one at the geometric center of the grain pile, and one each at the core heat-generating layer 80cm away from the surface on the east, west, south and north sides. The parameters are set to automatically record the temperature every 30 minutes.

[0025] The criteria for determining the precise intervention window are: the average temperature at the five sites (east, west, south, north, and center) rises to 46.8°C, white disc-shaped mycelia are observed when the grain pile is pulled apart 5cm from the surface, and an apple aroma and an amino acid-soy sauce precursor aroma can be smelled.

[0026] The specific parameters for spraying the microbial compound regulator and fermenting in the cellar are as follows: adjust the pressure of the sprayer pressure regulating valve to 0.2MPa, and the nozzle orifice diameter to 0.5mm to present a fan-shaped atomization surface; The dedicated tracked fully automatic compost turner for distiller's grains has a travel speed of 2m / min and a drum rotation speed of 220rpm; the spray flow rate is controlled to allow each ton of distiller's grains to adsorb 3000mL of microbial compound regulator. After turning and spraying, the pile is reassembled and kept warm for 24 hours. When the surface temperature of the pile stabilizes at 52.3°C, a loader is moved into the pit and compacted. The surface is covered with 10cm of sealing mud and transferred to anaerobic fermentation pit for 30 days.

[0027] When the obtained raw wine was diluted to an alcohol content of 53% vol, solid-phase microextraction and gas chromatography-mass spectrometry analysis showed that the content of tetramethylpyrazine was 8.92 mg / L, ethyl acetate was 2.42 g / L, ethyl lactate was 1.72 g / L, and the total ester content was 4.65 g / L. The ratio of ethyl acetate to ethyl lactate was 1.41. The wine exhibited a prominent soy sauce aroma and a long-lasting fragrance in the empty glass.

[0028] (1) Experimental materials: Microbial strains: Bacillus licheniformis and Pichia pastoris Reagents: tryptone, yeast extract, sodium chloride, deionized water, yeast extract, peptone, glucose, deionized water.

[0029] (2) Equipment: high pressure steam sterilizer, constant temperature shaking incubator, ultra-clean workbench, multi-channel microcomputer temperature recorder, high precision electric sterile spray device, special tracked fully automatic turning machine for distiller's grains, gas chromatography-mass spectrometry, solid phase microextraction device.

[0030] (3) Experimental steps: 1. Culture medium preparation: Preparation of modified LB liquid culture medium: Tryptone: 10.0g (Biochemical reagent grade, BR, purity) ); Yeast extract: 5.0g (biochemical reagent grade, BR); Sodium chloride: 10.0g (analytical grade, AR grade, purity) ); Accurately weigh 10.00g of biochemical reagent-grade tryptone, 5.00g of biochemical reagent-grade yeast extract, and 10.00g of analytical grade sodium chloride using an electronic balance. Pour all ingredients into a 1000mL beaker. Add 850mL of deionized water to the beaker, place a magnetic stir bar inside, and put the beaker on the magnetic stirrer. Turn on the stirrer at 400rpm until the solids are completely dissolved. Immerse the precision pH meter probe in the solution and add 1mol / L NaOH solution dropwise using a high-pressure burette until the pH value is precisely set to 7.3. Remove the stir bar, transfer the solution to a 1000mL graduated cylinder, and dilute to 1000mL with deionized water. Divide the solution into 10 equal 500mL Erlenmeyer flasks (100mL each), plug each Erlenmeyer flask with a medical cotton plug, wrap the outer layer with double layers of kraft paper, and secure with a high-temperature resistant rubber band.

[0031] Preparation of modified YPD liquid culture medium: Yeast extract: 10.0g (Biochemical reagent grade, BR); Peptone: 20.0g (Biochemical reagent grade, BR); Glucose: 20.0g (analytical grade, AR grade); Preparation of Bottle A (basic ingredients): Weigh 10.00g of yeast extract and 20.00g of peptone, dissolve in 800mL of deionized water, stir well, no need to adjust the pH value, dispense into 10 500mL Erlenmeyer flasks (80mL each), and seal; Preparation of Bottle B (Sugar): Weigh 20.00g of analytical grade glucose, dissolve it in 100mL of deionized water, bring the volume up to 100mL, dispense it into 10 50mL Erlenmeyer flasks (10mL per flask), and seal the flasks.

[0032] 2. Preparation of reagents and materials: sterile saline ( %NaCl): 9.0g of industrial grade or analytical grade NaCl is dissolved in 1000mL of purified water as a cell washing solution and resuspension dilution solution.

[0033] Sorghum for brewing: High-quality glutinous sorghum that meets the national standard GB / T8231, requiring a total starch content of [missing information]. The proportion of amylopectin in total starch , moisture .

[0034] High-temperature Daqu: High-temperature Daqu (sauce aroma type) prepared using traditional techniques from the core production area of ​​Maotai Town. The required powder content is 60% to 70% that passes through a 20-mesh sieve (0.85mm aperture).

[0035] Analytical grade sodium chloride: Before use, it is baked in a muffle furnace at 450°C for 4 hours to completely eliminate background organic impurities that may interfere with GC-MS detection.

[0036] Chromatographically pure internal standard: 4-methyl-2-pentanol (CAS No.: 108-11-2) or 2-octanol, purity (Chromatographic grade, GC grade).

[0037] GC-MS carrier gas: high-purity helium ( ),purity .

[0038] 3. High-pressure steam sterilization: Check the distilled water level in the sterilizer to ensure it is within the normal range. Place the prepared LB bottles and YPD-A bottles in the upper sterilization basket and the YPD-B bottles in the lower basket. Close the sterilizer door, tighten the safety handwheel, turn on the power, and select the liquid sterilization mode. The main program heats up to 121°C, exhausts the cold air, and maintains a pressure of 0.12MPa for 20 minutes. Then, it cools down and depressurizes naturally. When the pressure gauge of the sterilizer returns to zero and the temperature drops below 60°C, wear heat-resistant gloves to remove the triangular bottle and move it into the ultra-clean workbench. Under the protection of an alcohol lamp flame in a clean bench, pour 10 mL of sterilized glucose solution (bottle B) into 80 mL of YPD base material (bottle A), shake well, and prepare complete YPD liquid culture medium (90 mL per bottle).

[0039] 4. Primary seed culture: Turn on the sterile fan and UV lamp of the laminar flow 30 minutes in advance. Before operation, wipe your hands and the outer wall of the alcohol lamp with 75% alcohol cotton balls, and light the alcohol lamp in the center of the workbench to form a sterile operating area with a diameter of 10cm; Take out the preserved BL-06 culture tube, heat the inoculation loop over an alcohol lamp flame until it is red-hot, cool it against the inner wall of sterile stock solution or blank medium, pick up a loopful of colonies, quickly place it in 100mL LB medium, gently shake the inoculation loop to detach it, pass the bottle mouth over a flame 3 times, plug it with cotton plug, and use the same method to pick up PK-02 colonies and inoculate them into 90mL LYPD medium. Place the BL-06 bottle in a shaker and set the parameters to 37°C and 180 rpm for continuous incubation for 24 hours. Place the PK-02 bottle into another shaker, set the parameters to temperature 30°C and rotation speed 150 rpm, and incubate continuously for 24 hours.

[0040] 5. Secondary seed liquid propagation: Two hours before the end of the first stage of culture, prepare 10 fresh 100mL LB medium bottles and 10 90mL LYPD medium bottles. Under the flame protection of a clean bench, 5 mL of BL-06 primary seed culture was aspirated with a sterile pipette and added to a new LB medium (5% inoculum). A total of 10 bottles were transferred. Similarly, 4.5 mL of PK-02 primary seed culture was aspirated with a sterile pipette and added to a new YPD medium. A total of 10 bottles were transferred. Continue culturing for 18 hours using the same temperature and rotation speed parameters as the primary culture stage; Samples were observed under a microscope; BL-06 appeared as regular, long rods, free of contaminating bacteria. The bacterial concentration was determined using a micro-counting chamber. PK-02 exhibits an oval budding state, with a cell concentration of... .

[0041] 6. Compound formulation of microbial regulators: Aliquot 1000 mL of the secondary cultured Bacillus subtilis culture and 900 mL of yeast culture into 50 mL sterile centrifuge tubes. Place them in a benchtop refrigerated centrifuge and set the parameters as follows: speed 5000 rpm, temperature 4°C, centrifugation time 15 min. After centrifugation, carefully discard the supernatant in a clean bench. Add an equal volume of 0.9% sterile saline to the centrifuge tubes and resuspend (wash) them with a shaker. Repeat the above centrifugation steps once. The two bacterial cells were resuspended separately in sterile physiological saline, and diluted to obtain a count of 1 in the Bacillus licheniformis resuspension. The count of Pichia pastoris resuspension was... Take 2000 mL of Bacillus licheniformis resuspension and 1000 mL of Pichia pastoris resuspension, pour them into a sterilized 5L stainless steel spray tank, and stir with a sterile stirring rod for 3 minutes to ensure complete mixing. The volume ratio in the compound emulsion is 2:1.

[0042] 7. Workshop stacking and fermentation: After the sorghum mash is steamed and removed from the still, water is poured in, and 12% Daqu powder is mixed in, it is manually or by a stacking machine gathered into a trapezoidal grain pile with a height of 1.6m. Immediately, the PT100 long rod probe of the 16-channel temperature recorder is driven into the grain pile. There is one at the geometric center of the grain pile and one at each of the core heat-generating layers 80cm away from the surface on the east, west, south, and north sides. Data is automatically recorded every 30 minutes. When the average temperature at the five locations (east, west, south, north, and center) rises to 46.8°C (allowable error) When the grain pile is pulled apart and a distinct white disc-shaped mycelium can be seen 5cm from the surface, and a refreshing apple aroma and a faint amino acid-based soy sauce-like precursor aroma can be smelled, it is immediately determined to be the precise intervention window period. A stainless steel sprayer containing a compound regulator is mounted on the front crossbeam of a tracked fully automatic compost turner. The sprayer pressure regulating valve is adjusted to 0.2MPa, the electric switch is turned on, and the nozzle (0.5mm orifice diameter) is tested to see if it presents a stable micron-level fan-shaped atomization surface. The compost turner is started and moved forward at a travel speed of 2m / min and a drum speed of 220rpm. At the moment when the compost turner breaks up and throws the mash, the atomized regulator is evenly attached to the surface of the mash particles under high pressure. The total spraying flow rate is controlled so that each ton of mash ultimately adsorbs 3000mL of regulator. After turning and spraying, immediately use a bag or grab bucket to gather the mash back into a pile, maintaining its original height for secondary heat preservation and development. At this time, the introduced local functional bacteria quickly take over the ecology of the grain pile. Continue to monitor for 24 hours. When the surface temperature of the pile rises sharply and stabilizes at 52.3°C, it indicates that the Maillard reaction and aroma enrichment have reached their peak. Immediately move the mash into a stainless steel or stone-walled pit, compact it, and cover the surface with 10cm of special sealing mud for strong aroma or soy sauce aroma. Then, it enters the 30-day anaerobic fermentation stage.

[0043] 8. Control group setup: Control group 1: Low-proportion high-concentration regulation mode; Bacillus licheniformis liquid: Pichia pastoris liquid volume ratio 1:2, compound regulator sprayed per ton of mash On the third day of fermentation, when the core temperature reached 45.2°C, the pile was turned and sprayed. After 24 hours of secondary fermentation, the temperature at the top of the pile reached 50.5°C, and then it was put into the pit.

[0044] Control group 2: Balanced and optimized regulatory mode; Bacillus licheniformis liquid: Pichia pastoris liquid volume ratio 2:1, compound regulator sprayed per ton of mash On the fourth day of fermentation, when the core temperature reached 46.8°C, the pile was turned and sprayed precisely. This formula fully stimulated the interaction between strains. After 24 hours of secondary fermentation, the surface of the grain pile was covered with dense white mycelium, and the temperature at the top of the pile reached 52.3°C. The aroma of soy sauce was extremely rich, and the grain was then put into the cellar.

[0045] Control group 3: High proportion of high-temperature biased mode; Bacillus licheniformis liquid: Pichia pastoris liquid volume ratio 4:1, compound regulator sprayed per ton of mash The spraying was delayed until the 4.5th day of fermentation, when the core temperature reached 48.0°C. Due to the high proportion of Bacillus and its strong heat resistance, the temperature at the top of the pile rose sharply to 54.1°C after 24 hours of secondary fermentation, exhibiting a distinct roasted aroma, and then it was placed in the pit.

[0046] 9. Flavor component detection: The injection port temperature of the gas chromatograph-mass spectrometer was set to 250°C, the split ratio was set to 20:1, the carrier gas was set to high-purity helium, the flow rate was 1.0 mL / min, the temperature was maintained at 40°C for 2 min, the temperature was increased to 100°C at 5°C / min, and then increased to 230°C at 8°C / min and held for 10 min. Take the original spirit samples obtained from distillation in each example, dilute with deionized water to an alcohol content of 53% vol, take 5 mL of the diluted sample and place it in a 15 mL headspace vial, add 1.5 g of sodium chloride, insert the extraction head, equilibrate in a 50°C constant temperature water bath for 10 min, and then perform adsorption extraction for 30 min. Insert the extraction head into the GC-MS inlet, perform thermal desorption for 5 minutes, and then start the instrument to acquire data. Quantitatively analyze the contents of tetramethylpyrazine, ethyl acetate, ethyl hexanoate, and ethyl lactate using the internal standard method.

[0047] (4) Experimental results and analysis:

[0048] The tetramethylpyrazine (TMP) content in control groups 2 and 3 increased by 177.8% and 194.4% respectively compared with the traditional process, confirming that the targeted addition of a high proportion of local Bacillus licheniformis during the high-temperature window of 46.8°C to 48.0°C can adapt to and enhance the Maillard reaction and precursor metabolism during the stacking fermentation stage. Control group 2 introduced Pichia kudria to synergistically produce esters during the later stages of pile fermentation, resulting in a moderate increase in ethyl acetate content and successfully raising the ethyl acetate / milk ratio to the golden ratio of 1.41. This not only made the wine more elegant and delicate but also enhanced the lingering aroma in the empty glass. Although control group 3 had the highest tetramethylpyrazine content, the excessive proportion of Bacillus led to excessive acid production in the later stage of fermentation, resulting in a rebound of ethyl lactate and a strong burnt and bitter taste. Control group 2 reached the ecological balance point of co-evolution of the microbial community, and the physicochemical indicators and sensory experience both reached the highest level. In conclusion, the optimal ratio of the two strains in the control group is 2:1, and the optimal window temperature is 46.8°C.

[0049] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A fermentation control process for Maotai-flavor liquor based on microorganisms in the brewing environment, characterized in that, This includes sterilizing modified LB liquid medium and modified YPD liquid medium, and then inoculating them with Bacillus licheniformis and Pichia pastoris, respectively, for primary seed culture. The obtained primary seed culture was transferred to the corresponding fresh culture medium at the set inoculation amount for secondary seed culture propagation. The expanded secondary bacterial solution was subjected to freeze centrifugation, washing and sterilization, and bacterial resuspension. It was then aseptically mixed according to a set volume ratio to obtain a microbial composite regulator, which was then packaged into a stainless steel spray tank. After the sorghum lees are distilled, they are cooled, water is poured on them, and high-temperature Daqu powder is mixed in. They are then piled into a trapezoidal grain pile, and probes are inserted into different spatial locations of the grain pile to set up a multi-channel temperature monitoring network. When the average temperature of the grain pile reaches the window period temperature, and specific mycelia grow on the surface of the grain pile and produce a fruity aroma, start the turning and spraying linkage equipment to evenly spray the microbial compound regulator into the mash. After spraying, the mash is piled up again for secondary heat preservation and development. When the temperature stabilizes, the mash is moved into the pit, compacted, and covered with sealing mud, and then enters the anaerobic fermentation stage.

2. The fermentation control process for Maotai-flavor liquor based on microorganisms in the brewing environment according to claim 1, characterized in that, The modified LB liquid culture medium has the following formulation: 10.0 g / L tryptone, 5.0 g / L yeast extract, and 10.0 g / L sodium chloride, with the pH adjusted to 7.3 using 1 mol / L NaOH solution. The modified YPD liquid culture medium has the following formula: yeast extract 10.0 g / L, peptone 20.0 g / L, and glucose 20.0 g / L.

3. The fermentation control process for Maotai-flavor liquor based on microorganisms in the brewing environment according to claim 1, characterized in that, The specific steps for preparing the modified YPD liquid culture medium are as follows: dissolve yeast extract and peptone in deionized water to prepare bottle A as the base material, and dissolve glucose in deionized water to prepare bottle B as the sugar material; The base material in bottle A and the sugar material in bottle B are placed separately in a high-pressure steam sterilizer for sterilization. After sterilization, under the flame protection of an ultra-clean workbench, the liquid in bottle B is poured into the liquid in bottle A and mixed and shaken well.

4. The fermentation control process for Maotai-flavor liquor based on microorganisms in the brewing environment according to claim 1, characterized in that, The specific parameters for sterilization and primary seed culture are as follows: high-pressure steam sterilization is performed at 121°C and 0.12MPa for 20 minutes, and the sample is transferred to a clean bench when the temperature drops below 60°C. The Bacillus licheniformis BL-06 was cultured with continuous shaking at 37°C and 180 rpm for 24 h. The Pichia pastoris PK-02 was cultured with continuous shaking at 30°C and 150 rpm for 24 h.

5. The fermentation control process for Maotai-flavor liquor based on microorganisms in the brewing environment according to claim 1, characterized in that, The specific parameters for the secondary seed culture propagation are as follows: pipette the primary seed culture and transfer it at an inoculation rate of 5%, and continue to culture for 18 hours according to the temperature and rotation speed parameters of the primary seed culture. The propagation endpoint indicators were: Bacillus licheniformis exhibited regular, elongated rod-like shapes, and the concentration of cells was determined by microscopic counting. ; Pichia pastoris exhibits an oval budding state; microscopic cell count shows the concentration of cells. .

6. The fermentation control process for Maotai-flavor liquor based on microorganisms in the brewing environment according to claim 1, characterized in that, The formulation of the microbial compound regulator includes: placing the secondary bacterial culture in a benchtop refrigerated centrifuge and centrifuging at 5000 rpm and 4°C for 15 min, discarding the supernatant, adding 0.9% sterile physiological saline to resuspend and wash, and repeating the refrigerated centrifugation once; The centrifuged and washed Bacillus licheniformis resuspension and Pichia pastoris resuspension were aseptically stirred in a 5L stainless steel spray tank at a volume ratio of 2:1 for 3 minutes.

7. The fermentation control process for Maotai-flavor liquor based on microorganisms in the brewing environment according to claim 6, characterized in that, The multi-channel temperature monitoring network is arranged as follows: sorghum lees are gathered into a trapezoidal pile with a height of 1.6m, and the PT100 long rod probe of the 16-channel temperature recorder is driven into the grain pile. The points are arranged including one at the geometric center of the grain pile, and one each at the core heat-generating layer 80cm away from the surface on the east, west, south, and north sides. The parameters are set to automatically record the temperature every 30 minutes.

8. The fermentation control process for Maotai-flavor liquor based on microorganisms in the brewing environment according to claim 1, characterized in that, The criteria for determining the precise intervention window period are: the average temperature at the five sites (east, west, south, north, and center) rises to 46.8°C, white disc-shaped mycelia are observed when the grain pile is pulled apart 5cm from the surface, and an apple aroma and an amino acid-soy sauce precursor aroma can be smelled.

9. The fermentation control process for Maotai-flavor liquor based on microorganisms in the brewing environment according to claim 1, characterized in that, The specific parameters for spraying the microbial compound regulator and fermenting in the cellar are as follows: adjust the pressure of the sprayer pressure regulating valve to 0.2MPa, and set the nozzle orifice diameter to 0.5mm to present a fan-shaped atomization surface; The dedicated tracked fully automatic compost turner for distiller's grains has a travel speed of 2m / min and a drum rotation speed of 220rpm; the spray flow rate is controlled to allow each ton of distiller's grains to adsorb 3000mL of microbial compound regulator. After turning and spraying, the pile is reassembled and kept warm for 24 hours. When the surface temperature of the pile stabilizes at 52.3°C, a loader is moved into the pit and compacted. The surface is covered with 10cm of sealing mud and transferred to anaerobic fermentation pit for 30 days.

10. A fermentation control process for soy sauce-flavored Baijiu based on microorganisms in the brewing environment, as described in claims 1-9, is characterized in that... When the obtained raw wine was diluted to an alcohol content of 53% vol, solid-phase microextraction and gas chromatography-mass spectrometry analysis showed that the content of tetramethylpyrazine was 8.92 mg / L, ethyl acetate was 2.42 g / L, ethyl lactate was 1.72 g / L, and the total ester content was 4.65 g / L. The ratio of ethyl acetate to ethyl lactate was 1.

41. The wine exhibited a prominent soy sauce aroma and a long-lasting fragrance in the empty glass.

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