A high-yield pyrazine complex microbial inoculant, inoculation process, fermentation product and application

Abstract

The present application relates to a kind of high-yield pyrazine complex microbial inoculant, inoculation process, fermentation product and application.The complex microbial inoculant includes A group of yeast bacteria capable of producing pyrazine precursor metabolites, B group of pyrazine-producing functional bacteria promoting pyrazine synthesis and accumulation, and C group of functional bacteria with substrate degradation enzyme activity and promoting flavor substance synthesis and accumulation.Inoculation process includes: first access A group of microorganisms to the interior of substrate to colonize and pre-culture in mixing stage;Subsequently, B group and C group of microorganisms are accessed to the surface layer and crack microenvironment of substrate to colonize;With the segmented temperature rising procedure including high temperature stage above 60 DEG C, fermentation is carried out.The present application significantly improves the content of flavor substances such as tetramethylpyrazine in fermentation product by the separation and colonization of three groups of specific functional microorganisms in space-time and metabolic synergy, effectively enhances the characteristics of sauce flavor and douchi flavor.

Description

Technical Field

[0001] This invention relates to the field of solid-state fermentation and directional regulation of flavor substances in baijiu (Chinese liquor), and particularly to a high-yield pyrazine compound microbial agent, inoculation process, fermentation products, and applications. Background Technology

[0002] The typical flavor formation process of Maotai-flavor liquor and related high-temperature solid-state fermented foods involves complex processes such as multi-strain synergistic metabolism, substrate degradation, amino acid transformation, and Maillard reactions. Among them, pyrazine compounds are one of the important flavor components constituting the roasted aroma, nutty aroma, fermented black bean aroma, and Maotai aroma characteristics, and their content and composition have an important influence on the flavor intensity, layering, and harmony of the final product.

[0003] In traditional high-temperature solid-state fermentation systems, the fermentation environment is typically characterized by low moisture content, limited mass transfer, significant local temperature gradients, uneven oxygen distribution, and complex microecological structures. Especially at temperatures above 60℃, the survival and metabolic activities of the microbial community undergo significant changes, leading to marked differences in the roles of different functional bacteria at different stages and spatial locations (such as the interior, surface, and fissure microenvironment of the material). Existing technologies for enhancing pyrazine flavor compounds often employ single-strain fortification, simple compound inoculation, or single-stage inoculation. For example, CN120484900A discloses a method for enhancing pyrazine compounds in high-temperature Daqu (a type of Chinese liquor) based on mixed-culture fermentation. This method uses a mixture of four strains, including Kluyveromyces martensii, mixed with wheat slurry, and adds pyrazine synthesis precursors in batches during fermentation. However, under high-temperature solid-state fermentation conditions, existing technologies often have the following shortcomings: First, existing simple compound bacterial agents lack precise ratio optimization based on metabolic competition mechanisms. When the proportion of precursor-producing strains is too high, the precursor generation, system acidification, and nutrient competition in the early stages of fermentation will exceed the carrying capacity of pyrazine-producing functional bacteria, resulting in a mismatch between precursor supply and pyrazine synthesis and substrate release, making it difficult to simultaneously meet the needs of substrate degradation, precursor generation, and efficient pyrazine accumulation. Second, existing single-use inoculation methods fail to adapt to the temperature requirements of different bacterial groups in the solid-state system. The differentiated requirements of temperature, oxygen, and nutrient environment cause aerobic pyrazine-producing and flavor-enhancing functional bacteria to enter the substrate internal environment with low mass transfer, uneven oxygen distribution, and insufficient accumulation of precursor substances prematurely, resulting in a mismatch between the dominant period of functional bacteria and the target metabolic window. Finally, the functional division and compatibility between existing fermentation process parameters and compound microbial agents are unclear, especially the lack of a dedicated multi-stage heating program that matches the metabolic succession of the microbial community (such as cell recovery, substrate degradation, and high-temperature pyrazine production), making it difficult to achieve repeatable and scalable flavor enhancement effects in high-temperature solid-state fermentation.

[0004] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a high-yield pyrazine compound microbial agent, inoculation process, fermentation products, and applications, which significantly increase the content of pyrazine substances (especially key pyrazines such as tetramethylpyrazine) in high-temperature solid-state fermentation systems; enhance the sensory characteristics of soy sauce aroma, such as fermented black bean flavor and soy sauce flavor, and improve flavor stability; promote the synergistic enhancement or targeted reconstruction of multiple flavor substances such as furans, ketones, aldehydes, esters, acids, and alcohols, and some key indicators show high increases.

[0006] To achieve the above objectives, the first aspect of the present invention provides a compound microbial agent composition for high-temperature solid-state fermentation, the compound microbial agent composition comprising group A microorganisms, group B microorganisms and group C microorganisms; Among them, Group A microorganisms are yeasts that can produce pyrazine precursor metabolites; Group B microorganisms are pyrazine-producing functional bacteria that promote pyrazine synthesis and accumulation; Group C microorganisms are functional bacteria that possess substrate-degrading enzyme activity and promote the synthesis and accumulation of pyrazines and flavor compounds. Based on the sum of the effective viable counts of group A, group B, and group C microorganisms introduced into the solid fermentation substrate, the proportion of group A microorganisms is 0.5%~50%, the proportion of group B microorganisms is 5%~80%, and the proportion of group C microorganisms is 5%~80%.

[0007] According to a preferred embodiment, based on the proportion of each component in the total number of live bacteria inoculated, group A microorganisms account for 0.8% to 1.2%, group B microorganisms account for 65% to 75%, and group C microorganisms account for 24% to 34%.

[0008] According to a preferred embodiment, the proportions of group A microorganisms, group B microorganisms, and group C microorganisms are approximately 1%, 68%~70%, and 29%~31%, respectively. More preferably, the proportions of group A microorganisms, group B microorganisms, and group C microorganisms are 1%, 69%, and 30%, respectively.

[0009] According to a preferred embodiment, Group A microorganisms are *Saccharomyces ferruginea* T-1-4 with accession number CCTCC M 20261287; Group B microorganisms are selected from one or more combinations of *Bacillus sonoran* 45-3 with accession number CCTCC M 20261281, *Bacillus sonoran* GS2-45-29 with accession number CCTCC M 20261286, and *Bacillus licheniformis* 37 with accession number CCTCC M 20261284; Group C microorganisms are selected from one or more combinations of *Bacillus belyssus* G1-G2 with accession number CCTCC M 20261283, *Bacillus amyloliquefaciens* 00-N3 with accession number CCTCC M 20261285, and *Bacillus belyssus* C-19 with accession number CCTCC M 20261282.

[0010] A second aspect of the present invention provides an inoculation process for high-temperature solid-state fermentation, which employs the aforementioned compound microbial agent composition. The inoculation process includes the following steps: During the mixing stage of the solid fermentation substrate, group A microorganisms are introduced by mixing, allowing group A microorganisms to enter the interior of the solid fermentation substrate, colonize, and undergo pre-culture. After pre-culturing, the microorganisms of group B and group C were introduced into the surface and fissure microenvironment of the solid fermentation substrate by spraying or atomizing. Continue culturing and use a segmented heating program adapted to the fermentation process for high-temperature solid-state fermentation until fermentation is complete.

[0011] According to a preferred embodiment, in the pre-culture step, the pre-culture time of group A microorganisms is 1 to 3 days.

[0012] According to a preferred embodiment, the total inoculum amount of the compound microbial agent composition is 5%~20%; the moisture content of the solid fermentation substrate is 38%~42%; and the concentration of the inoculated bacterial solution is 10. 6 ~10 8 CFU / mL.

[0013] According to a preferred embodiment, the segmented heating process includes several heating stages, including a high-temperature stage of 60°C or higher.

[0014] A third aspect of the present invention provides a fermentation product obtained by the aforementioned high-temperature solid-state fermentation inoculation process.

[0015] The fourth aspect of the present invention provides the application of the aforementioned compound microbial agent composition, the aforementioned inoculation process, or the aforementioned fermentation product in improving the content of flavor substances in the solid-state fermentation system of baijiu.

[0016] According to a preferred embodiment, the application specifically involves increasing the content of tetramethylpyrazine and / or trimethylpyrazine, and enhancing the flavor characteristics of fermented black beans and soy sauce.

[0017] Compared with the prior art, the present invention has at least the following beneficial technical effects: (1) A highly efficient synergistic metabolic micro-ecosystem was constructed, breaking through the yield bottleneck of traditional pyrazine synthesis. This invention achieves a complete synergistic metabolic chain of "precursor supply - substrate release - pyrazine generation" through the functional complementarity of group A (pyrazine-producing precursor yeast), group B (pyrazine-producing functional bacteria), and group C (substrate-degrading functional bacteria); in particular, the specific proportion range of group A, group B, and group C in the total number of live bacteria inoculated (preferably 1%, 69%, and 30%) is limited. This critical ratio effectively avoids system acidification and nutrient competition caused by excessive precursor production in the early stage of fermentation. An ideal dynamic balance is formed between limited precursor supply, dominant colonization of pyrazine-producing functional bacteria, and synergistic substrate degradation, thereby significantly increasing the yield of key pyrazine flavor substances in the fermentation product (total pyrazine content can reach 10283.71 μg / kg) and effectively enhancing typical sensory characteristics such as fermented black bean flavor and soy sauce flavor.

[0018] (2) The inoculation process, which couples spatiotemporal separation colonization with multi-step heating, significantly improves the reproducibility of fermentation control and flavor stability. This invention employs a segmented inoculation process, prioritizing the colonization of group A microorganisms within the substrate to ensure initial precursor supply. Subsequently, groups B and C microorganisms are inoculated on the surface and in the microenvironment of fissures, spatially preventing aerobic pyrazine-producing and flavor-enhancing functional bacteria from prematurely entering a low-mass-transfer, low-oxygen environment. Simultaneously, the multi-step segmented heating program, including a high-temperature phase above 60°C, ensures precise alignment between the colonization dominance period, metabolic activity window, and environmental temperature curve of each group of functional bacteria. This method effectively overcomes the functional period mismatch problem caused by existing single-stage mixed inoculation and natural heating methods. It not only promotes the stable enrichment of target substances such as tetramethylpyrazine and trimethylpyrazine but also synergistically enhances the overall content of various volatile flavor compounds such as furans, ketones, aldehydes, esters, acids, and alcohols.

[0019] In existing technologies, although certain bacteria (such as Bacillus subtilis) can accumulate large amounts of pyrazine precursors such as acetoin through their own metabolic pathways under forced mass transfer and high dissolved oxygen conditions in liquid deep fermentation (as shown in CN101955980A and CN101445786A), in traditional solid-state fermentation systems (such as the simple mixed-culture direct inoculation process used in CN109090483A), due to the significant oxygen concentration decay gradient and gas-liquid-solid multiphase mass transfer resistance within the solid substrate, aerobic Bacillus distributed within the substrate in a conventional one-time mixing manner suffers severe inhibition of its aerobic precursor synthesis pathway under low oxygen and limited mass transfer conditions in a closed environment. This results in a severe shortage of precursor substances in the deep part of the solid system during the early stages of fermentation.

[0020] To overcome the limitations of microenvironmental mass transfer inherent in solid-state fermentation, this invention pre-embeds and embeds Group A microorganisms (yeast) into the deep interior of the solid-state fermentation substrate through forced mixing during the mixing stage. This allows Group A microorganisms to generate and accumulate large quantities of pyrazine precursors under microaerophilic conditions in the early stages of fermentation, based on sufficient contact with carbohydrates within the substrate. Subsequently, this invention uses spraying or atomization to attach Group B and Group C microorganisms to the surface layer and fissure microenvironment of the substrate, which are closer to the external environment than the deep interior layers. This constructs a microecological misaligned distribution structure with a clear internal division of labor within the three-dimensional space of the solid-state substrate.

[0021] Along the timeline of the fermentation process and the material diffusion path within the substrate, precursor substances generated by group A microorganisms in the core micro-anaerobic zone diffuse continuously from the inside to the outer surface driven by the concentration difference. Simultaneously, group B microorganisms, in an oxygen-rich state on the surface and in the fissures, with the cooperation of group C microorganisms in degrading macromolecular substrates and releasing nutrients, capture and transform precursor substances diffused from the inside to the upstream surface through efficient oxidative condensation. Thus, while avoiding competition for nutrients in the space during the early stages of fermentation, a synergistic chain of internal precursor supply, surface substrate release, and interface product synthesis is achieved.

[0022] Furthermore, this invention uses the total number of live bacteria inoculated as a benchmark and limits the proportions of groups A, B, and C within a specific ratio window (preferably 1%, 69%, and 30%). This critical ratio, combined with the aforementioned spatial isolation strategy, enables the micro-ecological system to maintain a stable dynamic synergistic steady state even under extreme heat stress conditions at temperatures above 60°C. This achieves a technical effect that is far superior to the simple physical mixing or single liquid fermentation techniques of the prior art.

[0023] Instructions for the preservation of biological materials: The microbial strain involved in this invention has been deposited at the China Center for Type Culture Collection (CCTCC; address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, China). Specific deposit information is as follows: Bacillus sonorensis 45-3, deposited on June 11, 2026, with accession number CCTCC NO: M 20261281; Bacillus velezensis C-19, deposited on June 11, 2026, with accession number CCTCC NO: M 20261282; Bacillus velezensis G1-G2, deposited on June 11, 2026, with accession number CCTCC NO: M 20261283; Bacillus licheniformis 37, deposited on June 11, 2026, with accession number CCTCC NO: M 20261284; Bacillus amyloliquefaciens 00-N3, deposited on June 11, 2026, with accession number CCTCC NO: M 20261285; Bacillus sonorensis GS2-45-29, deposited on June 11, 2026, with accession number CCTCC NO: M 20261286; Cyberlindnera fabianii T-1-4, deposited on June 11, 2026, with accession number CCTCC NO: M 20261287. Attached Figure Description

[0024] Figure 1 A shows the sensory evaluation results of bacterial strains in the compound microbial agent; Figure 1 B is a graph showing the sensory evaluation results of fungal strains in the compound inoculant.

[0025] Figure 2 The figure shows the results of single-factor optimization of solid-state fermentation conditions for compound microbial agents.

[0026] Figures 3-5 Three-dimensional plot and contour plot of the conditional response surface for high-yield pyrazine fermentation of compound microbial agents.

[0027] Figure 6 The figure shows the results of comprehensive optimization of the fermentation process parameters of the compound microbial agent based on the expectation function.

[0028] Figure 7 A heatmap comparing the effects of segmented inoculation (spatiotemporal separation) and single inoculation on pyrazine products.

[0029] Figure 8 Optimize the ramp diagram for the numerical distribution ratio of compound microbial agents A / B / C groups.

[0030] Figure 9 A ternary contour plot showing the effect of the ratio of compound microbial agents A / B / C on the total pyrazine content.

[0031] Figure 10 This is a comparison chart of the composition and relative content of volatile flavor compounds between the control group and the experimental group.

[0032] Figure 11 This is a flowchart of the method in this application. Detailed Implementation

[0033] The following is a detailed explanation with reference to the accompanying drawings.

[0034] Example 1 This example illustrates the preparation of the compound microbial agent.

[0035] (i) Activation of strains involved in the construction of compound microbial agents Sonoran Bacillus 45-3, Belize Bacillus C-19, Belize Bacillus G1-G2, Bacillus licheniformis 37, Bacillus amyloliquefaciens 00-N3, Sonoran Bacillus GS2-45-29, and Fibrian Seberlindnerella vaginalis T-1-4, preserved according to this invention, were each aseptically picked from -80℃ glycerol storage tubes and inoculated into 2 mL centrifuge tubes containing 1 mL of the corresponding liquid culture medium; wherein, TSB medium was used for bacteria and YPD medium was used for yeast. The centrifuge tubes were placed in a constant temperature shaker for incubation; the bacterial culture conditions were 37℃, 180 rpm, and the yeast culture conditions were 28℃, 180 rpm, for 24-48 h, until the culture medium became obviously turbid. The activated bacterial solution was transferred to an Erlenmeyer flask containing 200 mL of fresh liquid culture medium at an inoculation rate of 10% (v / v), and cultured under the same conditions as above until the late logarithmic growth phase (usually 24 h) to obtain the seed culture for later use.

[0036] (II) Sensory evaluation of solid-state fermentation products of strains 1. Preparation of solid-state fermentation culture medium and experimental setup The crushed wheat was used to prepare a solid-state fermentation medium, and the moisture content was adjusted to 38% using sterile deionized water. After mixing, the medium was allowed to stand for 2 hours to fully moisten it. 100.0 g of the medium was weighed and placed in a 300 mL tissue culture flask, and autoclaved at 121℃ for 30 min. After sterilization, 10 mL of seed culture was aseptically inoculated into the cooled solid-state medium (inoculation volume of 10% v / m) in a clean bench, and the medium was thoroughly mixed with the bacteria using a sterile glass rod. A blank control was set up, consisting of medium without seed culture but with an equal volume of sterile water. Three biological replicates were set up for each strain and control.

[0037] 2. Solid-state fermentation culture and sensory evaluation After inoculation, the tissue culture bottles were placed in a constant temperature incubator for static incubation; the bacterial culture temperature was 37℃, and the yeast / mold culture temperature was 28℃, for 5-7 days. Immediately after the incubation, 5 trained evaluators familiar with the characteristics of soy sauce aroma independently evaluated the samples in a dedicated sensory evaluation room; a 5-point intensity scale (0: none; 1: weak; 2: moderate; 3: strong; 4: strong) was used to score the soy sauce aroma, caramel aroma, roasted aroma, mellow aroma, fermented aroma, floral and fruity aroma, oily aroma, sour aroma, and off-odors, and descriptive aroma characteristics were recorded.

[0038] 3. Sensory evaluation results See sensory evaluation results Figure 1 A and Figure 1 B. Different strains exhibited differentiated characteristic parameters in the solid-state fermentation samples regarding aromas such as soy sauce, caramel, roasting, mellow, fermentation, floral and fruity, oily, acidic, and off-flavors. For example... Figure 1 As shown in Figure A, the highest score for the soy sauce aroma dimension was 4.5 (Bacillus vesicularis G1-G2), the highest score for the caramel aroma dimension was 4.6 (Bacillus vesicularis C-19), the highest score for the roasted aroma dimension was 4.1 (Bacillus sonoran GS2-45-29 and Bacillus licheniformis 37 tied), the highest score for the mellow aroma dimension was 3.5 (Bacillus vesicularis C-19), the highest score for the floral and fruity aroma dimension was 4.6 (Bacillus amyloliquefaciens 00-N3), and the highest score for the oily aroma dimension was 4.3 (Bacillus amyloliquefaciens 00-N3). Among the negative aromas, the highest score for the sour aroma dimension was 0.8 (Bacillus vesicularis G1-G2). Furthermore... Figure 1 The aroma profile of *Saccharomyces cerevisiae* T-1-4, shown in sample B, is mainly concentrated in the dimensions of alcohol, floral and fruity aromas, and oily aromas, while acidic aromas and off-odors are maintained at a low level. These results indicate that the strain types in the above-mentioned compound inoculants can achieve complementarity and coordination of the peak and compositional proportions of aroma attributes in the solid-state fermentation system, providing strain resources and theoretical basis for the subsequent construction of compound inoculants.

[0039] (III) Analysis of enzyme production characteristics of strains 1. Qualitative analysis of enzyme production using the clear zone method This embodiment provides a method for initial screening of enzyme production characteristics of bacterial strains, which uses the clear zone method to screen proteases, amylases and cellulases on plates, specifically including the following.

[0040] The screening media for analyzing the enzyme production characteristics of the strains include screening media for protease, amylase, and cellulase. The formulations and sterilization conditions of the media are as follows: (1) Protease screening plates (skim milk medium, g / L): peptone 10.0, beef extract 3.0, NaCl 5.0, skim milk powder 10.0, agar 15.0, pH 7.0, sterilized at 115℃ for 20 min; (2) Amylase screening plates (soluble starch medium, g / L): soluble starch 10.0, peptone 5.0, yeast extract 5.0, NaCl 5.0, agar 15.0, pH 7.0, sterilized at 121℃ for 20 min; (3) Cellulase screening plates (CMC-Na medium, g / L): CMC-Na 10.0, (NH4)2SO4 2.0, K2HPO4 1.0, MgSO4·7H2O 0.5g of peptone, 5.0g of agar, pH 7.0, sterilized at 121°C for 20 min.

[0041] The activated bacterial solution was concentrated by centrifugation and the bacterial cell concentration was adjusted to OD. 600 ≈1.0; 2 μL of bacterial suspension was inoculated onto the corresponding screening plate. Three bacterial and yeast strains were inoculated onto each plate. Bacteria were cultured at 37℃ for 24–48 h, and fungi at 28℃ for 48–72 h. For protease assays, direct observation was performed after culture, with a clear transparent zone around the colony as the positive criterion. For amylase assays, Lugol's iodine solution was poured onto the plate surface, allowed to stand for 1–2 min, and then excess iodine solution was discarded. A colorless transparent zone around the colony with a blue background was used as the positive criterion. For cellulase assays, 0.1% Congo red solution was poured onto the plate for staining for 30 min, the stain was discarded, and 1 mol / L NaCl was added for destaining for 30 min. A clear zone around the colony was used as the positive criterion.

[0042] 2. Results of qualitative analysis of enzyme production As shown in Table 1 of the qualitative screening results using the clear zone method, all six tested strains were amylase-positive, indicating that they all possess starch degradation capabilities. Specifically, *Bacillus belyceta var.* G1-G2 and *Bacillus belyceta var.* C-19 were positive for amylase, cellulase, and protease, indicating that these two strains have comprehensive enzyme production capabilities. *Bacillus sonoran* 45-3 and *Bacillus licheniformis* 37 were amylase and cellulase-positive but protease-negative, indicating that they primarily exhibit polysaccharide degradation-related enzyme production characteristics. *Bacillus amyloliquefaciens* 00-N3 was amylase and protease-positive but cellulase-negative. *Bacillus sonoran* GS2-45-29 was only amylase-positive. These results demonstrate that different strains exhibit varying enzyme production profiles. The clear zone method described in this embodiment can achieve rapid qualitative differentiation of the enzyme production characteristics of strains, providing a basis for subsequent target strain screening and strain combination.

[0043] Table 1. Qualitative analysis results of enzyme production by strains of compound microbial agent.

[0044] 3. Quantitative determination of enzyme activity in fermentation broth This embodiment further provides a method for re-screening superior strains from the initial screening, which uses fermentation supernatant as crude enzyme solution, and determines the saccharifying enzyme activity by DNS method and the liquefying enzyme activity by iodine colorimetric method, specifically including the following.

[0045] The initially screened superior strains were inoculated into 50 mL of enzyme-producing liquid culture medium and cultured on a shaker until the peak enzyme production period was reached. 10 mL of fermentation broth was centrifuged at 4℃ and a centrifugal force of 8000×g (g is the acceleration due to gravity) for 15 min. The supernatant was taken as crude enzyme solution, stored at 4℃, and the determination was completed within 24 h.

[0046] Prepare glucose standard solutions of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL. Add 1.0 mL of each standard solution to 1.5 mL of DNS reagent, incubate in a boiling water bath for 5 min, cool, and then bring the volume to 25 mL. Measure the absorbance at 540 nm and plot a standard curve. Mix 0.5 mL of appropriately diluted crude enzyme solution with 0.5 mL of 2% soluble starch solution (prepared with pH 4.5 acetate-sodium acetate buffer). After mixing, react in a water bath at 35℃ for 10 min. Replace the crude enzyme solution with an equal volume of pH 4.5 buffer for the blank control. Add 1.5 mL of DNS reagent to terminate the reaction and develop color. Subsequent treatment is the same as for the standard curve. Measure the absorbance at 540 nm. The saccharifying enzyme activity (U / mL) is calculated using the following formula: .

[0047] Where C is the glucose concentration (mg / mL) obtained from the standard curve, N is the dilution factor, V is the total volume of the reaction system (mL), t is the reaction time (min), and 0.5 is the enzyme volume in the reaction system (mL).

[0048] Prepare a soluble starch solution of 0–4 g / L. Take 1 mL of starch solution at 0.5 g / L intervals, mix it with 5 mL of dilute iodine solution and 10 mL of distilled water, and measure the absorbance at 660 nm to plot an absorbance-starch concentration standard curve. Add 20 mL of 4 g / L soluble starch solution to a 150 mL Erlenmeyer flask and preheat in a 60℃ water bath for 5 min. The 4 g / L soluble starch solution is prepared with pH 6.0 phosphate buffer. Add 1.0 mL of appropriately diluted crude enzyme solution and start timing immediately, reacting at 60℃ for 10 min. Immediately after the reaction, add 1.0 mL of the reaction solution to a test tube containing 5 mL of dilute iodine solution and 10 mL of distilled water, mix well to terminate the reaction, and measure the absorbance of sample A at 660 nm. The blank control A is treated with inactivated enzyme solution using the same steps. Calculate the amount of liquefied starch from the standard curve based on the difference between (blank A and sample A), and calculate the liquefying enzyme activity (U / mL) using the following formula: .

[0049] ΔC is the concentration of liquefied starch (mg / mL), N is the dilution factor, 20 is the total substrate volume (mL), t is the reaction time (min), and 1.0 is the enzyme volume in the reaction system (mL).

[0050] 4. Quantitative determination of enzyme activity in compound microbial agents As shown in Table 2, the quantitative determination results revealed significant differences in saccharifying and liquefying enzyme activities among the strains. *Bacillus belyssica* C-19 exhibited the best overall performance, with a saccharifying enzyme activity of 7249.276 U / mL and a liquefying enzyme activity of 1.4107 U / mL. Furthermore, *Bacillus sonoran* GS2-45-29 and *Bacillus amyloliquefaciens* 00-N3 also showed high starch degradation-related enzyme activities. Specifically, *Bacillus sonoran* GS2-45-29 had a saccharifying enzyme activity of 4228.07 U / mL and a liquefying enzyme activity of 1.3368 U / mL, while *Bacillus amyloliquefaciens* 00-N3 had a saccharifying enzyme activity of 1524.60 U / mL and a liquefying enzyme activity of 1.3441 U / mL. The above results indicate that Bacillus belyss C-19 exhibits high enzyme activity levels in both saccharification and liquefaction, making it a preferred strain for subsequent compounding or application evaluation; other high-activity strains can be used for further screening and combination optimization.

[0051] Table 2. Quantitative determination of enzyme activity in fermentation broth of compound microbial agents

[0052] (iv) Determination of the strain's ability to produce pyrazine (flavor) (HS-SPME-GC-MS) This embodiment provides a method for determining the pyrazine-producing ability of a bacterial strain. It employs headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) for qualitative and internal standard quantitative / semi-quantitative analysis of pyrazine compounds in fermentation samples. Specifically, 2.000 g of solid-state fermentation sample or 5.00 mL of supernatant / bacterial sludge suspension after centrifugation of liquid fermentation is accurately weighed and placed in a 20 mL headspace sample vial. 1 μL of internal standard working solution (2-octanol) with a concentration of 0.3 g / L is precisely added to each vial. The vial is then sealed with a polytetrafluoroethylene / silicone septum and vortexed for 30 s. The vials are then equilibrated in a 60°C water bath or heating platform for 15 min. An aged 50 / 30 μm DVB / CAR / PDMS extraction fiber is exposed to the headspace of the vial, and adsorption extraction is performed at 60°C for 40 minutes. min; after extraction, the extraction fiber was quickly retrieved and immediately inserted into the GC injection port for thermal desorption injection; gas chromatography conditions were as follows: Agilent DB-WAX column (60 m × 0.25 mm × 0.25 μm), injection port temperature 250℃, splitless injection, carrier gas high-purity helium, constant flow 1.0 mL / min, column temperature program 40℃ held for 5 min, increased to 100℃ at 5℃ / min, then increased to 230℃ at 10℃ / min and held for 10 min, total run time 40.5 min; mass spectrometry conditions were as follows: ion source temperature 230℃, transfer line temperature 250℃, electron impact ionization (EI) with energy 70 eV, data acquisition using full scan mode (m / z (35-350) is used for qualitative analysis and selected ion monitoring mode (SIM) is used for quantitative analysis. Qualitative analysis is performed by comparing the retention time of the sample chromatographic peaks, mass spectrometry information, and NIST mass spectrometry library search results with the matching degree of pyrazine standards. An internal standard method is used to establish a standard curve with the peak area ratio of the target analyte to the internal standard as the ordinate and the target analyte concentration as the abscissa. The peak area ratio of the target analyte to the internal standard in the sample is substituted into the regression equation to calculate the content of pyrazine substances. The results are expressed in μg / kg (solid sample) or μg / L (liquid sample), and the total pyrazine content is calculated.

[0053] Table 3. Determination of pyrazine production capacity of strains in compound bacterial agent

[0054] Table 3 shows that there are significant differences in pyrazine production capacity among different strains. *Bacillus sonoran* GS2-45-29 exhibited the strongest pyrazine production capacity, with a tetramethylpyrazine content of 1411.31 μg / kg and a total pyrazine content of 1573.56 μg / kg. *Bacillus amyloliquefaciens* 00-N3 and *Bacillus belesiensis* C-19 also showed good pyrazine production capacity and can be considered as candidate strains for high pyrazine production. Based on the strains' enzyme activity characteristics, pyrazine production capacity, and aroma evaluation, they were functionally grouped. Group A consists of yeasts capable of producing pyrazine precursor metabolites (including 2,3-butanediol, acetoin, and 2-hydroxy-3-hexanone); Group B consists of pyrazine-producing functional bacteria that promote pyrazine synthesis and accumulation; and Group C consists of functional bacteria that possess substrate-degrading enzyme activity and promote the synthesis and accumulation of pyrazine and flavor compounds. Figure 1 Based on the results of the pyrazine-producing capacity assay, T-1-4 was identified as a group A yeast strain capable of producing pyrazine precursor metabolites. According to the assay results in Tables 1 to 3, Sonoran Bacillus 45-3, Sonoran Bacillus GS2-45-29, and Licheniformis 37 were classified into group B, while Belize Bacillus G1-G2, Amyloliquefaciens 00-N3, and Belize Bacillus C-19 were classified into group C. These results demonstrate that the HS-SPME-GC-MS method described in this embodiment can effectively achieve qualitative and quantitative evaluation of the pyrazine-producing capacity of strains and can provide a basis for grouping and compounding functional strains.

[0055] (V) Functional Bacterial Screening and Identification SOP To enable those skilled in the art to screen strains with corresponding functions from high-temperature daqu, fermented mash of sauce-flavored baijiu, samples from pile fermentation environments, or other high-temperature solid-state fermentation environments, this embodiment further provides standard operating procedures for screening and identifying functional bacteria in groups A, B, and C, specifically including the following steps.

[0056] S1. Sample Collection and Candidate Strain Isolation Samples were collected from high-temperature Daqu (a type of starter culture), fermented mash of Maotai-flavor liquor, samples from the pile fermentation environment, or their surrounding ecological environment. The samples were added to sterile physiological saline, shaken to mix, and then serially diluted and spread onto isolation media. Bacterial isolation was performed using TSB, LB, or nutrient agar media, incubated at 37℃ for 24–48 h; yeast isolation was performed using YPD or malt extract agar media, incubated at 28℃ for 24–72 h. Single colonies with different morphologies were picked, and candidate strains were obtained after streak purification.

[0057] S2. Activation of candidate strains and standardization of inoculum Candidate bacteria were inoculated into TSB liquid medium and cultured at 37°C and 180 rpm for 24–48 h; candidate yeasts were inoculated into YPD liquid medium and cultured at 28°C and 180 rpm for 24–48 h. After reaching the logarithmic growth phase or when the culture medium became significantly turbid, the bacterial cells were collected by centrifugation and resuspended in sterile physiological saline. The bacterial suspension was then adjusted to OD0.05. 600 The concentration is 0.8~1.2, preferably about 1.0, as a standardized inoculum for subsequent screening.

[0058] Screening of S3 and A group pyrazine-producing precursor yeasts Candidate yeasts were inoculated into a precursor generation screening medium and cultured. The precursor generation screening medium used glucose, pyruvate, or wheat saccharification broth as the carbon source and yeast extract, peptone, or an amino acid mixture as the nitrogen source; the preferred medium was: 20.0 g / L glucose, 10.0 g / L yeast extract, 20.0 g / L peptone, 2.0–5.0 g / L sodium pyruvate, pH 5.5–6.5. The inoculation amount of candidate yeast was 1%–10%, preferably 5%, and cultured at 28–37°C and 180 rpm for 24–72 h.

[0059] After cultivation, the fermentation broth was centrifuged, and the supernatant was used for the detection of acetoin and / or 2,3-butanediol. Initial screening for acetoin could be performed using the Voges-Proskauer colorimetric reaction. The fermentation supernatant was added to α-naphthol ethanol solution and KOH solution, and the absorbance was measured at 540 nm after color development. Uninoculated culture medium was used as a blank control. When sample A... 540 Not less than 0.10, and sample A 540 When the concentration is more than twice that of the blank control, the candidate yeast is considered to have the ability to produce acetoin. Further confirmatory testing for one or more of acetoin and 2,3-butanediol is performed using HS-SPME-GC-MS. If the candidate yeast can stably detect acetoin and / or 2,3-butanediol, and its peak area or equivalent concentration is higher than that of the uninoculated control, it is designated as a candidate strain in group A.

[0060] Screening of pyrazine-producing functional bacteria in groups S4 and B Candidate bacteria or candidate functional bacteria were inoculated into a solid-state wheat fermentation model for pyrazine production screening. The solid-state wheat fermentation model consisted of crushed wheat with water added to adjust the moisture content to 35%–45%, preferably approximately 38%–40%, sterilized at 121°C for 30 min, and then cooled for later use. A standardized bacterial suspension of the candidate bacteria was inoculated into the solid substrate at an inoculation rate of 5%–15%, preferably 10%, and cultured at 37°C or under stepped temperature increases for 5–14 days. After culture, LC-MS was used to detect pyrazine compounds such as tetramethylpyrazine, trimethylpyrazine, 2,3-dimethylpyrazine, 2,5-dimethylpyrazine, and 2-ethyl-3,5-dimethylpyrazine.

[0061] Uninoculated solid substrate was used as a blank control. Candidate bacteria were classified as Group B candidate strains if they met one of the following conditions: First, the total pyrazine content in the fermentation sample was higher than that of the blank control, and the total pyrazine content was not less than 50 μg / kg; second, at least one of tetramethylpyrazine, trimethylpyrazine, or other target pyrazines was stably detected, and its peak area or equivalent content was more than twice that of the blank control; third, after being combined with Group A precursor yeast or Group C substrate-degrading functional bacteria, the total pyrazine content was increased by more than 20% relative to the control group without the candidate bacteria. Strains meeting the above conditions could be classified into the pyrazine-producing functional bacteria component that promotes pyrazine synthesis and accumulation.

[0062] Screening of substrate-degrading and flavor-enhancing functional bacteria in groups S5 and C Candidate bacteria were inoculated onto screening plates for protease, amylase, and cellulase, respectively, for qualitative analysis of enzyme function.

[0063] The protease screening plates were prepared using skim milk medium, which, by g / L, consisted of: 10.0 g of peptone, 3.0 g of beef extract, 5.0 g of NaCl, 10.0 g of skim milk powder, and 15.0 g of agar, with a pH of 7.0, and were sterilized at 115°C for 20 min.

[0064] The amylase screening plates were prepared using soluble starch medium, which, by g / L, consisted of: 10.0 g of soluble starch, 5.0 g of peptone, 5.0 g of yeast extract, 5.0 g of NaCl, and 15.0 g of agar, at pH 7.0, and were sterilized at 121°C for 20 min.

[0065] Cellulase screening plates were prepared using CMC-Na medium, which, by g / L, contained: CMC-Na 10.0, (NH4)2SO4 2.0, K2HPO4 1.0, MgSO4·7H2O 0.5, peptone 5.0, agar 15.0, pH 7.0, and were sterilized at 121℃ for 20 min.

[0066] After activating the candidate bacteria, a bacterial suspension was prepared and the bacterial concentration was adjusted to OD0.05. 600 The concentration is 0.8–1.2, preferably about 1.0; 2 μL of bacterial suspension is inoculated onto the above screening plate. Bacteria are cultured at 37°C for 24–48 h, and yeasts or fungi are cultured at 28°C for 48–72 h.

[0067] Qualitative determination was performed after culture: protease was positive when a clear transparent zone appeared around the colony; amylase was positive when a colorless transparent zone appeared around the colony and the background was blue after adding Lugol's iodine solution; cellulase was positive when a transparent zone appeared around the colony after Congo red staining and decolorization with NaCl.

[0068] Candidate bacteria that are positive for at least one of the three qualitative screenings (amylase, protease, and cellulase) and can promote the formation of pyrazine compounds or flavor characteristics such as soy sauce aroma, caramel aroma, roasted aroma, alcoholic aroma, floral and fruity aroma, and oily aroma in a solid-state fermentation model can be designated as group C candidate functional bacteria. Preferably, candidate bacteria that are positive for at least two enzyme functions, or that are positive for only one enzyme function but can increase the content of total pyrazine or key flavor compounds in subsequent compound fermentation, are designated as group C preferred strains.

[0069] S6, Solid-state fermentation sensory and flavor re-screening Candidate bacteria from groups A, B, and C, obtained through initial screening (S3-S5), were inoculated into a solid-state wheat fermentation model for cultivation. After cultivation, the samples were evaluated by smell and volatile flavor compounds were detected. Sensory evaluation indicators included aroma (soy sauce, caramel, roast, mellow, fermented, floral and fruity, oily, sour, and off-odors). An intensity scale of 0-4 or 0-5 points was preferred for evaluation. Candidate bacteria should exhibit at least one higher level of aroma (soy sauce, caramel, roast, mellow, floral and fruity, or oily) than the blank control, with no significant increase in negative odors such as sourness or off-odors.

[0070] S7, Functional Grouping Determination After the above screening, yeast strains capable of producing one or more pyrazine precursor metabolites, such as acetoin and 2,3-butanediol, were classified into Group A; strains capable of promoting the synthesis and accumulation of tetramethylpyrazine, trimethylpyrazine, or total pyrazine in solid-state fermentation systems were classified into Group B; and strains possessing the ability to degrade at least one substrate, such as amylase, saccharifying enzyme, liquefying enzyme, protease, or cellulase, and capable of promoting the accumulation of pyrazine and flavor compounds, were classified into Group C. The yeast strains from Group A, functional strains from Group B, and functional strains from Group C obtained through the screening and verification steps were combined to obtain the high-pyrazine-producing compound microbial agent described in this invention.

[0071] Example 2 This embodiment illustrates the optimal solid-state fermentation conditions for complex microbial communities.

[0072] (I) Experimental detection of solid-state fermentation conditions 1. Solid-state fermentation experimental conditions Using wheat for koji making as raw material, distilled water was added according to a preset material-to-water ratio and thoroughly mixed. The mixture was then moistened at room temperature for 2 hours. The moistened material was dispensed into 300 mL tissue culture bottles (100 g per bottle), and subsequently sterilized by autoclaving at 121℃ for 30 minutes. After cooling, the obtained seed liquid was inoculated into the solid-state fermentation medium at a preset total inoculation amount. After inoculation, the mixture was thoroughly mixed and cultured under specified conditions for 14 days. During the culture process, each strain was added in equal proportions according to a set ratio. In the analysis of process influencing factors, based on actual production conditions and application feasibility, the proportion of compound microbial agents, total inoculation amount, water addition amount, bacterial concentration, and temperature program were determined as influencing factors on the pyrazine content. First, single-factor experiments were conducted on the total inoculation amount, water addition amount, bacterial concentration, and temperature program. When changing a single factor, the other factors remained at level 2, and the temperature program was set at a preset level. The single-factor levels included: total inoculation amount 5%, 10%, 15%, 20%; water addition amount 32%, 40%, 48%, 56%; bacterial concentration 10%. 5 10 6 10 7 10 8 The temperature programs are 30℃→37℃→45℃→52℃→55℃→58℃→58℃, 34℃→42℃→48℃→52℃→56℃→60℃→60℃, 37℃→48℃→52℃→55℃→59℃→63℃→63℃, and 39℃→52℃→55℃→58℃→62℃→65℃→65℃ (each temperature point is maintained for 2 days). Among them, the stepped temperature increase program of 37℃→48℃→52℃→55℃→59℃→63℃→63℃ is the preferred temperature program. This program is not simply a natural temperature increase of the fermentation system to the high-temperature endpoint, but a functional window-type temperature control program adapted to the functional division of groups A, B, and C in the compound microbial agent of this invention. Specifically, the 37℃ stage is mainly used for cell recovery in the early stage of fermentation, preferential colonization of group A in the substrate, and the formation of pyrazine precursors such as acetoin and 2,3-butanediol; the 48℃~55℃ stage is mainly used for the adaptation of groups B and C to the heated environment, substrate degradation of group C in the solid substrate, and the transition of precursors to pyrazine products; the 59℃~63℃ stage is mainly used for functional bacteria tolerant to the high-temperature solid-state fermentation environment to produce pyrazine and enhance flavor, and to promote the accumulation of pyrazine flavor compounds such as tetramethylpyrazine and trimethylpyrazine. Based on the single-factor experiments, response surface methodology was further used for optimization. The optimal conditions were determined according to the Box-Behnken central composite design principle, using pyrazine production content as the response value for response surface analysis. Design-Expert software was used for model fitting and parameter optimization to obtain the optimal preparation conditions.

[0073] 2. Qualitative determination of pyrazine-producing capacity In the quantitative determination of pyrazine production capacity, the solid-state fermentation sample and the extraction solvent were mixed at a material-to-liquid ratio of 1:1.5. The extraction solvent was a 70% methanol aqueous solution containing 0.1% formic acid. After ultrasonic extraction for 40 min, solid-liquid separation was achieved by centrifugation at 10,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm microporous membrane and used as the test solution. Quantitative analysis was performed using the external standard method. A calibration curve was established using a series of concentration standards to quantify the target compound. The concentration range of the pyrazine compound standard solution was 19.53125~15,000 μg·L⁻¹. -1 A linear regression model was constructed based on this. Instrumental analysis was performed using a triple quadrupole mass spectrometer system (Shimadzu LCMS-8045) equipped with a Gemini C6-Phenyl column (4.6 mm × 250 mm, 5 μm). Chromatographic separation employed gradient elution with phase A and phase B. Phase A consisted of an aqueous solution containing 0.1% trifluoroacetic acid and 0.1% formic acid, while phase B consisted of methanol. The gradient program was as follows: 0–22 min: 93% → 80% A; 22–33 min: 80% → 93% A; 33–40 min: 93% → 30% A; 40–47 min: 30% → 93% A; 47–50 min: 93% A. The column temperature was 40℃ and the flow rate was 0.6 mL / min. -1 The injection volume was 20 μL; mass spectrometry detection was performed in electrospray ionization (ESI) positive ion mode. + The ion spraying was performed at a voltage of 4.0 kV, a desolvation tube temperature of 250°C, and a heating module temperature of 400°C. The target compound was detected using multiple reaction monitoring (MRM) mode. The detection specificity and sensitivity were improved by optimizing the collision energy and ion pair parameters.

[0074] (II) Experimental Results of Solid-State Fermentation Conditions 1. Results of univariate analysis This embodiment first establishes a quantitative analytical method for 24 pyrazine compounds. A series of standard solutions with concentrations ranging from 19.53125 to 15000 μg / L were prepared for each of the 24 pyrazine standards. The LC-MS method described above was used to determine the concentrations sequentially, and standard curves were established. The results are shown in Table 4. The standard curves for each target analyte showed good linearity, with coefficients of determination R0... 2 The value ranges from 0.9938 to 0.9995, which meets the requirements for quantitative analysis of pyrazine compounds in fermentation samples.

[0075] Table 4 Standard curves for 24 pyrazine compounds

[0076] Single-factor experiments were used to preliminarily optimize the fermentation conditions of the compound microbial agent. For example... Figure 2The results showed that: (1) the total inoculum amount had an effect on pyrazine yield that first increased and then decreased, with the highest yield at 10%; (2) the amount of water added also had an effect on pyrazine yield that first increased and then decreased, reaching a peak at 40%; (3) the bacterial concentration had a significant parabolic effect on pyrazine yield, with the highest yield at 10%. 7 The highest yield was achieved at CFU / mL (approximately 4800 μg / kg); (4) The temperature program showed a clear optimal value for pyrazine yield, with the highest yield (approximately 4550 μg / kg) under temperature program 3.0 (37℃→48℃→52℃→55℃→59℃→63℃→63℃). The above results indicate that each factor has a suitable range, and both excessively low and excessively high temperatures are detrimental to pyrazine accumulation. Among them, the temperature program has a significant impact on pyrazine accumulation, indicating that there is a compatibility between the stepped heating program used in this invention and the compound microbial agent. This stepped heating program enables the precursor generation, substrate release, pyrazine synthesis, and flavor accumulation at high temperatures to be sequential in time, thus distinguishing it from simple natural heating or extensive high-temperature fermentation processes.

[0077] 2. Box-Behnken Response Surface Analysis Further, a four-factor, three-level Box-Behnken response surface methodology was employed, using total pyrazine content as the response value, to evaluate the effects of total inoculum amount (5%, 10%, 15%), water addition amount (32%, 40%, 48%), and strain concentration (10%). 6 10 7 10 8 System optimization was performed using CFU / mL and temperature programs (3 seven-step heating regimes), with a total of 27 experiments (see Table 5). The results showed that within the set range of factors, the total pyrazine content fluctuated from 695.799 to 5338.91 μg / kg, with an average of approximately 2367.35 μg / kg, indicating that different combinations of conditions significantly affected pyrazine formation. Among these, the optimal conditions were: total inoculum amount 10%, water addition 40%, and strain concentration 10... 7 The combination of CFU / mL and temperature program 37℃→48℃→52℃→55℃→59℃→63℃→63℃ performed best, with the total pyrazine content reaching 5338.91 μg / kg in Experiment 12. Repeated experiments under the same center point conditions (such as Experiments 2 and 15) yielded similar results, indicating that this combination of conditions has good repeatability and stability. A summary analysis of pyrazines with high detection frequencies and above the limit of quantitation in 27 experimental groups revealed that 2,3-dimethylpyrazine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, trimethylpyrazine, and tetramethylpyrazine were the main detected components.

[0078] Table 5 Response Surface Optimization Scheme and Results

[0079] Quadratic polynomial regression and analysis of variance (Table 6) on the response surface data showed that the model was highly significant overall (F=19.00, p<0.0001), indicating that the established model could effectively describe the relationship between each factor and the total pyrazine content; among them, the linear terms of strain concentration (C) and temperature program (D) were highly significant (p≤0.0001), which are key factors affecting pyrazine formation; the quadratic terms of each factor (A) 2 B 2 C 2 D 2 All values ​​were highly significant (p < 0.0001), indicating a significant curve effect in the response surface methodology; the lack-of-fit term was not significant (p = 0.0503), indicating a reasonable model fit. Model statistical parameters show: the coefficient of determination R0... 2 The coefficient of determination (COP) is 0.9568, the correction coefficient of determination (COP) is 0.9065, the prediction coefficient of determination (COP) is 0.7529, the precision is 16.49, and the coefficient of variation (CV) is 17.24%, indicating that the model has good fit, stability, and predictive ability. Response surface 3D plot and contour plot (…) Figures 3-5 Further analysis showed that the total pyrazine content exhibited a distinct dome-shaped quadratic relationship with each factor, with the optimal region concentrated near the moderate levels of each factor; among them, there was a certain synergistic trend between strain concentration and temperature program combination (CD), consistent with the results of analysis of variance.

[0080] Table 6. Response surface methodology and significance test of fermentation conditions for high-yield pyrazine-based compound microbial agents.

[0081] Based on the comprehensive optimization results of the expectation function ( Figure 6 The recommended process parameters obtained are: inoculum size approximately 12.9%, water addition approximately 40.3%, and bacterial strain concentration approximately 1.5 × 10⁻⁶. 7 With a CFU / mL concentration and a stepped temperature program between medium and high levels, the model predicted a total pyrazine content of 4982.54 μg / kg under these conditions, with an expectation factor of 0.869. These results demonstrate that the analytical and optimization method established in this embodiment can be effectively used to optimize the fermentation process conditions for high-yield pyrazine-containing compound microbial agents.

[0082] Example 3 This embodiment illustrates the application of segmented inoculation (space-time separation) high-temperature solid-state fermentation.

[0083] (a) Methods This embodiment further provides a high-temperature solid-state fermentation method with segmented inoculation based on Embodiment 2. The compound microbial agent is introduced in two stages according to a spatiotemporal separation method, based on the optimal conditions of the optimized process in Embodiment 2, to achieve the matching of colonization and dominance periods of different microbial communities in different microenvironments. For example... Figure 11The flowchart shown illustrates the high-temperature solid-state fermentation method as follows: During the mixing stage, group A bacterial solution is added to the solid substrate and thoroughly mixed to allow group A to enter the substrate and preferentially colonize, thus providing a supply of pyrazine precursor substances in the early stages of fermentation. After culturing the system for 2 days, group B and group C bacterial solutions are inoculated onto the surface and microenvironment of the fermentation material using spraying or atomization, allowing them to colonize in the surface and microporous areas. The subsequent solid-state fermentation process is then completed following the stepwise temperature increase procedure described in Example 2. The comparative example is a solid-state fermentation method using the same non-segmented, single-inoculation method as Example 2, simultaneously introducing groups A, B, and C into the fermentation system. The pyrazine production capacity was determined using the same qualitative method as in Example 2.

[0084] Furthermore, the segmented inoculation method of this invention is not a simple simulation of the natural spatial distribution of microorganisms in traditional solid-state fermentation, but rather an active regulation based on the differences in fermentation stage, spatial niche, and target metabolic function among three groups of functional bacteria (Groups A, B, and C). Specifically, Group A consists of yeast components capable of producing pyrazine precursor metabolites, and its main function is in the early stage of fermentation. Introducing Group A into the solid substrate during the mixing stage allows it to preferentially colonize when initial moisture is relatively sufficient, temperature is relatively low, and nutrients within the substrate have not yet been largely consumed, thus generating pyrazine-forming precursors such as acetoin and 2,3-butanediol, thereby establishing a precursor supply basis in the early stage of fermentation. Group B consists of pyrazine-producing functional bacteria components that promote pyrazine synthesis and accumulation, while Group C consists of functional bacteria components that possess substrate-degrading enzyme activity and promote the synthesis and accumulation of pyrazine and flavor compounds. If groups B and C are mixed with group A at the beginning of fermentation and incorporated entirely into the material, groups B and C will prematurely enter an internal environment characterized by low mass transfer, uneven oxygen distribution, and insufficient accumulation of precursor substances. This can easily lead to a missynchronization between their pyrazine production and substrate degradation processes and the precursor supply phase of group A, resulting in an ineffective connection between the three stages of precursor supply, substrate release, and pyrazine synthesis. This situation may not necessarily manifest as complete cell death, but it will cause a mismatch between the dominant phase of the target functional bacteria and the target metabolic window, thereby reducing the accumulation efficiency of pyrazine compounds.

[0085] (II) Results like Figure 7As shown, under the same substrate conditions and the same stepwise heating program, the treatment groups (T1~T3) that adopted the segmented inoculation method of this embodiment (first inoculated into group A, cultured for 2 days, and then inoculated into groups B and C) showed an overall increasing trend in pyrazine products compared with the control group (CK1~CK3) which adopted the single inoculation method. In the standardized heat map, most target components changed from the blue / light-colored area of ​​the control group to the orange-red area of ​​the treatment group, especially the tetramethylpyrazine and trimethylpyrazine components, which showed more obvious increases. Specifically, the tetramethylpyrazine content increased from 4605.97–5198.65 μg / kg in the control group to 5576.12–5878.83 μg / kg in the treatment group; the trimethylpyrazine content increased from 126.01–147.56 μg / kg in the control group to 266.01–309.28 μg / kg in the treatment group; and the cumulative content increased from 4902.09–5432.24 μg / kg in the control group to 6138.76–6458.27 μg / kg in the treatment group, with the highest cumulative content in group T3 at 6458.27 μg / kg. These results indicate that the segmented inoculation high-temperature solid-state fermentation method of this invention can effectively promote the colonization and synergistic metabolism of the complex microbial community in different microenvironments, and improve pyrazine production capacity and the accumulation level of the target pyrazine. This segmented inoculation method is not a simple copy of the natural spatial distribution phenomenon in the traditional fermentation process. Instead, it actively allocates and sequentially connects the precursor supply effect of Group A in the early stage of fermentation with the pyrazine synthesis, substrate degradation and flavor synergy of Groups B and C in the later stages. As a result, under the same substrate and the same step temperature increase conditions, it achieves a higher pyrazine accumulation effect than the single inoculation method.

[0086] Example 4 This embodiment illustrates how the mixing design of the compound microbial agent determines the optimal ratio of A / B / C.

[0087] (a) Mixing design and testing This embodiment, based on Embodiments 2 and 3, optimizes the ratio of groups A, B, and C in the compound microbial agent using a mixing design. Its key feature is that, while maintaining the total inoculum and other solid-state fermentation conditions unchanged, only the composition ratio of the three groups (A, B, and C) in the total inoculum is changed. Here, A, B, and C are the proportion coefficients of each group in the compound microbial agent, satisfying A+B+C=1. The compound microbial agent is prepared according to the ratios listed in Table 7, and inoculated and cultured according to the fermentation conditions of Embodiment 2 and / or the segmented inoculation (spatiotemporal separation) method of Embodiment 3, followed by the subsequent fermentation process. After fermentation, the optimal ratio of A / B / C is determined using the pyrazine-producing substance content as a response index. The pyrazine-producing capacity is tested using the same method as in Embodiment 2.

[0088] Table 7 Mixing Design Test Scheme (A / B / C Ratio)

[0089] (II) Results of the mixing experiment While keeping the total inoculum amount, water addition amount, bacterial concentration, temperature program, and inoculation process constant, only the composition ratio of groups A, B, and C in the total viable count of the compound microbial agent was changed. Solid-state fermentation experiments were conducted according to 31 mixing design schemes shown in Table 7, with the total pyrazine content at the fermentation endpoint used as the response index. The results showed that the total pyrazine content varied significantly under different A / B / C ratios. The total pyrazine content in the 31 experiments ranged from 4421.91 to 10283.70 μg / kg, with the highest value differing from the lowest by approximately 2.33 times. This indicates that the composition ratio of the compound microbial agent has a significant impact on pyrazine production, and high pyrazine production cannot be obtained by arbitrary mixing. The highest measured value was found in Experiment 2, corresponding to ratios A=0.009494, B=0.694052, and C=0.296454, with a total pyrazine content of 10283.70 μg / kg, highly consistent with the optimal ratio of A:B:C≈1:69:30. Further observation of adjacent ratios revealed that when group A was maintained at approximately 1%, group B at 65%–72%, and group C at 27%–34%, the total pyrazine content remained at a high level. For example, when A=0.01, B=0.65, and C=0.34, the total pyrazine content was 9031.42 μg / kg; when A=0.01, B=0.68, and C=0.31, the total pyrazine content was 9853.75 μg / kg; when A=0.01, B=0.70, and C=0.29, the total pyrazine content was 10169.10 μg / kg; and when A=0.01, B=0.72, and C=0.27, the total pyrazine content was 9732.53 μg / kg. These data indicate that the high-response region is concentrated within a relatively narrow ratio window: approximately 1% in group A, approximately 65%–72% in group B, and approximately 27%–34% in group C. Furthermore, when the ratio deviates from this high-response window, the total pyrazine content decreases significantly. Even with group A remaining at approximately 1%, when group B further increased to 75% and group C decreased to 24%, the total pyrazine content decreased to 8957.23 μg / kg; when group B increased to 80% and group C decreased to 19%, the total pyrazine content further decreased to 7763.63 μg / kg. These results indicate that a higher proportion of group B pyrazine-producing bacteria is not necessarily better, and the substrate degradation and flavor synergistic effects of group C are equally necessary for maintaining high pyrazine production.

[0090] On the other hand, when the proportion of group A increased significantly, the total pyrazine content decreased significantly even though groups B or C remained at a high level. For example, when group A was approximately 9%, the total pyrazine content of each group ranged from 5872.62 to 6722.39 μg / kg; when group A was 20%, the total pyrazine content ranged from 4985.35 to 6854.64 μg / kg; and when group A increased to approximately 50%, the total pyrazine content ranged from 4421.91 to 6371.61 μg / kg. The results indicate that while group A plays a role in supplying pyrazine precursors in the early stages of fermentation, excessively high proportions do not further increase the total pyrazine content. Instead, they weaken the matching relationship between precursor supply from group A, pyrazine synthesis from group B, and substrate release / flavor synergy from group C. This is because an excessively high proportion of group A would cause the precursor generation, system acidification, and nutrient competition in the early stages of fermentation to exceed the carrying capacity of groups B and C, resulting in a mismatch between precursor supply and pyrazine synthesis and substrate release. In contrast, A:B:C ≈ 1:69:30 achieves a dynamic balance between limited precursor supply, dominant colonization of pyrazine-producing bacteria, and synergistic substrate degradation, thus exhibiting a significant nonlinear high-response characteristic. Therefore, the A:B:C ≈ 1:69:30 ratio of this invention is not a conventional linear adjustment of the mixed bacteria ratio, but rather a high-response ratio point that dynamically matches the precursor supply, substrate release, and pyrazine synthesis in a solid-state fermentation system. A clear high-response zone is formed near this ratio, and the total pyrazine content decreases to varying degrees when deviating from this zone, indicating that this ratio has significant criticality and nonlinear synergistic characteristics. This can be used to demonstrate that the compound bacterial agent ratio of this invention has a non-obvious technical effect compared to ordinary mixed-culture fermentation schemes.

[0091] Furthermore, the experimental data in Table 7 were fitted with a mixing model and the model was selected (Table 8). The results of the sequential model sum of squares analysis showed that the increments of the linear, quadratic, and quartic terms were all significant (p<0.05). As shown in Table 9, a special quartic mixing model was used to model and optimize the total pyrazine content. The analysis of variance showed that the model was highly significant overall (p<0.001); among them, the interaction terms between groups B and C (BC term) and the higher-order interaction terms (A... 2 BC, AB 2 C) all showed significant results (p<0.05), indicating a clear nonlinear synergistic effect among groups A, B, and C in the fermentation system. The coefficient of determination R of this model is... 2 The adjusted coefficient of determination R is 0.9606. 2 The value of 0.9261 indicates that the model has good fitting accuracy and predictive ability.

[0092] Based on the above model, the total pyrazine content was numerically optimized, resulting in multiple candidate schemes that met the target requirements. Combined with... Figure 8The results shown in the numerical optimization ramp diagram indicate that the overall expectation of one of the preferred schemes is 0.905, with the corresponding group allocation ratios as follows: A=0.009342, B=0.677171, C=0.313487 (A+B+C=1), and the predicted total pyrazine content is 9725.8 μg / kg.

[0093] Depend on Figure 9 (Ternary contour map) As can be seen, the high-value area of ​​total pyrazine content is mainly located in the region where the proportion of component A is close to the lower limit; when component B is at a high level and component C is at a medium level, a clear high-response zone is formed. The preferred range of compound microbial agent ratio can be summarized as: A=0.0093~0.0101; B=0.65~0.72; C=0.27~0.34. More preferably, the compound microbial agent ratio is: A≈0.01, B≈0.68~0.70, C≈0.29~0.31.

[0094] In a preferred embodiment, A / B / C = 0.009342 / 0.677171 / 0.313487, or equivalent approximate ratios (e.g., 0.01 / 0.68 / 0.31, 0.01 / 0.69 / 0.30, 0.01 / 0.70 / 0.29) are used for compounding to obtain a higher pyrazine-producing capacity.

[0095] Table 8 Results of the Sequential Model Sum of Squares Analysis

[0096] Table 9. Analysis of variance for the special quartic model

[0097] It should be noted that although deviations from the optimal ratio lead to a decrease in total pyrazine content, even at the aforementioned extreme boundary ratios (e.g., the lowest total pyrazine content of 4421.91 μg / kg corresponding to 50% of group A), the total pyrazine content is still significantly higher than that of the uninoculated blank control group (approximately 1147.61 μg / kg), achieving an increase of nearly 300%. Experimental results indicate that within the ratio parameter range of 0.5% to 50% of group A, the composite microbial agent described in this invention has a significant promoting effect on the accumulation of pyrazine compounds in the solid-state fermentation substrate, effectively increasing pyrazine yield compared to the control group.

[0098] Example 5 This embodiment illustrates the effect of optimized compound microbial agents on flavor compounds.

[0099] (a) Methods This embodiment, based on the aforementioned embodiments (e.g., Embodiments 2 to 4), provides a method for detecting and quantifying flavor substances in fermentation endpoint samples. The method is characterized by detecting and quantifying volatile flavor substances in the fermentation endpoint samples, and statistically comparing the flavor substances according to compound categories. Specifically, fermentation endpoint samples are collected, and volatile flavor detection methods are used to perform qualitative and semi-quantitative analysis of the flavor substances in the samples. The preferred detection method is HS-SPME-GC-MS, and the specific method refers to the determination of pyrazine (flavor) production capacity of the strain in Embodiment 1. The obtained flavor substance detection results are categorized and statistically analyzed, and the categories include at least pyrazines, furans, ketones, aldehydes, esters, acids, and alcohols. The detection results of the comparative sample are used as a reference for comparative analysis.

[0100] (II) Results In this embodiment, volatile flavor compounds were detected and semi-quantitatively analyzed in the fermentation endpoint samples, and then categorized, statistically analyzed, and compared according to compound type. The results showed that, compared with the control group, the composition and relative content of volatile flavor compounds in the fermentation endpoint samples of the experimental group changed significantly, demonstrating a better flavor enhancement effect. Figure 10 As shown, statistical analysis was conducted on esters, pyrazines, alcohols, acids, aldehydes, ketones, and furans. The total content of volatile flavor compounds in the blank group was 33279.40 μg / kg, while the total content in the experimental group was 54296.41 μg / kg, representing an increase of approximately 63.15% compared to the blank group. This indicates that the method described in this embodiment can significantly improve the overall level of volatile flavor compounds in the fermentation endpoint sample. In terms of category distribution, the content of esters in the experimental group increased from 19327.18 μg / kg to 28809.37 μg / kg, an increase of approximately 49.06%; alcohols increased from 11339.11 μg / kg to 14366.16 μg / kg, an increase of approximately 26.70%; and pyrazines significantly increased from 1147.61 μg / kg to 10231.61 μg / kg, an increase of approximately 791.56% (approximately 8.92 times). Regarding the changes in flavor structure proportions, the proportion of pyrazines in the total volatile flavor compounds in the experimental group significantly increased from approximately 3.45% in the control group to approximately 18.84%, indicating that this embodiment not only increased the total amount of volatile flavor compounds but also significantly reshaped the flavor composition structure, making pyrazines an important component. Esters remained the dominant flavor category, and their relative content further increased, indicating that the experimental group maintained a high level of basic aroma compounds while enhancing characteristic flavors.

[0101] Analysis of the monomers with a certain proportion of pyrazines (Table 10) shows that the experimental group significantly promoted the formation of various pyrazine compounds. Among them, tetramethylpyrazine increased from 1017.79875 μg / kg to 9737.15625 μg / kg, an increase of approximately 856.69% (approximately 9.57 times), making it the main contributing component to the increase in pyrazines in the experimental group; trimethylpyrazine increased from 83.9325 μg / kg to 265.25175 μg / kg, an increase of approximately 216.03%; 2,3-dimethylpyrazine increased from 9.81975 μg / kg to 110.53425 μg / kg, an increase of approximately 1025.63%; and 2,6-dimethylpyrazine increased from 3.71325 to 24.582, an increase of approximately 562.01%.

[0102] Table 10 Comparison of pyrazine content between the blank group and the experimental group (μg / kg)

[0103] In summary, the method described in this embodiment can significantly increase the total amount of volatile flavor compounds in the fermentation endpoint sample, especially significantly promote the generation of pyrazine flavor compounds and increase their proportion in the overall flavor; at the same time, esters and alcohols remain at a high level and further increase, while acids are relatively reduced, which is conducive to forming a fermentation endpoint sample with richer flavor, more distinct layers and better harmony. The above results show that this embodiment has significant technical effects in improving the flavor quality of the endpoint sample. Specifically, compared with the blank control group without inoculation of compound microbial agent, the experimental group of this invention achieved a significant improvement in at least one of the following indicators: (1) Total pyrazine content increased by ≥300%, preferably ≥500%; (2) Tetramethylpyrazine and / or trimethylpyrazine content increased by ≥200%, preferably ≥500%; (3) The total amount of at least one of furans, ketones, aldehydes, esters, acids and alcohols increased by ≥30%, and the content of at least one key monomer compound increased by ≥200%. Combining the results of the segmented inoculation in Example 3 and the results of the mixing design in Example 4, it can be seen that the technical effect of the present invention cannot be simply obtained by single-strain enhancement or one-time compound inoculation, but is formed by the combined effect of the functional division of groups A, B, and C, the optimal ratio of 1:69:30, the segmented inoculation from the inside to the outside, and the stepwise temperature increase program. Meanwhile, the fermentation product obtained by the process described in this invention has a stable total pyrazine content of ≥9000 μg / kg, and under the optimal ratio, it can reach a high yield level of ≥9500 μg / kg.

[0104] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; terms such as "preferredly," "according to a preferred embodiment," or "optionally" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept.

Claims

1. A compound microbial agent composition for high-temperature solid-state fermentation, characterized in that, The compound microbial agent composition contains group A microorganisms, group B microorganisms, and group C microorganisms; Among them, the microorganisms in group A are yeasts that can produce pyrazine precursor metabolites; The microorganisms in group B are pyrazine-producing functional bacteria that promote the synthesis and accumulation of pyrazine. The microorganisms in Group C are functional bacteria that possess substrate-degrading enzyme activity and promote the synthesis and accumulation of pyrazines and flavor compounds. Based on the sum of the effective viable counts of the group A, group B, and group C microorganisms introduced into the solid fermentation substrate, the proportion of group A microorganisms is 0.5% to 50%, the proportion of group B microorganisms is 5% to 80%, and the proportion of group C microorganisms is 5% to 80%.

2. The compound microbial agent composition according to claim 1, characterized in that, Based on the proportion of each component in the total number of live bacteria inoculated, the microorganisms in group A account for 0.8% to 1.2%, the microorganisms in group B account for 65% to 75%, and the microorganisms in group C account for 24% to 34%.

3. The compound microbial agent composition according to claim 1 or 2, characterized in that, The proportions of group A, group B, and group C microorganisms were 1%, 69%, and 30%, respectively.

4. The compound microbial agent composition according to any one of claims 1 to 3, characterized in that, The microorganisms in Group A are *Saccharomyces ferruginea* T-1-4 with accession number CCTCC M 20261287; the microorganisms in Group B are selected from one or more combinations of *Bacillus sonoran* 45-3 with accession number CCTCC M 20261281, *Bacillus sonoran* GS2-45-29 with accession number CCTCC M 20261286, and *Bacillus licheniformis* 37 with accession number CCTCC M 20261284; the microorganisms in Group C are selected from one or more combinations of *Bacillus belyssus* G1-G2 with accession number CCTCC M 20261283, *Bacillus amyloliquefaciens* 00-N3 with accession number CCTCC M 20261285, and *Bacillus belyssus* C-19 with accession number CCTCC M 20261282.

5. An inoculation process for high-temperature solid-state fermentation, characterized in that, The inoculation process using the compound microbial agent composition according to any one of claims 1 to 4 includes the following steps: During the mixing stage of the solid fermentation substrate, the microorganisms of group A are introduced by mixing, so that the microorganisms of group A enter the interior of the solid fermentation substrate, colonize and pre-culture. After the pre-culture, the microorganisms of group B and group C are introduced into the surface and fissure microenvironment of the solid fermentation substrate by spraying or atomizing and colonizing. Continue culturing and use a segmented heating program adapted to the fermentation process for high-temperature solid-state fermentation until fermentation is complete.

6. The inoculation process according to claim 5, characterized in that, In the pre-culture step, the pre-culture time of the microorganisms in group A is 1 to 3 days.

7. The inoculation process according to claim 5 or 6, characterized in that, The total inoculum amount of the compound microbial agent composition is 5%~20%; the moisture content of the solid fermentation substrate is 38%~42%; and the concentration of the inoculated bacterial solution is 10. 6 ~10 8 CFU / mL.

8. The inoculation process according to any one of claims 5 to 7, characterized in that, The segmented heating process includes several heating stages, with a high-temperature stage above 60°C.

9. A fermentation product, characterized in that, The fermentation product is obtained by inoculation process of high-temperature solid-state fermentation as described in any one of claims 5 to 8.

10. The application of the compound microbial agent composition according to any one of claims 1 to 4, the inoculation process according to any one of claims 5 to 8, or the fermentation product according to claim 9 in improving the content of flavor substances in the solid-state fermentation system of Baijiu.

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