A method for temperature-controlled Daqu bacteria to promote metabolite diffusion

Abstract

This invention relates to a method for temperature-controlled fermentation of Daqu (a type of starter culture) microorganisms to promote metabolite diffusion. The method involves: first, preparing a primary fermentation liquid containing viscosity-reducing bacteria and a fermentation liquid containing porosiform bacteria; then, wetting crushed wheat, steaming, cooling, and transferring it to a sterile disc fermentation machine, spreading the mixture, uniformly mixing in Aspergillus niger, and fermenting at a temperature increase of 3°C to 7°C every 10-15 hours. The fermentation process involves turning the koji and mixing in the obtained primary fermentation liquid containing viscosity-reducing bacteria for continued fermentation at 35°C to 40°C, followed by ventilation and loosening until the koji is produced; mixing the resulting material with mother koji and the obtained secondary fermentation liquid containing porosiform bacteria to form koji blocks; and then, after sealed fermentation at a controlled temperature, ventilating and air-drying the koji blocks, continuing to seal and increasing the temperature by 2°C to 4°C daily for 7-10 days to produce the final koji. This invention achieves targeted regulation of the Daqu microorganisms through stepwise temperature increases combined with slow-release of the microorganisms, reducing the stickiness of the koji blocks by approximately 30%. Under these conditions, the acidity and saccharification capacity of the koji blocks can be diffused uniformly.

Description

Technical Field

[0001] This invention relates to the field of brewing starter culture technology, and in particular to a method for temperature-controlled starter culture to promote the diffusion of metabolites. Background Technology

[0002] High-starch grain raw materials are widely used in baijiu brewing. However, after cooking, saccharification, or gelatinization, these materials have high viscosity, which hinders oxygen flow and material diffusion during fermentation, easily leading to regional koji burning or incomplete substrate metabolism. To address these issues, current production methods often involve increasing the particle size of the raw materials or introducing industrial enzyme systems such as proteases and glucans to reduce viscosity. Larger raw material particles inevitably lead to substrate decomposition or incomplete utilization; while industrial enzyme systems for viscosity reduction have drawbacks such as high cost, immediate effects, and enzyme activity being severely limited by fermentation temperature.

[0003] However, as the source of enzyme secretion, microorganisms can more conveniently and efficiently regulate the yeast environment in situ during brewing, but the metabolic activity of microorganisms is also affected by the temperature of the yeast starter. Therefore, there is an urgent need in this field for a method to regulate the yeast starter flora to promote the diffusion of metabolites. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for promoting the diffusion of metabolites by temperature-controlled Daqu bacteria.

[0005] This invention is achieved through the following technical solution:

[0006] The first objective of this invention is to provide a method for promoting the diffusion of metabolites by temperature-controlled *Corydalis yanhusuo* microorganisms, comprising the following steps:

[0007] (1) Mix Pediococcus pentosacchari and Bacillus subtilis with slow-release buffer to prepare Pediococcus pentosacchari suspension and Bacillus subtilis suspension respectively; mix the obtained Pediococcus pentosacchari suspension with soybean oil containing soybean lecithin to form an O / W emulsion; mix the obtained O / W emulsion with Bacillus subtilis suspension to obtain primary fermentation viscosity-reducing bacteria solution;

[0008] (2) Moisten the crushed wheat with water, steam it, cool it to 10℃~20℃ and then transport it to a sterile disc koji-making machine. Spread the material to a thickness of 20cm~30cm, mix it evenly with Aspergillus niger, and ferment it for 1.5 days~2 days by raising the temperature by 3℃~7℃ every 10h~15h. Turn the koji and mix it with the initial fermentation viscosity-reducing bacteria liquid obtained in step (1) and keep it warm for 1 day~3 days. Raise the temperature to 35℃~40℃ and keep it warm for 1 day~3 days. After inoculation, ventilate and loosen the koji until the koji is produced.

[0009] (3) The fused Weissella and Saccharomyces cerevisiae were co-inoculated into the culture medium and cultured under acidic conditions for 7 to 14 days. The complexed bacterial film and bacterial sludge were collected. The obtained complexed bacterial film and bacterial sludge were resuspended in the slow-release buffer solution and then transferred to soybean oil containing soybean lecithin. The mixture was mixed to obtain the post-fermentation pore-forming bacterial culture solution.

[0010] (4) Mix the mother koji and the post-fermentation pore-forming bacteria liquid obtained in step (3) into the koji-producing material obtained in step (2) to make koji blocks; ferment the koji blocks in a sealed environment at 25℃~35℃ for 2 to 3 days, then ventilate and air-dry them to remove mold, continue to seal them and increase the temperature by 2℃~4℃ every day for 7 to 10 days to produce koji.

[0011] In one embodiment of the present invention, the sustained-release buffer solution comprises the following components by weight: 1 to 10 parts trehalose, 0.1 to 1 part chitosan, 0.001 to 0.01 parts calcium chloride, and 1 part sodium chloride.

[0012] In one embodiment of the present invention, in step (1), the concentration of the Pediococcus pentosaceus suspension is 10. 6 cfu / L ~ 10 7 cfu / L; the concentration of the Bacillus subtilis suspension is 10. 6 cfu / L ~ 10 7 cfu / L.

[0013] In one embodiment of the present invention, in step (1), by weight, the primary fermentation viscosity-reducing bacteria include 1 to 3 parts of Pediococcus pentosaceus suspension, 3 to 5 parts of soybean oil containing soybean lecithin, and 7 to 10 parts of Bacillus subtilis suspension.

[0014] In one embodiment of the present invention, in step (2), the amount of Aspergillus niger added is 0.1wt% to 0.5wt%.

[0015] In one embodiment of the present invention, in step (2), the amount of the initial fermentation viscosity-reducing bacterial culture added is 0.1wt% to 1wt%.

[0016] In one embodiment of the present invention, in step (3), the culture medium is MRS culture medium;

[0017] By weight, the complexed bacterial film and bacterial sludge in the post-fermentation pore-forming inoculum solution are 1 to 5 parts; the slow-release buffer solution is 10 to 50 parts; and the soybean oil containing soybean lecithin is 50 to 100 parts.

[0018] The soybean oil is soybean oil containing 5 wt% soybean phospholipids; the Bacillus subtilis suspension also contains 5 wt% Tween 80.

[0019] In one embodiment of the present invention, in step (3), the amount of the mother koji added is 0.5wt% to 5wt%; the amount of the post-fermentation pore-forming bacteria inoculum added is 0.5wt% to 5wt%.

[0020] The post-fermentation pore-forming bacteria are a fusion of Weissella and Saccharomyces cerevisiae, which are cross-fed substrates and have high gas production capacity.

[0021] In one embodiment of the present invention, in step (3), the moisture content of the slab is 30wt% to 40wt%.

[0022] A second objective of this invention is to provide the application of the method in the brewing of baijiu (Chinese liquor).

[0023] The technical solution of the present invention has the following advantages compared with the prior art:

[0024] This invention provides a method for promoting the diffusion of metabolites by temperature-controlled Daqu (fermented koji) microbial community. This invention achieves targeted regulation of Daqu microbial community by gradually increasing the temperature and combining it with slow release of microbial strains, avoiding the colonization inhibition between microbial strains that inhibit the degradation effects of Bacillus subtilis and pentose granules on cellulose and pentoses, respectively. With temperature control, the adhesion of koji blocks can be reduced by about 30%. Under these conditions, the acidity and saccharification ability of koji blocks can be diffused uniformly. Attached Figure Description

[0025] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0026] Figure 1 This is a comparison of acidity and saccharification power at the center and edge of the curved block in an embodiment of the present invention;

[0027] Figure 2 This is a comparison of the adhesion of curved blocks in the embodiments of the present invention. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0029] This invention provides a method for temperature-controlled *Aspergillus oryzae* flora to promote metabolite diffusion, comprising the following steps:

[0030] (1) Sustained-release buffer: By weight, 1 to 10 parts of trehalose, 0.1 to 1 part of chitosan, 0.001 to 0.01 parts of calcium chloride, and 1 part of sodium chloride are placed in 100 parts of PBS buffer with pH=6 and mixed well. The mixture is then treated at 121°C and 0.1 MPa for 20 min.

[0031] (2) Primary fermentation viscosity-reducing bacteria: Pediococcus pentosaceus and Bacillus subtilis were placed in a slow-release buffer to prepare 10 7 CFU / L bacterial suspension. By weight, 1 to 3 parts of Pediococcus pentosaceus suspension are mixed with 3 to 5 parts of soybean oil (containing 5 wt% soybean lecithin) to form an O / W emulsion, which is then transferred to 7 to 10 parts of Bacillus subtilis suspension (containing 5 wt% Tween 80) and mixed to form a W / O / W emulsion.

[0032] (3) Low-temperature heating disc koji making: Crushed wheat is moistened with 30-40 wt% water, steamed at 0.1 MPa for 10 min, and then the pressure is increased to 0.2 MPa for 20 min. Cool to 15℃ and transport to a sterile disc koji making machine. Spread the material to a thickness of 20cm-30cm, and evenly mix in 0.1 wt%-0.5 wt% Aspergillus niger. Ferment at 5℃ every 12 hours for 1.5-2 days. Turn the koji and mix in 0.1 wt%-1 wt% of the initial fermentation viscosity-reducing bacteria. Keep it warm for 1-3 days. Increase the temperature to 35℃-40℃ and keep it warm for 1-3 days. After inoculation, ventilate and loosen the koji every 8 hours until the koji is produced.

[0033] (4) Post-fermentation pore-forming bacteria: Fusion Weissella and Saccharomyces cerevisiae were co-inoculated on commercial MRS medium and cultured at a constant pH of 5 and 30°C for 7 to 14 days. By weight, 1 to 5 parts of complexed bacterial film and bacterial sludge were collected, resuspended in 10 to 50 parts of slow-release buffer, and then transferred to 50 to 100 parts of soybean oil (containing 5 wt% soybean lecithin) and mixed to form an O / W emulsion.

[0034] (5) Medium-temperature fermentation of koji blocks: After the koji is produced from the disc, 0.5wt% to 5wt% of mother koji and 0.5wt% to 5wt% of post-fermentation porosiform bacteria are mixed into the material, and the moisture content is adjusted to 30wt% to 40wt% to form koji blocks, which are then transferred to the koji room. The koji room is sealed and kept at 30℃ for 2 days of fermentation, then ventilated to allow mold to set. The room is then sealed again and the temperature is increased by 2℃ every day for 10 days to produce koji.

[0035] Furthermore, the sustained-release buffer consists of 8 parts by weight of trehalose, 1 part of chitosan, 0.005 parts of calcium chloride, and 1 part of sodium chloride, mixed in 100 parts of pH=6 PBS buffer.

[0036] Furthermore, the primary fermentation bacteria for reducing viscosity are Bacillus subtilis, a cellulose-degrading bacterium, and Pediococcus pentosaccharide-degrading bacteria.

[0037] Furthermore, the method for preparing the primary fermentation viscosity-reducing bacteria strain is as follows: *Pediococcus pentosaceus* and *Bacillus subtilis* are placed in a slow-release buffer to prepare 10... 7CFU / L bacterial suspension. By weight, 2 parts of Pediococcus pentosaceus suspension were mixed with 4 parts of soybean oil (containing 5 wt% soybean lecithin) to form an O / W emulsion, which was then transferred to 8 parts of Bacillus subtilis suspension (containing 5 wt% Tween 80) and mixed to form a W / O / W emulsion.

[0038] Further, the low-temperature heating disc koji-making step is as follows: crushed wheat is moistened with 35wt% water, steamed at 0.1MPa for 10 minutes, and then the pressure is increased to 0.2MPa for 20 minutes. The mixture is cooled to 15℃ and transported to a sterile disc koji-making machine, with a 20cm thick layer of material. 0.3wt% Aspergillus niger is evenly mixed in, and fermentation is carried out at a temperature increase of 5℃ every 12 hours for 2 days. The koji is then turned and mixed with 0.5wt% of primary fermentation viscosity-reducing bacteria for incubation for 2 days. The temperature is then increased to 35℃ for incubation for 3 days. After inoculation, the koji is loosened by ventilation every 8 hours until the koji is produced.

[0039] Furthermore, the post-fermentation pore-forming bacteria are a fusion of Weissella and Saccharomyces cerevisiae that are cross-fed with substrates and have high gas production capacity.

[0040] Furthermore, the method for preparing the post-fermentation pore-forming bacteria involves co-inoculating *Westernella* and *Saccharomyces cerevisiae* onto commercial MRS medium and culturing at a constant pH of 5 and 30°C for 14 days. Five parts by weight of the complexed bacterial film and sludge are collected, resuspended in 20 parts of slow-release buffer, and then transferred to 50 parts of soybean oil (containing 5 wt% soybean lecithin) and mixed to form an O / W emulsion.

[0041] Furthermore, the method for fermenting the medium-temperature koji blocks is as follows: after the koji is extracted from the disc, 5 wt% of mother koji and 5 wt% of post-fermentation porosiform bacteria are mixed into the material, and the moisture content is adjusted to 40 wt% to form koji blocks, which are then transferred to the koji room. The koji room is sealed and kept at 30°C for 2 days of fermentation, then ventilated to allow mold to set. The room is then sealed again and the temperature is increased by 2°C daily for 10 days to produce the koji.

[0042] Materials used in this invention:

[0043] Pediococcus pentosaceus (CICC 22228), Bacillus subtilis (CICC 10335), Aspergillus niger (CICC 41254), Fusionia fussima (CICC 23465), and Saccharomyces cerevisiae (CICC 1406) were all purchased from the China Industrial Microbial Culture Collection Center.

[0044] Trehalose, chitosan, calcium chloride, sodium chloride, Tween 80, and MRS culture medium were all purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.

[0045] Mother koji: Red Heart Koji, Liangshan Changjiu Biological Koji Making Co., Ltd.;

[0046] Soybean lecithin was purchased from Guangzhou Huayu Biotechnology Co., Ltd.

[0047] Unless otherwise specified, all other raw materials and equipment are commercially available.

[0048] Example 1

[0049] This embodiment provides a method for temperature-controlled *Corydalis yanhusuo* microorganisms to promote metabolite diffusion, including the following steps:

[0050] (1) Sustained-release buffer: By weight, 8 parts of trehalose, 1 part of chitosan, 0.005 parts of calcium chloride, and 1 part of sodium chloride are placed in 100 parts of PBS buffer with pH=6 and mixed well. The mixture is then treated at 121℃ and 0.1MPa for 20 min.

[0051] (2) Primary fermentation viscosity-reducing bacteria: Pediococcus pentosaceus and Bacillus subtilis were placed in a slow-release buffer to prepare 10 7 CFU / L bacterial suspension. By weight, 2 parts of Pediococcus pentosaceus suspension were mixed with 4 parts of soybean oil (containing 5 wt% soybean lecithin) to form an O / W emulsion, which was then transferred to 8 parts of Bacillus subtilis suspension (containing 5 wt% Tween 80) and mixed to form a W / O / W emulsion.

[0052] (3) Low-temperature heating disc koji making: Crushed wheat is moistened with 35wt% water, steamed at 0.1MPa for 10min, and then the pressure is increased to 0.2MPa for 20min. Cool to 15℃ and transport to a sterile disc koji making machine. Spread the material to a thickness of 20cm, and evenly mix in 0.3wt% Aspergillus niger. Ferment at 5℃ every 12h for 2 days. Turn the koji and mix in 0.5wt% primary fermentation viscosity-reducing bacteria to keep it warm for 2 days. Increase the temperature to 35℃ and keep it warm for 3 days. After inoculation, ventilate and loosen the koji every 8h until the koji is produced.

[0053] (4) Post-fermentation pore-forming bacteria: Fusion Weissella and Saccharomyces cerevisiae were co-inoculated on commercial MRS medium and cultured at a constant pH of 5 and 30°C for 14 days. Five parts by weight of complexed bacterial film and bacterial sludge were collected, resuspended in 20 parts of slow-release buffer, and then transferred to 50 parts of soybean oil (containing 5 wt% soybean lecithin) and mixed to form an O / W emulsion.

[0054] (5) Medium-temperature fermentation of starter culture blocks: After the starter culture is produced from the disc, 5 wt% mother starter culture and 5 wt% post-fermentation porosiform bacteria are mixed into the material, and the moisture content is adjusted to 40 wt% to form starter culture blocks, which are then transferred to the starter culture room. The starter culture room is sealed and kept at 30℃ for 2 days of fermentation, then ventilated to allow mold to dissipate. The room is then sealed again and the temperature is increased by 2℃ every day for 10 days of fermentation to produce starter culture. The finished starter culture can be used directly for brewing or as mother starter culture.

[0055] Example 2 (non-gradual heating)

[0056] This embodiment provides a method for promoting the diffusion of metabolites by Aspergillus oryzae, including the following steps:

[0057] (1) Sustained-release buffer: By weight, 8 parts of trehalose, 1 part of chitosan, 0.005 parts of calcium chloride, and 1 part of sodium chloride are placed in 100 parts of PBS buffer with pH=6 and mixed well. The mixture is then treated at 121℃ and 0.1MPa for 20 min.

[0058] (2) Primary fermentation viscosity-reducing bacteria: Pediococcus pentosaceus and Bacillus subtilis were placed in a slow-release buffer to prepare 10 7 CFU / L bacterial suspension. By weight, 2 parts of Pediococcus pentosaceus suspension were mixed with 4 parts of soybean oil (containing 5 wt% soybean lecithin) to form an O / W emulsion, which was then transferred to 8 parts of Bacillus subtilis suspension (containing 5 wt% Tween 80) and mixed to form a W / O / W emulsion.

[0059] (3) Disc koji making: Crushed wheat is moistened with 35wt% water, steamed at 0.1MPa for 10min, and then the pressure is increased to 0.2MPa for 20min. Cool to 35℃ and transport to a sterile disc koji making machine. Spread the material to a thickness of 20cm, evenly mix in 0.3wt% Aspergillus niger and keep warm for 2 days for fermentation. Turn the koji and mix in 0.5wt% primary fermentation viscosity-reducing bacteria and keep warm for 5 days for fermentation. After inoculation, ventilate and loosen the koji every 8 hours until the koji is produced.

[0060] (4) Post-fermentation pore-forming bacteria: Fusion Weissella and Saccharomyces cerevisiae were co-inoculated on commercial MRS medium and cultured at a constant pH of 5 and 30°C for 14 days. Five parts by weight of complexed bacterial film and bacterial sludge were collected, resuspended in 20 parts of slow-release buffer, and then transferred to 50 parts of soybean oil (containing 5 wt% soybean lecithin) and mixed to form an O / W emulsion.

[0061] (5) Medium-temperature fermentation of starter culture blocks: After the starter culture is produced from the disc, 5 wt% mother starter culture and 5 wt% post-fermentation porosiform bacteria are mixed into the material, and the moisture content is adjusted to 40 wt% to form starter culture blocks, which are then transferred to the starter culture room. The starter culture room is sealed and kept at 30℃ for 2 days of fermentation, then ventilated to remove mold, and then sealed and fermented at 50℃ for 10 days to produce starter culture. The finished starter culture can be used directly for brewing or as mother starter culture.

[0062] Example 3 (Non-sequential sustained release)

[0063] This embodiment provides a method for temperature-controlled *Corydalis yanhusuo* microorganisms to promote metabolite diffusion, including the following steps:

[0064] (1) Sustained-release buffer: By weight, 8 parts of trehalose, 1 part of chitosan, 0.005 parts of calcium chloride, and 1 part of sodium chloride are placed in 100 parts of PBS buffer with pH=6 and mixed well. The mixture is then treated at 121℃ and 0.1MPa for 20 min.

[0065] (2) Primary fermentation viscosity-reducing bacteria: Pediococcus pentosaceus and Bacillus subtilis were placed in a slow-release buffer to prepare 10 7CFU / L bacterial suspension. By weight, 2 parts of Pediococcus pentosaceus suspension and 8 parts of Bacillus subtilis suspension were diluted with 4 parts of PBS buffer and mixed thoroughly.

[0066] (3) Low-temperature heating disc koji making: Crushed wheat is moistened with 35wt% water, steamed at 0.1MPa for 10min, and then the pressure is increased to 0.2MPa for 20min. Cool to 15℃ and transport to a sterile disc koji making machine. Spread the material to a thickness of 20cm, and evenly mix in 0.3wt% Aspergillus niger. Ferment at 5℃ every 12h for 2 days. Turn the koji and mix in 0.5wt% primary fermentation viscosity-reducing bacteria to keep it warm for 2 days. Increase the temperature to 35℃ and keep it warm for 3 days. After inoculation, ventilate and loosen the koji every 8h until the koji is produced.

[0067] (4) Post-fermentation pore-forming bacteria: Fusion Weissella and Saccharomyces cerevisiae were co-inoculated on commercial MRS medium and cultured at a constant pH of 5 and 30°C for 14 days. Five portions of complexed bacterial film and bacterial sludge were collected by weight, resuspended in 20 portions of slow-release buffer, and then transferred to 50 portions of PBS buffer for dilution and mixing.

[0068] (5) Medium-temperature fermentation of starter culture blocks: After the starter culture is produced from the disc, 5 wt% mother starter culture and 5 wt% post-fermentation porosiform bacteria are mixed into the material, and the moisture content is adjusted to 40 wt% to form starter culture blocks, which are then transferred to the starter culture room. The starter culture room is sealed and kept at 30℃ for 2 days of fermentation, then ventilated to allow mold to dissipate. The room is then sealed again and the temperature is increased by 2℃ every day for 10 days of fermentation to produce starter culture. The finished starter culture can be used directly for brewing or as mother starter culture.

[0069] Example 4 (without adding anti-viscosity bacteria)

[0070] This embodiment provides a method for temperature-controlled *Corydalis yanhusuo* microorganisms to promote metabolite diffusion, including the following steps:

[0071] (1) Low-temperature heating disc koji making: Crushed wheat is moistened with 35wt% water, steamed at 0.1MPa for 10min, and then the pressure is increased to 0.2MPa for 20min. Cool to 15℃ and transport to a sterile disc koji making machine. Spread the material to a thickness of 20cm, and evenly mix in 0.3wt% Aspergillus niger. Ferment at 5℃ every 12h for 2 days, turn the koji and keep it warm for another 2 days, then raise the temperature to 35℃ and keep it warm for 3 days. After inoculation, ventilate and loosen the koji every 8h until the koji is produced.

[0072] (2) Medium-temperature fermentation of starter culture blocks: After the starter culture is produced from the disc, 5 wt% of mother starter culture is mixed into the material and the moisture content is adjusted to 40 wt% to form starter culture blocks, which are then transferred to the starter culture room. The starter culture room is sealed and kept at 30℃ for 2 days of fermentation. After that, it is ventilated and allowed to dry out. The room is then sealed again and the temperature is increased by 2℃ every day for 10 days of fermentation to produce starter culture. The finished starter culture can be used directly for brewing or as mother starter culture.

[0073] Performance testing

[0074] Based on the standard QB / T 4257—2011 "General Analytical Methods for Brewing Daqu", the acidity and saccharification power of the daqu samples at the center (sample size 3×3×3cm) and edge (3×3×3cm from the leftmost side towards the center) were compared (sample number 10, results are presented as average). The viscosity of the daqu was determined using the method described by Zhao Dong et al. in "Determination of Parameters for TPA Analysis and Preliminary Determination of Physical Properties of Fermented Grains at Different Levels" (measured after sample mixing, sample number 10, results are presented as average).

[0075] like Figure 1 and Figure 2 The stepwise temperature control of fermentation is crucial to the viability of the microbial strains in this invention. The optimal temperature for *Bacillus subtilis* is the low-temperature stage (25℃~30℃), for *Pediococcus pentosaceus* the medium-low temperature stage (30℃~35℃), and for *Weissella fusionis* and *Saccharomyces cerevisiae* the medium-high temperature stage (37℃~42℃). Therefore, stepwise temperature control is beneficial for the fermentation and regulation of viscosity reduction and porosity enhancement. Furthermore, this technology uses emulsion technology to regulate the sequence of action of the microorganisms, avoiding colonization inhibition between strains. Examples 1 and 4 compare the degradation effects of *Bacillus subtilis* and *Pediococcus pentosaceus* on cellulose and pentoses, respectively. Combined with temperature control, this reduces the viscosity of the koji blocks by approximately 30%. Under these conditions, the acidity and saccharification capacity of the koji blocks can be diffused uniformly.

[0076] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for promoting metabolite diffusion by temperature-controlled *Daqu* microorganisms, characterized in that, Includes the following steps: (1) Mix Pediococcus pentosacchari and Bacillus subtilis with slow-release buffer to prepare Pediococcus pentosacchari suspension and Bacillus subtilis suspension respectively; mix the obtained Pediococcus pentosacchari suspension with soybean oil containing soybean lecithin, and then mix it with Bacillus subtilis suspension to obtain primary fermentation viscosity-reducing bacteria solution; (2) The crushed wheat is moistened with water, steamed, cooled and then transported to a sterile disc koji-making machine. The material is spread and mixed evenly with Aspergillus niger. The temperature is increased by 3℃~7℃ every 10 h~15 h to ferment for 1.5 to 2 days. The koji is turned and mixed with the initial fermentation viscosity-reducing bacteria liquid obtained in step (1) for 1 to 3 days of heat preservation and fermentation. The temperature is increased to 35℃~40℃ for 1 to 3 days of heat preservation and fermentation. After inoculation, ventilation is provided to loosen the koji until the koji is produced. (3) The fused Weissella and Saccharomyces cerevisiae were co-inoculated into the culture medium and cultured. The complexed bacterial film and bacterial sludge were collected. The obtained complexed bacterial film and bacterial sludge were resuspended in the slow-release buffer and then transferred to soybean oil containing soybean lecithin. The mixture was then mixed to obtain the post-fermentation pore-forming bacterial culture solution. (4) Mix the mother koji and the post-fermentation pore-forming bacteria liquid obtained in step (3) into the koji-producing material obtained in step (2) to make koji blocks; after the koji blocks are sealed and kept warm for 2 to 3 days, ventilate them, continue to seal them and raise the temperature by 2℃ to 4℃ every day for 7 to 10 days to produce koji. The *Pediococcus pentosaceus* is *Pediococcus pentosaceus* CICC 22228; *Bacillus subtilis* is *Bacillus subtilis* CICC10335; *Weissella fusionis* is *Weissella fusionis* CICC 23465; and *Saccharomyces cerevisiae* is *Saccharomyces cerevisiae* CICC 1406. In step (1), the concentration of the Pediococcus pentosaceus suspension is 10. 6 cfu / L~10 7 cfu / L; the concentration of the Bacillus subtilis suspension is 10. 6 cfu / L~10 7 cfu / L; In step (1), by weight, the primary fermentation viscosity-reducing bacteria strain contains 1 to 3 parts of Pediococcus pentosaceus suspension, 3 to 5 parts of soybean oil containing soybean phospholipids, and 7 to 10 parts of Bacillus subtilis suspension.

2. The method according to claim 1, characterized in that, The sustained-release buffer solution comprises, by weight, 1 to 10 parts trehalose, 0.1 to 1 part chitosan, 0.001 to 0.01 parts calcium chloride, and 1 part sodium chloride.

3. The method according to claim 1, characterized in that, In step (2), the amount of Aspergillus niger added is 0.1wt%~0.5wt%.

4. The method according to claim 1, characterized in that, In step (2), the amount of the initial fermentation viscosity-reducing bacteria culture solution added is 0.1 wt%~1 wt%.

5. The method according to claim 1, characterized in that, In step (3), the culture medium is MRS culture medium; By weight, the complexed bacterial film and bacterial sludge in the post-fermentation pore-forming inoculum solution are 1 to 5 parts; the slow-release buffer solution is 10 to 50 parts; and the soybean oil containing soybean lecithin is 50 to 100 parts.

6. The method according to claim 1, characterized in that, In step (4), the amount of the mother koji added is 0.5 wt% to 5 wt%; the amount of the post-fermentation pore-forming bacteria inoculum added is 0.5 wt% to 5 wt%.

7. The method according to claim 1, characterized in that, In step (4), the moisture content of the slab is 30 wt%~40 wt%.

8. The application of the method according to any one of claims 1-7 in the brewing of baijiu (Chinese liquor).

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