Liquid culture method of Daqu

Abstract

The application discloses a liquid culture method of Daqu, and relates to the technical field of liquor brewing, which comprises the steps of liquid seed source preparation, gelatinization treatment, initial culture, stage-by-stage culture, microporous membrane filtration, reverse osmosis dehydration and embedding granulation; a culture additive is added in the gelatinization culture medium, the culture additive comprises beta-glucanase, xylanase, calcium chloride, magnesium sulfate heptahydrate, glycerol, soybean lecithin, ectoine and hydroxypropyl cellulose; polyvinylpyrrolidone suspension is added in the stage-by-stage culture process; through the above treatment, the raw material particles in the culture process are dispersed, the culture system is stable, and the subsequent membrane separation process is matched with each other; and the obtained liquid Daqu is subjected to microporous membrane filtration, reverse osmosis concentration, embedding and spray granulation to obtain a Daqu product; the application can improve the stability of the liquid culture process, and improve the adaptability of the post-treatment process of the culture product, and is suitable for the large-scale preparation of Daqu.

Description

Technical Field

[0001] This invention relates to the field of brewing technology, specifically to a liquid culture method for Daqu (a type of starter culture). Background Technology

[0002] Daqu (a type of starter culture) is an important basic fermentation material in traditional brewing processes. It contains a variety of microorganisms and related enzyme systems, which can participate in processes such as saccharification of raw materials, fermentation, and the formation of flavor substances. Traditional Daqu production usually adopts a solid-state cultivation method, that is, using grain raw materials as a culture medium, and then stacking them for cultivation after natural or artificial inoculation, so that microorganisms can multiply in a solid environment and form Daqu products with certain enzyme activity and fermentation functions.

[0003] With the development of modern brewing industry, liquid culture technology has been gradually applied to the preparation of Daqu (a type of starter culture). Compared with traditional solid-state culture, liquid culture allows for continuous control of the culture process by adjusting the culture temperature, stirring, aeration conditions, and culture medium composition, which is beneficial for improving the automation and stability of the production process. However, since the Daqu culture system usually contains grain raw material particles, starch degradation products, proteins, polysaccharides, and various microbial components, problems still exist in the liquid culture process, such as complex culture system, uneven dispersion of solid particles, significant changes in the viscosity of the culture medium, and difficulty in stably controlling the growth state of microorganisms.

[0004] Existing methods for liquid culture of Daqu (a type of Chinese liquor) typically improve the culture effect by adjusting the composition of the culture medium, optimizing the culture temperature and culture time. However, in actual culture, the complex colloidal structure formed after the raw materials are gelatinized may affect the fluidity and mass transfer process of the culture medium. At the same time, microbial reproduction, accumulation of metabolites, and temperature changes during culture may cause changes in the state of the culture system, affecting the stability of the culture process.

[0005] In addition, after liquid culture, post-processing steps such as filtration, concentration, drying, or granulation are usually required to obtain Daqu products that are easy to store and transport. Because the culture medium contains microorganisms, incompletely degraded raw material particles, and soluble or colloidal substances, existing filtration and concentration processes are easily affected by changes in the composition of the culture medium, resulting in problems such as insufficient separation efficiency, complex post-processing, and the need to improve the stability of the obtained product.

[0006] Therefore, it is still necessary to provide a liquid culture method suitable for Daqu production to improve the stability of the culture process, enhance the compatibility of the culture system with subsequent separation, concentration and granulation processes, and thus obtain a Daqu product with stable performance. Summary of the Invention

[0007] To address the challenges of existing liquid culture methods for Daqu (a type of Chinese koji), such as high viscosity of the gelatinized medium, easy aggregation of fine particles, insufficient stability of enzyme preparations and cells during stirring, aeration, and staged heating, as well as the tendency of culture additives to remain or increase filtration load during subsequent membrane separation, making it difficult to simultaneously achieve mass transfer, temperature control, microbial enrichment, and post-culture separation, this invention provides a liquid culture method for Daqu.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a liquid culture method for Daqu (a type of Chinese koji), comprising the following steps: S1, Liquid Seed Source Preparation Daqu liquid seed source was prepared using Daqu.

[0009] Specifically, Daqu (a type of starter culture) is mixed with water at 28-35℃ at a weight ratio of 1:8 to 1:15, kept at 28-35℃ for 8-12 hours to absorb water, and then stirred and pulverized to obtain a suspension. When no less than 95% of the material in the obtained suspension can pass through a 40-mesh sieve, the pulverization is stopped, the suspension under the sieve is collected, and Daqu liquid seed source is obtained.

[0010] S2, Gelatinization treatment The raw materials for Daqu (a type of Chinese liquor) cultivation are mixed with water and gelatinized to obtain a gelatinized culture medium. After the gelatinized culture medium is cooled, a culture additive is added.

[0011] The raw material for Daqu cultivation consists of 70-75 wt% barley and the remainder peas, with the sum of the mass percentages of barley and peas being 100 wt%.

[0012] The raw material for Daqu culture is pulverized to pass through a 120-mesh sieve. The pulverized raw material is mixed with water at a weight ratio of 1:8 to 1:15, heated and stirred to gelatinize. During the gelatinization process, samples are taken for iodine reaction detection. Gelatinization is stopped when the gelatinized material does not react with iodine to form a blue color, and the gelatinized culture medium is obtained.

[0013] Cool the gelatinized culture medium to 30-32°C, add culture additives, and treat at 30-32°C for 30-60 minutes.

[0014] The culture additives include β-glucanase, xylanase, calcium chloride, magnesium sulfate heptahydrate, glycerol, soybean lecithin, ectoine, and hydroxypropyl cellulose.

[0015] Based on the dry matter mass of the Daqu culture raw material used in step S2, the amount of β-glucanase added is 30~80 U / g dry matter, and the amount of xylanase added is 50~150 U / g dry matter.

[0016] Based on the total volume of the gelatinized culture medium after adding the culture additive in step S2 and before inoculating the Daqu liquid seed source, the concentration of calcium chloride is 0.10~0.30 g / L, the concentration of magnesium sulfate heptahydrate is 0.05~0.20 g / L, the concentration of glycerol is 1~3 g / L, the concentration of soybean lecithin is 0.10~0.30 g / L, the concentration of ectoine is 0.5~2.0 g / L, and the concentration of hydroxypropyl cellulose is 0.2~0.8 g / L.

[0017] The viscosity of the 2wt% aqueous solution of the hydroxypropyl cellulose is 2~10 mPa·s, and the cloud point of the hydroxypropyl cellulose in the gelatinized medium after the addition of culture additives is 39~44℃.

[0018] In this invention, the dry matter mass of the Daqu (a type of starter culture) raw material is the mass of the Daqu raw material after deducting its moisture content. The enzyme activity units of β-glucanase and xylanase are determined according to the enzyme activity determination methods of the corresponding enzyme preparations, and the same enzyme activity determination conditions are used in the same set of examples and comparative examples.

[0019] The culture additive is prepared according to the following steps: A1. Hydroxypropyl cellulose is added to water for dispersion and hydration to obtain a hydroxypropyl cellulose dispersion. A2. Add ectoine, glycerol, calcium chloride, magnesium sulfate heptahydrate and water-dispersed soybean lecithin to the hydroxypropyl cellulose dispersion obtained in step A1, and mix to obtain a mixture. A3. Add β-glucanase and xylanase to the mixture obtained in step A2, mix well, and obtain the culture additive.

[0020] Preferably, in step A1, hydroxypropyl cellulose is added to water at a temperature not higher than 25°C to prepare a dispersion with a hydroxypropyl cellulose content of 0.5~1.0wt%, and hydrated for 30~60 minutes.

[0021] Preferably, in step A3, β-glucanase and xylanase are added when the temperature of the mixture obtained in step A2 is not higher than 35°C, and then mixed for 5-15 minutes.

[0022] S3, Initial Cultivation The Daqu liquid seed source obtained in step S1 was inoculated into the gelatinized culture medium obtained in step S2, and initial culture was carried out under stirring and ventilation and oxygen supply conditions to obtain the initial culture medium.

[0023] The inoculation amount of the Daqu liquid seed source is 30%~35% of the gelatinized culture medium volume. After inoculation, the culture temperature is controlled at 28~35℃, and the culture is carried out for 24~30h under continuous stirring and aeration to obtain the initial culture medium.

[0024] S4. Phased training The initial culture medium obtained in step S3 is subjected to staged heating culture. During the staged heating culture process, polyvinylpyrrolidone suspension is added and the staged heating culture is continued to obtain liquid Daqu culture mash.

[0025] The phased cultivation includes the following steps: S41. Adjust the temperature of the initial culture medium obtained in step S3 to 33~35℃ and culture it for 24~32h under stirring and ventilation and oxygen supply conditions. S42. Adjust the temperature of the culture medium obtained in step S41 to 35~39℃ and culture it for 48~56h under stirring and ventilation conditions. After the culture in step S43 and S42 is completed, before adjusting the temperature of the culture medium to 39~42℃, add polyvinylpyrrolidone suspension to the culture medium obtained in step S42, then adjust the temperature of the culture medium to 39~42℃ and culture for 24~36h under stirring and ventilation and oxygen supply conditions. S44. Detect the total number of bacteria in the culture medium obtained in step S43; when the total number of bacteria is less than 250 million / mL, extend the culture time of step S43 and detect it again; when the total number of bacteria is not less than 250 million / mL, adjust the temperature of the culture medium to 42~45℃, and culture for 24~36h under stirring and ventilation and oxygen supply conditions to obtain liquid Daqu culture mash.

[0026] The polyvinylpyrrolidone suspension was prepared according to the following method: Porous cross-linked polyvinylpyrrolidone particles were added to water and dispersed and hydrated under stirring conditions to obtain a polyvinylpyrrolidone suspension.

[0027] The median particle size D50 of the porous cross-linked polyvinylpyrrolidone particles is 20~80μm, the D10 is not less than 5μm, and the mass proportion of particles with a particle size less than 2μm is not higher than 1%.

[0028] The polyvinylpyrrolidone suspension contains 5-10 wt% porous cross-linked polyvinylpyrrolidone particles and has a hydration time of 15-120 min.

[0029] After the polyvinylpyrrolidone suspension is prepared, it is stirred before use; when the suspension settles, it is stirred again until the particles are evenly dispersed before adding the culture medium obtained in step S42.

[0030] In step S43, the amount of polyvinylpyrrolidone suspension added meets the following condition: The concentration of porous cross-linked polyvinylpyrrolidone particles in the culture medium after addition was 0.2~1.0 g / L; The mass ratio of porous cross-linked polyvinylpyrrolidone particles to hydroxypropyl cellulose added in step S2 is 0.5:1 to 2:1.

[0031] S5, Microporous membrane filtration The liquid Daqu culture mash obtained in step S4 is subjected to microporous membrane cross-flow filtration, and the filtrate is collected.

[0032] Specifically, the liquid Daqu culture mash obtained in step S4 is sent to a cross-flow filtration device and filtered using a microporous membrane with a pore size of 2μm. The liquid Daqu culture mash circulates along the surface of the microporous membrane, and the filtrate that permeates through the microporous membrane is collected.

[0033] S6, Reverse Osmosis Dehydration The filtrate obtained in step S5 is subjected to reverse osmosis dehydration to obtain Daqu concentrate.

[0034] Specifically, the filtrate obtained in step S5 is sent to a reverse osmosis membrane device for dehydration. When the water content of the retentate is not higher than 80 wt%, the reverse osmosis dehydration is terminated to obtain Daqu concentrate.

[0035] S7, Encapsulation Granulation Add an encapsulating agent to the Daqu concentrate obtained in step S6 and perform spray granulation to obtain the Daqu product.

[0036] The encapsulating agent is dextrin, and the amount of dextrin added is 5% to 7% of the mass of the Daqu concentrate obtained in step S6.

[0037] Dextrin is added to the Daqu concentrate and mixed evenly. The resulting mixture is then sent to a spray granulation device for atomization and drying. The resulting granules are collected to obtain the Daqu product.

[0038] The viscosity of the 2wt% aqueous solution of hydroxypropyl cellulose was determined using a rotational viscometer. During the determination, the hydroxypropyl cellulose was prepared into a 2wt% aqueous solution, and its dynamic viscosity was measured at 25°C.

[0039] The turbidity point of the hydroxypropyl cellulose in the gelatinized medium was determined as follows: The gelatinized medium, after the addition of the culture additive in step S2 and before inoculation with the Daqu liquid seed source, was taken and gradually heated under stirring. The absorbance of the gelatinized medium at a wavelength of 600 nm was measured at different temperatures. The temperature at which the absorbance began to continuously increase was determined as the turbidity point. All samples used the same heating rate, sampling volume, and detection path length.

[0040] The D10, D50, and proportion of particles with a diameter less than 2 μm of the porous cross-linked polyvinylpyrrolidone particles were determined using a laser particle size analyzer. D10 is the particle size corresponding to a cumulative particle size distribution reaching 10%, and D50 is the particle size corresponding to a cumulative particle size distribution reaching 50%.

[0041] The moisture content was determined by the loss on drying method, which involves taking a quantitative sample, drying it to a constant weight, and calculating the moisture content of the sample based on the mass difference before and after drying.

[0042] Compared with the prior art, the present invention has the following beneficial effects: This invention, based on the processes of preparing liquid seed sources for barley koji (a type of starter culture), gelatinization, staged cultivation, microporous membrane filtration, reverse osmosis dehydration, encapsulation, and spray granulation, adds a culture additive to the gelatinization medium and incorporates a polyvinylpyrrolidone (PVP) suspension during the staged cultivation process. The β-glucanase and xylanase in the culture additive reduce the viscous structure formed by non-starch polysaccharides in the barley and pea gelatinization system. Calcium chloride, magnesium sulfate heptahydrate, glycerol, and soybean lecithin maintain the dispersion of the enzyme preparation and the stability of the culture system. Ectoin and hydroxypropyl cellulose maintain the stability of the cells and enzymes, and improve the dispersion of solid particles in the culture medium, respectively. The porous cross-linked PPVP particles form composite particles with hydroxypropyl cellulose during the heated cultivation stage and are retained during subsequent microporous membrane filtration. Therefore, this invention can balance the fluidity of the gelatinized culture medium, the uniformity of dispersion in the initial culture, the stability of the staged culture, and the compatibility of the culture additives with the subsequent membrane treatment process. Furthermore, the resulting filtrate can be concentrated via reverse osmosis, encapsulated with dextrin, and spray-granulated to produce a microparticle-shaped Daqu product that is easy to store, transport, and use. Liquid culture also facilitates the adjustment of culture temperature, stirring, and ventilation conditions, and avoids the pulverization process before use of the Daqu product.

[0043] This invention introduces ectoine, hydroxypropyl cellulose with specific viscosity and cloud point, and porous cross-linked polyvinylpyrrolidone particles into the Daqu liquid culture system in sequence according to the culture stages. This allows the culture additives to maintain dispersion and stability in the initial low-temperature culture stage, and to be transformed into composite particles that can be retained by the microporous membrane in the subsequent heating stage. This allows the stabilization treatment during the culture process to be connected with the separation treatment after the culture is completed.

[0044] This invention introduces ectoine into the culture additive. Ectoine can form a stable hydration environment with water molecules, reducing structural changes in the cell membrane, added enzymes, and enzymes formed during culture due to temperature changes, stirring, shearing, and gas-liquid interface interactions. When used in conjunction with β-glucanase and xylanase, ectoine can maintain the stability of the enzymes as the enzyme preparation reduces the viscosity of the culture medium, and minimize the adverse effects of increased stirring and aeration on the cells and enzymes.

[0045] This invention also introduces hydroxypropyl cellulose (HCP) with specific viscosity and cloud point into the culture additive. HCP maintains a hydrated and dispersed state at the initial culture temperature, and its molecular chains can distribute between the culture medium and raw material particles, reducing the re-agglomeration or rapid sedimentation of small raw material particles after enzyme treatment, thus ensuring more uniform contact between the liquid starter culture and the gelatinized culture medium. Due to the use of low-viscosity HCP and controlled addition, this component maintains particle dispersion without significantly counteracting the viscosity-reducing effects of β-glucanase and xylanase. When combined with HCP, ectoine can maintain the stability of the cells and enzymes while preserving the dispersion of particles in the culture medium, thus forming a synergistic effect.

[0046] As the culture temperature rises to the cloud point range of hydroxypropyl cellulose (HCP), the HCP molecular chains gradually lose some bound water and shrink and aggregate. Ectoin's regulation of the hydration state of the culture medium simultaneously affects both the dehydration and aggregation processes of HCP, potentially leading to the formation of numerous small aggregates after turbidity occurs. Although these aggregates have undergone temperature-responsive changes, their particle size may still be insufficient for stable retention by a 2 μm microporous membrane, resulting in the problem of HCP aggregating but being difficult to separate from the filtrate.

[0047] Therefore, this invention incorporates porous cross-linked polyvinylpyrrolidone (PVP) particles before hydroxypropyl cellulose (HCP) undergoes temperature-responsive aggregation. HCP molecules contain hydroxyl groups, and PCP contains carbonyl groups capable of interacting with these hydroxyl groups. As the culture temperature increases, small aggregates of HCP adsorb onto the surface and pores of the PCP particles, further agglomerating around these particles to form composite particles larger than individual HCP aggregates. Since the PCP particles themselves are insoluble in water, the resulting composite particles can enter the membrane cutoff phase during subsequent 2μm microporous membrane cross-flow filtration, reducing the likelihood of HCP entering the reverse osmosis dehydration and spray granulation steps with the filtrate.

[0048] Therefore, ectoine, hydroxypropyl cellulose, and porous cross-linked polyvinylpyrrolidone particles form a synergistic effect: at lower temperatures, ectoine maintains the stability of the cells and enzymes, while hydroxypropyl cellulose maintains the dispersion of the raw material particles; during the warming cultivation stage, hydroxypropyl cellulose undergoes temperature-responsive aggregation, and the porous cross-linked polyvinylpyrrolidone particles adsorb and aggregate its small aggregates; after cultivation, the resulting composite particles are retained by microporous membrane filtration. This achieves the transformation of the culture additive from a low-temperature dispersed state to a high-temperature separable state, ensuring a continuous combination of stable initial cultivation, staged warming cultivation, and subsequent membrane filtration. Attached Figure Description

[0049] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some schematic diagrams of certain embodiments of the present invention, and therefore should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0050] Figure 1 The fluorescence distribution of hydroxypropyl cellulose before heating (A) and the fluorescence distribution of hydroxypropyl cellulose aggregates after heating (B) in Example 1 are shown. Figure 2 The fluorescence distribution of hydroxyethyl cellulose after heating in Comparative Example 2; Figure 3 The fluorescence distribution of hydroxypropyl cellulose in Comparative Example 3 after heating is shown. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Of course, the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0052] Unless otherwise specified, all chemical reagents and materials in this invention are purchased from the market or synthesized from raw materials purchased from the market.

[0053] The starter culture used is commercially available; the barley and peas are food-grade raw materials, which are pulverized before use; β-glucanase and xylanase are food-grade enzyme preparations; calcium chloride, magnesium sulfate heptahydrate, glycerol, and soybean lecithin are food-grade raw materials; hydroxypropyl cellulose is food-grade or pharmaceutical-grade hydroxypropyl cellulose with appropriate viscosity and cloud point parameters; porous cross-linked polyvinylpyrrolidone particles are food-grade insoluble polyvinylpyrrolidone; polyvinylpyrrolidone K30 is food-grade or pharmaceutical-grade water-soluble polyvinylpyrrolidone; and dextrin is food-grade dextrin.

[0054] Unless otherwise specified, none of the raw materials were further purified and were used directly in the preparation of the embodiments and comparative examples of this invention.

[0055] A liquid culture method for Daqu (a type of Chinese koji) includes the following steps: (1) Mix Daqu with water at 28~35℃ at a weight ratio of 1:8~1:15, keep warm at 28~35℃ for 8~12h to absorb water, then stir and crush to obtain a suspension; when no less than 95% of the material in the obtained suspension can pass through a 40-mesh sieve, stop crushing, collect the suspension under the sieve, and obtain Daqu liquid seed source.

[0056] (2) Preparation of culture additives: The amount of each component to be added is determined according to the dry matter mass of the Daqu culture raw material and the total volume of the gelatinized culture medium after the addition of the culture additive and before inoculation; hydroxypropyl cellulose with a viscosity of 2-10 mPa·s in 2 wt% aqueous solution and a cloud point of 39-44℃ in the gelatinized culture medium is added to water at a temperature not higher than 25℃ to prepare a dispersion with a hydroxypropyl cellulose content of 0.5-1.0 wt%, and stirred and hydrated for 30-60 min; ectoine, glycerol, calcium chloride, magnesium sulfate heptahydrate and water-dispersed soybean lecithin are added to the obtained hydroxypropyl cellulose dispersion and mixed evenly; the temperature of the obtained mixture is controlled at not higher than 35℃, and β-glucan is added. β-glucanase and xylanase are mixed for 5-15 minutes to obtain a culture additive. Based on the dry matter mass of the Daqu culture raw material, the amount of β-glucanase added is 30-80 U / g dry matter, and the amount of xylanase added is 50-150 U / g dry matter. Based on the total volume of the gelatinized culture medium after the addition of the culture additive and before inoculation, the concentrations are as follows: calcium chloride 0.10-0.30 g / L, magnesium sulfate heptahydrate 0.05-0.20 g / L, glycerol 1-3 g / L, soybean lecithin 0.10-0.30 g / L, ectoine 0.5-2.0 g / L, and hydroxypropyl cellulose 0.2-0.8 g / L.

[0057] (3) Barley and peas are used as raw materials for Daqu culture, wherein barley accounts for 70-75 wt% of the total mass of Daqu culture raw materials and peas are the remainder; the Daqu culture raw materials are crushed to pass through a 120-mesh sieve, and the two are mixed according to the weight ratio of Daqu culture raw materials to water of 1:8 to 1:15, heated and stirred to gelatinize; during the gelatinization process, samples are taken for iodine reaction detection, and the gelatinization is stopped when the gelatinized material does not react with iodine to form a blue color, and the gelatinized culture medium is obtained; the obtained gelatinized culture medium is cooled to 30-32°C, the culture additives prepared in step (2) are added, and the mixture is treated at 30-32°C for 30-60 min.

[0058] (4) Inoculate the Daqu liquid seed source obtained in step (1) into the gelatinized culture medium obtained in step (3). The inoculation amount of the Daqu liquid seed source is 30%~35% of the volume of the gelatinized culture medium after adding culture additives. After inoculation, the culture temperature is controlled at 28~35℃ and cultured for 24~30h under stirring and ventilation and oxygen supply conditions to obtain the initial culture medium.

[0059] (5) Adjust the temperature of the initial culture medium obtained in step (4) to 33~35℃ and culture it for 24~32h under stirring and ventilation and oxygen supply conditions; then adjust the temperature of the culture medium to 35~39℃ and culture it for 48~56h under stirring and ventilation and oxygen supply conditions.

[0060] (6) Preparation of polyvinylpyrrolidone suspension: Add porous cross-linked polyvinylpyrrolidone particles with a median particle size D50 of 20~80μm, a D10 of not less than 5μm and a particle size of less than 2μm in a mass ratio of not more than 1% to water, stir and disperse to prepare a suspension with a porous cross-linked polyvinylpyrrolidone particle content of 5~10wt%, and continue to hydrate for 15~120min to obtain polyvinylpyrrolidone suspension.

[0061] (7) After the culture at 35~39℃ in step (5) is completed, before the culture medium is heated to 39~42℃, add the polyvinylpyrrolidone suspension prepared in step (6) to the culture medium so that the concentration of porous cross-linked polyvinylpyrrolidone particles in the culture medium after addition is 0.2~1.0g / L, and the mass ratio of porous cross-linked polyvinylpyrrolidone particles to hydroxypropyl cellulose added in step (3) is 0.5∶1~2∶1; then adjust the temperature of the culture medium to 39~42℃ and culture for 24~36h under stirring and ventilation oxygen supply conditions.

[0062] (8) Detect the total number of bacteria in the culture medium obtained in step (7); when the total number of bacteria is less than 250 million / mL, extend the culture time of 39~42℃ in step (7) and test again; when the total number of bacteria is not less than 250 million / mL, adjust the temperature of the culture medium to 42~45℃, and culture for 24~36h under stirring and ventilation and oxygen supply conditions to obtain liquid Daqu culture mash.

[0063] (9) The liquid Daqu culture mash obtained in step (8) is sent into a cross-flow filtration device, and a microporous membrane with a pore size of 2μm is used for cross-flow filtration. The filtrate that passes through the microporous membrane is collected.

[0064] (10) The filtrate obtained in step (9) is sent to a reverse osmosis membrane device for dehydration. When the water content of the retentate is not higher than 80 wt%, the reverse osmosis dehydration is ended to obtain Daqu concentrate.

[0065] (11) Add dextrin to the Daqu concentrate obtained in step (10), wherein the amount of dextrin added is 5% to 7% of the mass of the Daqu concentrate; mix the Daqu concentrate after adding dextrin evenly, then perform spray granulation, collect the obtained particles, and obtain Daqu product.

[0066] The present invention will be further described below through specific embodiments.

[0067] Example 1 A liquid culture method for Daqu (a type of Chinese koji) includes the following steps: (1) Weigh 4 kg of mature Daqu, add 40 kg of water at 30℃, and keep warm at 30℃ for 10 h to absorb water; after the water absorption is completed, stir and crush to obtain Daqu suspension. Take a sample and screen it with a 40 mesh sieve. When no less than 95% of the material in the suspension can pass through the 40 mesh sieve, stop crushing, collect the suspension under the sieve, and obtain Daqu liquid seed source for later use.

[0068] (2) Weigh 50g of hydroxypropyl cellulose with a 2wt% aqueous solution viscosity of 6mPa·s and a cloud point of 42℃ in the gelatinized medium. Slowly add it to 6.62kg of water at 20℃ under stirring conditions, stir to disperse and hydrate for 45min to obtain hydroxypropyl cellulose dispersion.

[0069] Add 125g of ectoine, 200g of glycerol, 20g of calcium chloride and 12.5g of magnesium sulfate heptahydrate to the obtained hydroxypropyl cellulose dispersion in sequence; separately, add 20g of soybean lecithin to 500g of water for dispersion, and then add the obtained soybean lecithin dispersion to the hydroxypropyl cellulose dispersion, stir for 15min to obtain a mixed solution.

[0070] Adjust the temperature of the mixture to 30℃, add 44g of β-glucanase preparation with an enzyme activity of 10000U / g and 80g of xylanase preparation with an enzyme activity of 10000U / g, stir for 10 minutes to obtain the culture additive.

[0071] (3) Weigh 5.6 kg of barley and 2.4 kg of peas, mix them and then crush them so that all the raw materials for Daqu culture pass through a 120-mesh sieve. Add the crushed Daqu culture materials to 80 kg of water and heat them under stirring to gelatinize. During the gelatinization process, take a sample for iodine reaction detection. When the gelatinized material does not react with iodine to turn blue, stop the gelatinization and obtain the gelatinized culture medium.

[0072] The gelatinized culture medium was transferred into a culture tank equipped with a stirring device, a ventilation and oxygen supply device and a temperature control device. It was cooled to 32°C, and the culture additive prepared in step (2) was added. Sterile water was added to make the total volume of the gelatinized culture medium after adding the culture additive and before inoculation 100L. The mixture was stirred at 32°C for 30min.

[0073] (4) Inoculate 32.5L of the Daqu liquid seed source prepared in step (1) into the gelatinized culture medium obtained in step (3), adjust the culture temperature to 35℃, and culture for 24h under continuous stirring and ventilation to obtain the initial culture medium.

[0074] (5) Keep the temperature of the initial culture medium obtained in step (4) at 35°C and culture for 24 hours under continuous stirring and ventilation and oxygen supply; then raise the temperature of the culture medium to 39°C and culture for 48 hours under continuous stirring and ventilation and oxygen supply.

[0075] (6) Weigh 66g of porous cross-linked polyvinylpyrrolidone particles with a median particle size of 50μm, D10 of 10μm and a particle size of less than 2μm, and add them to 814g of water. Stir and disperse the particles at 25℃ and hydrate for 60min to obtain a polyvinylpyrrolidone suspension.

[0076] (7) After the culture at 39°C in step (5) is completed, add all the polyvinylpyrrolidone suspension prepared in step (6) while maintaining the stirring of the culture medium. Continue stirring for 20 minutes after the addition is completed.

[0077] After addition, the mass ratio of porous cross-linked polyvinylpyrrolidone particles to hydroxypropyl cellulose used in step (2) is 1.32:1, and the concentration of porous cross-linked polyvinylpyrrolidone particles in the culture medium is about 0.50 g / L.

[0078] The temperature of the culture medium was raised to 42°C, and the culture was carried out for 24 hours under continuous stirring and ventilation.

[0079] (8) After culturing for 24 hours, the total number of bacteria in the culture medium is detected. When the total number of bacteria is not less than 250 million / mL, the next culture stage is started. When the total number of bacteria is less than 250 million / mL, the culture cycle is extended by 6 hours. The culture is continued at 42℃ and the test is repeated until the total number of bacteria is not less than 250 million / mL.

[0080] The culture medium that meets the above requirements for total bacterial count is heated to 45°C and cultured for 24 hours under continuous stirring and ventilation to obtain liquid Daqu culture mash.

[0081] (9) Cool the liquid Daqu culture mash obtained in step (8) to 30°C and send it into a cross-flow filtration device. Use a microporous membrane with a pore size of 2 μm for cross-flow filtration. The cross-flow filtration temperature is 30°C, the cross-flow velocity is 1.5 m / s, the transmembrane pressure difference is 0.15 MPa, and collect the filtrate that has passed through the microporous membrane.

[0082] (10) The filtrate obtained in step (9) is subjected to reverse osmosis dehydration at 25°C and operating pressure of 2.0 MPa, and concentrated until the mass of the retentate is 20 kg and the water content is not higher than 80%, thus obtaining Daqu concentrate.

[0083] (11) Add 1.2 kg of dextrin to the 20 kg Daqu concentrate obtained in step (10), stir for 20 min to disperse the dextrin evenly, and obtain the material to be sprayed.

[0084] The obtained material to be sprayed is fed into a spray granulation equipment, and the inlet temperature is controlled at 110℃, the outlet temperature at 55℃, the feed rate at 4kg / h, and the atomization pressure at 0.5MPa for spray granulation. The obtained particles are collected to obtain the Daqu product.

[0085] Example 2 A liquid culture method for Daqu (a type of Chinese koji) includes the following steps: (1) Weigh 2 kg of mature Daqu, add 30 kg of water at 28℃, and keep warm at 28℃ for 12 h to absorb water. After the water absorption is completed, stir and crush to obtain Daqu suspension. Take a sample and screen it with a 40 mesh sieve. When no less than 95% of the material in the suspension can pass through the 40 mesh sieve, stop crushing and collect the suspension under the sieve to obtain Daqu liquid seed source for later use.

[0086] (2) Weigh 20g of hydroxypropyl cellulose with a viscosity of 2mPa·s in 2wt% aqueous solution and a cloud point of 39℃ in gelatinized medium. Slowly add it to 3.98kg of water at 20℃ under stirring conditions, stir to disperse and hydrate for 30min to obtain hydroxypropyl cellulose dispersion.

[0087] Add 50g of ectoine, 100g of glycerol, 10g of calcium chloride and 5g of magnesium sulfate heptahydrate to the obtained hydroxypropyl cellulose dispersion in sequence; separately, add 10g of soybean lecithin to 250g of water for dispersion, and then add the obtained soybean lecithin dispersion to the hydroxypropyl cellulose dispersion, stir for 15min to obtain a mixture.

[0088] Adjust the temperature of the obtained mixture to 30℃, add 16.5g of β-glucanase preparation with an enzyme activity of 10000U / g and 27.5g of xylanase preparation with an enzyme activity of 10000U / g, stir for 5 minutes to obtain the culture additive.

[0089] (3) In this embodiment, the weight of barley and peas is based on dry matter. Weigh 3.85 kg of barley and 1.65 kg of peas, mix them and then crush them so that all the raw materials for Daqu cultivation pass through a 120-mesh sieve.

[0090] Add 82.5 kg of water to 5.5 kg of crushed Daqu culture material and heat to gelatinize under stirring conditions; take samples for iodine reaction detection during gelatinization, and stop gelatinization when the gelatinized material does not react with iodine to form a blue color, thus obtaining gelatinized culture medium.

[0091] The gelatinized culture medium was transferred into a culture tank equipped with a stirring device, a ventilation and oxygen supply device and a temperature control device. It was cooled to 30°C, and the culture additive prepared in step (2) was added. Sterile water was added to make the total volume of the gelatinized culture medium after adding the culture additive and before inoculation 100L. The mixture was stirred at 30°C for 60min.

[0092] (4) Inoculate 30L of the Daqu liquid seed source prepared in step (1) into the gelatinized culture medium obtained in step (3), control the culture temperature at 28℃, and culture for 30h under continuous stirring and ventilation and oxygen supply to obtain the initial culture medium.

[0093] (5) Adjust the temperature of the initial culture medium obtained in step (4) to 33°C and culture it for 32 hours under continuous stirring and ventilation and oxygen supply; then adjust the temperature of the culture medium to 35°C and culture it for 56 hours under continuous stirring and ventilation and oxygen supply.

[0094] (6) Weigh 26.1g of porous cross-linked polyvinylpyrrolidone particles with a median particle size of 20μm, D10 of 5μm and a particle size of less than 2μm, and add them to 495.9g of water. Stir and disperse the particles at 25℃ and hydrate them for 15min to obtain a polyvinylpyrrolidone suspension.

[0095] (7) After the 35°C incubation in step (5) is completed, add all the polyvinylpyrrolidone suspension prepared in step (6) while maintaining the stirring of the culture medium. Continue stirring for 20 minutes after the addition is completed.

[0096] After addition, the mass ratio of porous cross-linked polyvinylpyrrolidone particles to hydroxypropyl cellulose used in step (2) is 1.305:1, and the concentration of porous cross-linked polyvinylpyrrolidone particles in the culture medium is 0.20 g / L.

[0097] The temperature of the culture medium was adjusted to 39°C, and the culture was carried out for 36 hours under continuous stirring and ventilation.

[0098] (8) Detect the total number of bacteria in the culture medium obtained in step (7). When the total number of bacteria is less than 250 million / mL, extend the culture cycle by 6 hours and continue to culture at 39°C and detect again; when the total number of bacteria reaches or exceeds 250 million / mL, adjust the temperature of the culture medium to 42°C and culture for 36 hours under continuous stirring and ventilation to obtain liquid Daqu culture mash.

[0099] (9) Cool the liquid Daqu culture mash obtained in step (8) to 30°C and send it into a cross-flow filtration device. Use a microporous membrane with a pore size of 2 μm for cross-flow filtration. The cross-flow filtration temperature is 30°C, the cross-flow velocity is 1.5 m / s, the transmembrane pressure difference is 0.15 MPa, and collect the filtrate that has passed through the microporous membrane.

[0100] (10) The filtrate obtained in step (9) is subjected to reverse osmosis dehydration at 25°C and operating pressure of 2.0 MPa until the water content of the retentate is 80%, thus obtaining Daqu concentrate.

[0101] (11) Take 20 kg of Daqu concentrate obtained in step (10), add 1 kg of dextrin, stir for 20 min to disperse the dextrin evenly, and obtain the material to be sprayed.

[0102] The obtained material to be sprayed is fed into a spray granulation equipment, and the inlet temperature is controlled at 110℃, the outlet temperature at 55℃, the feed rate at 4kg / h, and the atomization pressure at 0.5MPa for spray granulation. The obtained particles are collected to obtain the Daqu product.

[0103] Example 3 A liquid culture method for Daqu (a type of Chinese koji) includes the following steps: (1) Weigh 5 kg of mature Daqu, add 40 kg of water at 35℃, and keep warm at 35℃ for 8 hours to absorb water. After the water absorption is completed, stir and crush to obtain Daqu suspension. Take a sample and screen it through a 40-mesh sieve. When no less than 95% of the material in the suspension can pass through the 40-mesh sieve, stop crushing and collect the suspension under the sieve to obtain Daqu liquid seed source for later use.

[0104] (2) Weigh 80g of hydroxypropyl cellulose with a 2wt% aqueous solution viscosity of 10mPa·s and a cloud point of 44℃ in the gelatinized medium. Slowly add it to 7.92kg of water at 20℃ under stirring conditions, stir to disperse and hydrate for 60min to obtain hydroxypropyl cellulose dispersion.

[0105] Add 200g of ectoine, 300g of glycerol, 30g of calcium chloride and 20g of magnesium sulfate heptahydrate to the obtained hydroxypropyl cellulose dispersion in sequence; separately, add 30g of soybean lecithin to 500g of water for dispersion, and then add the obtained soybean lecithin dispersion to the hydroxypropyl cellulose dispersion, stir for 15min to obtain a mixture.

[0106] Adjust the temperature of the obtained mixture to 30℃, add 80g of β-glucanase preparation with an enzyme activity of 10000U / g and 150g of xylanase preparation with an enzyme activity of 10000U / g, stir for 15min to obtain the culture additive.

[0107] (3) In this embodiment, the weight of barley and peas is based on dry matter. Weigh 7.5 kg of barley and 2.5 kg of peas, mix them and then crush them so that all the raw materials for Daqu cultivation pass through a 120-mesh sieve.

[0108] Add 80 kg of water to 10 kg of crushed Daqu culture material and heat to gelatinize under stirring conditions; take samples for iodine reaction detection during gelatinization, and stop gelatinization when the gelatinized material does not react with iodine to form a blue color, thus obtaining gelatinized culture medium.

[0109] The gelatinized culture medium was transferred into a culture tank equipped with a stirring device, a ventilation and oxygen supply device and a temperature control device. It was cooled to 32°C, and the culture additive prepared in step (2) was added. Sterile water was added to make the total volume of the gelatinized culture medium after adding the culture additive and before inoculation 100L. The mixture was stirred at 32°C for 30min.

[0110] (4) Inoculate 35L of the Daqu liquid seed source prepared in step (1) into the gelatinized culture medium obtained in step (3), control the culture temperature at 35℃, and culture for 24h under continuous stirring and ventilation to obtain the initial culture medium.

[0111] (5) Keep the temperature of the initial culture medium obtained in step (4) at 35°C and culture for 24 hours under continuous stirring and ventilation and oxygen supply; then adjust the temperature of the culture medium to 39°C and culture for 48 hours under continuous stirring and ventilation and oxygen supply.

[0112] (6) Weigh 136.4g of porous cross-linked polyvinylpyrrolidone particles with a median particle size of 80μm, D10 of 20μm and a particle size of less than 2μm, and add them to 1.2276kg of water. Stir and disperse the particles at 25℃ and hydrate for 120min to obtain a polyvinylpyrrolidone suspension.

[0113] (7) After the culture at 39°C in step (5) is completed, add all the polyvinylpyrrolidone suspension prepared in step (6) while maintaining the stirring of the culture medium. Continue stirring for 20 minutes after the addition is completed.

[0114] After addition, the mass ratio of porous cross-linked polyvinylpyrrolidone particles to hydroxypropyl cellulose used in step (2) is 1.705:1, and the concentration of porous cross-linked polyvinylpyrrolidone particles in the culture medium is 1.00 g / L.

[0115] The temperature of the culture medium was adjusted to 42℃, and cultured for 24 hours under continuous stirring and ventilation.

[0116] (8) Detect the total number of bacteria in the culture medium obtained in step (7). When the total number of bacteria is less than 250 million / mL, extend the culture cycle by 6 hours, continue the culture at 42°C and test again; when the total number of bacteria reaches or exceeds 250 million / mL, adjust the temperature of the culture medium to 45°C and culture for 24 hours under continuous stirring and ventilation to obtain liquid Daqu culture mash.

[0117] (9) Cool the liquid Daqu culture mash obtained in step (8) to 30°C and send it into a cross-flow filtration device. Use a microporous membrane with a pore size of 2 μm for cross-flow filtration. The cross-flow filtration temperature is 30°C, the cross-flow velocity is 1.5 m / s, the transmembrane pressure difference is 0.15 MPa, and collect the filtrate that has passed through the microporous membrane.

[0118] (10) The filtrate obtained in step (9) is subjected to reverse osmosis dehydration at 25°C and operating pressure of 2.0 MPa until the water content of the retentate is 75%, thus obtaining Daqu concentrate.

[0119] (11) Take 20 kg of Daqu concentrate obtained in step (10), add 1.4 kg of dextrin, stir for 20 min to disperse the dextrin evenly, and obtain the material to be sprayed.

[0120] The obtained material to be sprayed is fed into a spray granulation equipment, and the inlet temperature is controlled at 110℃, the outlet temperature at 55℃, the feed rate at 4kg / h, and the atomization pressure at 0.5MPa for spray granulation. The obtained particles are collected to obtain the Daqu product.

[0121] Comparative Example 1 The only difference between this comparative example and Example 1 is that the ectoine in the culture additive is replaced with an equal mass of sucrose, while the other raw materials, amounts, and process conditions are the same as in Example 1.

[0122] A liquid culture method for Daqu (a type of Chinese koji) includes the following steps: (1) Weigh 4 kg of mature Daqu, add 40 kg of water at 30℃, and keep warm at 30℃ for 10 h to absorb water; after the water absorption is completed, stir and crush to obtain Daqu suspension. Take a sample and screen it with a 40 mesh sieve. When no less than 95% of the material in the suspension can pass through the 40 mesh sieve, stop crushing, collect the suspension under the sieve, and obtain Daqu liquid seed source for later use.

[0123] (2) Weigh 50g of hydroxypropyl cellulose with a 2wt% aqueous solution viscosity of 6mPa·s and a cloud point of 42℃ in the gelatinized medium. Slowly add it to 6.62kg of water at 20℃ under stirring conditions, stir to disperse and hydrate for 45min to obtain hydroxypropyl cellulose dispersion.

[0124] Add 125g of sucrose, 200g of glycerol, 20g of calcium chloride and 12.5g of magnesium sulfate heptahydrate to the obtained hydroxypropyl cellulose dispersion in sequence; separately, add 20g of soybean lecithin to 500g of water for dispersion, and then add the obtained soybean lecithin dispersion to the hydroxypropyl cellulose dispersion, stir for 15min to obtain a mixture.

[0125] Adjust the temperature of the mixture to 30℃, add 44g of β-glucanase preparation with an enzyme activity of 10000U / g and 80g of xylanase preparation with an enzyme activity of 10000U / g, stir for 10 minutes to obtain the culture additive.

[0126] (3) Weigh 5.6 kg of barley and 2.4 kg of peas, mix them and then crush them so that all the raw materials for Daqu culture pass through a 120-mesh sieve. Add the crushed Daqu culture materials to 80 kg of water and heat them under stirring to gelatinize. During the gelatinization process, take a sample for iodine reaction detection. When the gelatinized material does not react with iodine to turn blue, stop the gelatinization and obtain the gelatinized culture medium.

[0127] The gelatinized culture medium was transferred into a culture tank equipped with a stirring device, a ventilation and oxygen supply device and a temperature control device. It was cooled to 32°C, and the culture additive prepared in step (2) was added. Sterile water was added to make the total volume of the gelatinized culture medium after adding the culture additive and before inoculation 100L. The mixture was stirred at 32°C for 30min.

[0128] (4) Inoculate 32.5L of the Daqu liquid seed source prepared in step (1) into the gelatinized culture medium obtained in step (3), adjust the culture temperature to 35℃, and culture for 24h under continuous stirring and ventilation to obtain the initial culture medium.

[0129] (5) Keep the temperature of the initial culture medium obtained in step (4) at 35°C and culture for 24 hours under continuous stirring and ventilation and oxygen supply; then raise the temperature of the culture medium to 39°C and culture for 48 hours under continuous stirring and ventilation and oxygen supply.

[0130] (6) Weigh 66g of porous cross-linked polyvinylpyrrolidone particles with a median particle size of 50μm, D10 of 10μm and a particle size of less than 2μm, and add them to 814g of water. Stir and disperse the particles at 25℃ and hydrate for 60min to obtain a polyvinylpyrrolidone suspension.

[0131] (7) After the culture at 39°C in step (5) is completed, add all the polyvinylpyrrolidone suspension prepared in step (6) while maintaining the stirring of the culture medium. Continue stirring for 20 minutes after the addition is completed.

[0132] After addition, the mass ratio of porous cross-linked polyvinylpyrrolidone particles to hydroxypropyl cellulose used in step (2) is 1.32:1, and the concentration of porous cross-linked polyvinylpyrrolidone particles in the culture medium is about 0.50 g / L.

[0133] The temperature of the culture medium was raised to 42°C, and the culture was carried out for 24 hours under continuous stirring and ventilation.

[0134] (8) After culturing for 24 hours, the total number of bacteria in the culture medium is detected. When the total number of bacteria is not less than 250 million / mL, the next culture stage is started. When the total number of bacteria is less than 250 million / mL, the culture cycle is extended by 6 hours. The culture is continued at 42℃ and the test is repeated until the total number of bacteria is not less than 250 million / mL.

[0135] The culture medium that meets the above requirements for total bacterial count is heated to 45°C and cultured for 24 hours under continuous stirring and ventilation to obtain liquid Daqu culture mash.

[0136] (9) Cool the liquid Daqu culture mash obtained in step (8) to 30°C and send it into a cross-flow filtration device. Use a microporous membrane with a pore size of 2 μm for cross-flow filtration. The cross-flow filtration temperature is 30°C, the cross-flow velocity is 1.5 m / s, the transmembrane pressure difference is 0.15 MPa, and collect the filtrate that has passed through the microporous membrane.

[0137] (10) The filtrate obtained in step (9) is subjected to reverse osmosis dehydration at 25°C and operating pressure of 2.0 MPa, and concentrated until the mass of the retentate is 20 kg and the water content is not higher than 80%, thus obtaining Daqu concentrate.

[0138] (11) Add 1.2 kg of dextrin to the 20 kg Daqu concentrate obtained in step (10), stir for 20 min to disperse the dextrin evenly, and obtain the material to be sprayed.

[0139] The obtained material to be sprayed is fed into a spray granulation equipment, and the inlet temperature is controlled at 110℃, the outlet temperature at 55℃, the feed rate at 4kg / h, and the atomization pressure at 0.5MPa for spray granulation. The obtained particles are collected to obtain the Daqu product.

[0140] Comparative Example 2 The only difference between this comparative example and Example 1 is that hydroxypropyl cellulose is replaced with an equal mass of hydroxyethyl cellulose, wherein a 2wt% aqueous solution of the hydroxyethyl cellulose has a viscosity of 6 mPa·s and does not exhibit thermal turbidity in the range of 39~44℃. All other raw materials, dosages, and process conditions are the same as in Example 1.

[0141] A liquid culture method for Daqu (a type of Chinese koji) includes the following steps: (1) Weigh 4 kg of mature Daqu, add 40 kg of water at 30℃, and keep warm at 30℃ for 10 h to absorb water; after the water absorption is completed, stir and crush to obtain Daqu suspension. Take a sample and screen it with a 40 mesh sieve. When no less than 95% of the material in the suspension can pass through the 40 mesh sieve, stop crushing, collect the suspension under the sieve, and obtain Daqu liquid seed source for later use.

[0142] (2) Weigh 50g of hydroxyethyl cellulose with a 2wt% aqueous solution viscosity of 6mPa·s and no thermal response turbidity in the range of 39~44℃. Slowly add it to 6.62kg of water at 20℃ under stirring conditions, stir to disperse and hydrate for 45min to obtain hydroxyethyl cellulose dispersion.

[0143] Add 125g of ectoine, 200g of glycerol, 20g of calcium chloride and 12.5g of magnesium sulfate heptahydrate to the obtained hydroxyethyl cellulose dispersion in sequence; separately, add 20g of soybean lecithin to 500g of water for dispersion, and then add the obtained soybean lecithin dispersion to the hydroxyethyl cellulose dispersion, stir for 15min to obtain a mixed solution.

[0144] Adjust the temperature of the mixture to 30℃, add 44g of β-glucanase preparation with an enzyme activity of 10000U / g and 80g of xylanase preparation with an enzyme activity of 10000U / g, stir for 10 minutes to obtain the culture additive.

[0145] (3) Weigh 5.6 kg of barley and 2.4 kg of peas, mix them and then crush them so that all the raw materials for Daqu culture pass through a 120-mesh sieve. Add the crushed Daqu culture materials to 80 kg of water and heat them under stirring to gelatinize. During the gelatinization process, take a sample for iodine reaction detection. When the gelatinized material does not react with iodine to turn blue, stop the gelatinization and obtain the gelatinized culture medium.

[0146] The gelatinized culture medium was transferred into a culture tank equipped with a stirring device, a ventilation and oxygen supply device and a temperature control device. It was cooled to 32°C, and the culture additive prepared in step (2) was added. Sterile water was added to make the total volume of the gelatinized culture medium after adding the culture additive and before inoculation 100L. The mixture was stirred at 32°C for 30min.

[0147] (4) Inoculate 32.5L of the Daqu liquid seed source prepared in step (1) into the gelatinized culture medium obtained in step (3), adjust the culture temperature to 35℃, and culture for 24h under continuous stirring and ventilation to obtain the initial culture medium.

[0148] (5) Keep the temperature of the initial culture medium obtained in step (4) at 35°C and culture for 24 hours under continuous stirring and ventilation and oxygen supply; then raise the temperature of the culture medium to 39°C and culture for 48 hours under continuous stirring and ventilation and oxygen supply.

[0149] (6) Weigh 66g of porous cross-linked polyvinylpyrrolidone particles with a median particle size of 50μm, D10 of 10μm and a particle size of less than 2μm, and add them to 814g of water. Stir and disperse the particles at 25℃ and hydrate for 60min to obtain a polyvinylpyrrolidone suspension.

[0150] (7) After the culture at 39°C in step (5) is completed, add all the polyvinylpyrrolidone suspension prepared in step (6) while maintaining the stirring of the culture medium. Continue stirring for 20 minutes after the addition is completed.

[0151] After addition, the mass ratio of porous cross-linked polyvinylpyrrolidone particles to hydroxyethyl cellulose used in step (2) is 1.32:1, and the concentration of porous cross-linked polyvinylpyrrolidone particles in the culture medium is about 0.50 g / L.

[0152] The temperature of the culture medium was raised to 42°C, and the culture was carried out for 24 hours under continuous stirring and ventilation.

[0153] (8) After culturing for 24 hours, the total number of bacteria in the culture medium is detected. When the total number of bacteria is not less than 250 million / mL, the next culture stage is started. When the total number of bacteria is less than 250 million / mL, the culture cycle is extended by 6 hours. The culture is continued at 42℃ and the test is repeated until the total number of bacteria is not less than 250 million / mL.

[0154] The culture medium that meets the above requirements for total bacterial count is heated to 45°C and cultured for 24 hours under continuous stirring and ventilation to obtain liquid Daqu culture mash.

[0155] (9) Cool the liquid Daqu culture mash obtained in step (8) to 30°C and send it into a cross-flow filtration device. Use a microporous membrane with a pore size of 2 μm for cross-flow filtration. The cross-flow filtration temperature is 30°C, the cross-flow velocity is 1.5 m / s, the transmembrane pressure difference is 0.15 MPa, and collect the filtrate that has passed through the microporous membrane.

[0156] (10) The filtrate obtained in step (9) is subjected to reverse osmosis dehydration at 25°C and operating pressure of 2.0 MPa, and concentrated until the mass of the retentate is 20 kg and the water content is not higher than 80%, thus obtaining Daqu concentrate.

[0157] (11) Add 1.2 kg of dextrin to the 20 kg Daqu concentrate obtained in step (10), stir for 20 min to disperse the dextrin evenly, and obtain the material to be sprayed.

[0158] The obtained material to be sprayed is fed into a spray granulation equipment, and the inlet temperature is controlled at 110℃, the outlet temperature at 55℃, the feed rate at 4kg / h, and the atomization pressure at 0.5MPa for spray granulation. The obtained particles are collected to obtain the Daqu product.

[0159] Comparative Example 3 The only difference between this comparative example and Example 1 is that the insoluble porous cross-linked polyvinylpyrrolidone particles are replaced with an equal mass of water-soluble polyvinylpyrrolidone K30, and the polyvinylpyrrolidone suspension is replaced with a polyvinylpyrrolidone aqueous solution. All other raw materials, dosages, and process conditions are the same as in Example 1.

[0160] A liquid culture method for Daqu (a type of Chinese koji) includes the following steps: (1) Weigh 4 kg of mature Daqu, add 40 kg of water at 30℃, and keep warm at 30℃ for 10 h to absorb water; after the water absorption is completed, stir and crush to obtain Daqu suspension. Take a sample and screen it with a 40 mesh sieve. When no less than 95% of the material in the suspension can pass through the 40 mesh sieve, stop crushing, collect the suspension under the sieve, and obtain Daqu liquid seed source for later use.

[0161] (2) Weigh 50g of hydroxypropyl cellulose with a 2wt% aqueous solution viscosity of 6mPa·s and a cloud point of 42℃ in the gelatinized medium. Slowly add it to 6.62kg of water at 20℃ under stirring conditions, stir to disperse and hydrate for 45min to obtain hydroxypropyl cellulose dispersion.

[0162] Add 125g of ectoine, 200g of glycerol, 20g of calcium chloride and 12.5g of magnesium sulfate heptahydrate to the obtained hydroxypropyl cellulose dispersion in sequence; separately, add 20g of soybean lecithin to 500g of water for dispersion, and then add the obtained soybean lecithin dispersion to the hydroxypropyl cellulose dispersion, stir for 15min to obtain a mixed solution.

[0163] Adjust the temperature of the mixture to 30℃, add 44g of β-glucanase preparation with an enzyme activity of 10000U / g and 80g of xylanase preparation with an enzyme activity of 10000U / g, stir for 10 minutes to obtain the culture additive.

[0164] (3) Weigh 5.6 kg of barley and 2.4 kg of peas, mix them and then crush them so that all the raw materials for Daqu culture pass through a 120-mesh sieve. Add the crushed Daqu culture materials to 80 kg of water and heat them under stirring to gelatinize. During the gelatinization process, take a sample for iodine reaction detection. When the gelatinized material does not react with iodine to turn blue, stop the gelatinization and obtain the gelatinized culture medium.

[0165] The gelatinized culture medium was transferred into a culture tank equipped with a stirring device, a ventilation and oxygen supply device and a temperature control device. It was cooled to 32°C, and the culture additive prepared in step (2) was added. Sterile water was added to make the total volume of the gelatinized culture medium after adding the culture additive and before inoculation 100L. The mixture was stirred at 32°C for 30min.

[0166] (4) Inoculate 32.5L of the Daqu liquid seed source prepared in step (1) into the gelatinized culture medium obtained in step (3), adjust the culture temperature to 35℃, and culture for 24h under continuous stirring and ventilation to obtain the initial culture medium.

[0167] (5) Keep the temperature of the initial culture medium obtained in step (4) at 35°C and culture for 24 hours under continuous stirring and ventilation and oxygen supply; then raise the temperature of the culture medium to 39°C and culture for 48 hours under continuous stirring and ventilation and oxygen supply.

[0168] (6) Weigh 66g of water-soluble polyvinylpyrrolidone K30, add it to 814g of water, and stir at 25℃ for 60min to completely dissolve the water-soluble polyvinylpyrrolidone, so as to obtain a polyvinylpyrrolidone aqueous solution with a polyvinylpyrrolidone content of 7.5wt%.

[0169] (7) After the culture at 39°C in step (5) is completed, add all the polyvinylpyrrolidone aqueous solution prepared in step (6) while maintaining the stirring of the culture medium. Continue stirring for 20 minutes after the addition is completed.

[0170] After addition, the mass ratio of water-soluble polyvinylpyrrolidone to hydroxypropyl cellulose used in step (2) is 1.32:1, and the concentration of water-soluble polyvinylpyrrolidone in the culture medium is about 0.50 g / L.

[0171] The temperature of the culture medium was raised to 42°C, and the culture was carried out for 24 hours under continuous stirring and ventilation.

[0172] (8) After culturing for 24 hours, the total number of bacteria in the culture medium is detected. When the total number of bacteria is not less than 250 million / mL, the next culture stage is started. When the total number of bacteria is less than 250 million / mL, the culture cycle is extended by 6 hours. The culture is continued at 42℃ and the test is repeated until the total number of bacteria is not less than 250 million / mL.

[0173] The culture medium that meets the above requirements for total bacterial count is heated to 45°C and cultured for 24 hours under continuous stirring and ventilation to obtain liquid Daqu culture mash.

[0174] (9) Cool the liquid Daqu culture mash obtained in step (8) to 30°C and send it into a cross-flow filtration device. Use a microporous membrane with a pore size of 2 μm for cross-flow filtration. The cross-flow filtration temperature is 30°C, the cross-flow velocity is 1.5 m / s, the transmembrane pressure difference is 0.15 MPa, and collect the filtrate that has passed through the microporous membrane.

[0175] (10) The filtrate obtained in step (9) is subjected to reverse osmosis dehydration at 25°C and operating pressure of 2.0 MPa, and concentrated until the mass of the retentate is 20 kg and the water content is not higher than 80%, thus obtaining Daqu concentrate.

[0176] (11) Add 1.2 kg of dextrin to the 20 kg Daqu concentrate obtained in step (10), stir for 20 min to disperse the dextrin evenly, and obtain the material to be sprayed.

[0177] The obtained material to be sprayed is fed into a spray granulation equipment, and the inlet temperature is controlled at 110℃, the outlet temperature at 55℃, the feed rate at 4kg / h, and the atomization pressure at 0.5MPa for spray granulation. The obtained particles are collected to obtain the Daqu product.

[0178] I. Sample Preparation Samples were prepared according to the methods described in Examples 1-3 and Comparative Examples 1-3, respectively. Each group used the same batch of koji, barley, peas, enzyme preparations, and other raw materials. Three batches were prepared independently for each group, and each batch of samples was tested separately.

[0179] After the gelatinized culture medium was treated with culture additives, samples were taken to determine the uniformity of solid dispersion. A parallel sample of the culture medium without inoculation with Daqu liquid seed source was taken and treated according to the corresponding temperature, stirring and aeration procedures to determine the enzyme activity retention rate. Samples were taken after the 39~42℃ culture stage to determine the particle size of cellulose ether particles. During the microporous membrane filtration process, the feed liquid and filtrate were collected to determine the cellulose ether rejection rate and membrane flux retention rate.

[0180] II. Testing Methods 1. Uniformity of solid dispersion Take 1000 mL of gelatinized culture medium after adding culture additives and treatment, place it in a graduated cylinder with the same inner diameter, and let it stand at 32℃ for 2 hours. Take 20 mL samples from 5 cm below the liquid surface, the middle of the liquid column, and 5 cm above the bottom of the container.

[0181] Each sample was dried at 105℃ to constant weight. The solid concentration at each sampling location was calculated, and the coefficient of variation of the solid concentration at the three locations was calculated. The smaller the coefficient of variation, the more uniform the distribution of solid particles in the culture medium.

[0182] 2. Enzyme activity retention rate The assay was performed using parallel samples of culture medium without inoculation with Daqu liquid seed source. Initial enzyme activity was measured immediately after the enzyme preparation was added and mixed. Subsequently, the medium was treated according to the temperature, stirring, and aeration procedures of the corresponding examples or comparative examples. Enzyme activity was measured again after the 39-42°C incubation period was completed.

[0183] β-glucanase was determined using barley β-glucan substrate, and xylanase was determined using xylan substrate. Culture medium blanks and heat-inactivated sample blanks were included for each sample.

[0184] Enzyme activity retention rate is calculated according to the following formula: Enzyme activity retention rate = enzyme activity after treatment ÷ initial enzyme activity × 100%.

[0185] 3. Extended time required to reach the culture endpoint After completing the prescribed incubation period at 39–42°C, the total bacterial count in the culture medium was measured. When the total bacterial count was below 250 million CFU / mL, the culture was extended for 6 hours and the count was repeated. The extended time required for the total bacterial count to reach 250 million CFU / mL was recorded.

[0186] All groups used the same bacterial counting method and culture endpoint determination method.

[0187] 4. Particle size of cellulose ether particles after heating Parallel tracing was performed using small amounts of fluorescently labeled hydroxypropyl cellulose or hydroxyethyl cellulose. After the 35–39 °C incubation period, PVPP suspension or PVP K30 aqueous solution was added, and the temperature was raised for 20 min. Samples were then taken for observation using a fluorescence microscope.

[0188] At least 20 fields of view were randomly collected for each sample. Image analysis software was used to identify particles containing fluorescent cellulose ether, calculate the equivalent circle diameter of the particles, and obtain the D50 of the particles containing cellulose ether.

[0189] 5. Cellulose ether rejection rate Cross-flow filtration was performed using a ceramic microporous membrane with a pore size of 2 μm at 45℃, a cross-flow velocity of 1.5 m / s, and a transmembrane pressure difference of 0.15 MPa.

[0190] The total amount of cellulose ethers in the culture medium before filtration and the filtrate were determined separately, and the cellulose ether retention rate was calculated according to the following formula: Cellulose ether rejection rate = (Total cellulose ether before filtration - Total cellulose ether in filtrate) ÷ Total cellulose ether before filtration × 100%.

[0191] Examples 1-3, Comparative Examples 1 and 3 were used to determine hydroxypropyl cellulose, and Comparative Example 2 was used to determine hydroxyethyl cellulose.

[0192] 6. Flux retention rate of microporous membranes Using the same cross-flow filtration conditions as the cellulose ether rejection rate, the average membrane flux J0 was recorded from 0 to 5 minutes after the start of filtration and from 55 to 60 minutes after filtration. 60 .

[0193] Membrane flux is calculated using the following formula: J = V ÷ (A × t).

[0194] Membrane flux retention is calculated using the following formula: Membrane flux retention rate = J 60 ÷J0×100%.

[0195] III. Test Results Table 1 Experimental Test Results The coefficient of variation of solid concentration in Examples 1-3 was 4.9%-8.9%, indicating that hydroxypropyl cellulose can reduce the sedimentation and stratification of fine particles in the culture medium at the initial culture temperature.

[0196] The β-glucanase activity retention rates of Examples 1-3 were 71.8%-80.6%, and the xylanase activity retention rates were 68.7%-78.1%. Moreover, the total bacterial count reached 250 million / mL without the need for extended culture, indicating that the culture additive containing ectoine is beneficial for maintaining the stability of the enzyme preparation and the culture system.

[0197] After heating and adding PVPP, the D50 of the hydroxypropyl cellulose particles in Examples 1-3 reached 24.7-88.4 μm, the cellulose ether rejection rate reached 89.2%-97.1%, and the microporous membrane flux retention rate reached 74.5%-80.9%. This indicates that after heating and agglomeration, hydroxypropyl cellulose can form composite particles that can be retained by a 2 μm microporous membrane with the help of insoluble PVPP particles.

[0198] After replacing ectoin with an equal mass of sucrose, Comparative Example 1 showed similar solids dispersibility, heated particle size, cellulose ether rejection, and membrane flux retention to Example 1. However, the β-glucanase and xylanase activity retention rates of Comparative Example 1 decreased to 58.3% and 55.6%, respectively, and required an extended culture time of 12 hours to reach the specified total bacterial count. This indicates that sucrose cannot replace ectoin in maintaining the stability of the enzyme preparation and the culture process.

[0199] Comparative Example 2, where hydroxyethyl cellulose of similar viscosity was used instead of hydroxypropyl cellulose, showed a solids concentration variation coefficient similar to that of Example 1, indicating that hydroxyethyl cellulose can provide some particle dispersion at low temperatures. However, after heating, the D50 of the hydroxyethyl cellulose-containing particles was only 1.4 μm, the cellulose ether rejection rate was only 28.4%, and the microporous membrane flux retention rate decreased to 59.3%. These results indicate that a similar low-temperature viscosity is insufficient to replace the thermal response aggregation of hydroxypropyl cellulose within the specified temperature range.

[0200] In Comparative Example 3, after replacing the insoluble PVPP with water-soluble PVP K30, the initial particle dispersibility and enzyme activity retention were similar to those in Example 1. However, after heating, the D50 of the hydroxypropyl cellulose-containing particles was only 2.9 μm, the cellulose ether rejection rate was only 43.7%, and the microporous membrane flux retention rate decreased to 55.7%. These results indicate that soluble PVP K30 cannot provide the particle framework of insoluble PVPP, making it difficult to further form stable composite particles from small-sized hydroxypropyl cellulose aggregates.

[0201] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A liquid culture method for Daqu (a type of Chinese koji), characterized in that, Includes the following steps: S1. Preparation of liquid seed source: Liquid seed source of Daqu is prepared using Daqu; S2. Gelatinization treatment: The raw materials for Daqu culture are mixed with water and gelatinized to obtain a gelatinized culture medium. After the gelatinized culture medium is cooled, culture additives are added. S3. Initial culture: The Daqu liquid seed source obtained in step S1 is inoculated into the gelatinized culture medium obtained in step S2, and initial culture is carried out under stirring and ventilation and oxygen supply conditions to obtain the initial culture medium. S4. Staged culture: The initial culture medium obtained in step S3 is subjected to staged temperature increase culture. During the staged temperature increase culture process, polyvinylpyrrolidone suspension is added and the staged temperature increase culture continues to obtain liquid Daqu culture mash. S5. Microporous membrane filtration: The liquid Daqu culture mash obtained in step S4 is subjected to microporous membrane cross-flow filtration, and the filtrate is collected. S6. Reverse osmosis dehydration: The filtrate obtained in step S5 is subjected to reverse osmosis dehydration to obtain Daqu concentrate; S7. Encapsulation and granulation: Add an encapsulating agent to the Daqu concentrate obtained in step S6 and perform spray granulation to obtain the Daqu product.

2. The liquid culture method for Daqu (a type of Chinese liquor) according to claim 1, characterized in that: Step S1 includes: mixing Daqu with water at 28~35℃ at a weight ratio of 1:8~1:15 and keeping it warm to absorb water for 8~12 hours, then stirring and crushing. When no less than 95% of the material in the resulting suspension can pass through a 40-mesh sieve, the suspension under the sieve is collected to obtain the Daqu liquid seed source. In step S2, the raw material for Daqu cultivation consists of 70-75 wt% barley and the remainder peas. The raw material is pulverized to pass through a 120-mesh sieve, mixed with water at a weight ratio of 1:8 to 1:15, and then gelatinized. The gelatinization endpoint is defined as the gelatinized product not turning blue when reacting with iodine.

3. The liquid culture method for Daqu (a type of starter culture) according to claim 1, characterized in that, The culture additives include β-glucanase, xylanase, calcium chloride, magnesium sulfate heptahydrate, glycerol, soybean lecithin, ectoine, and hydroxypropyl cellulose; Based on the dry matter mass of the Daqu culture raw material used in step S2, the amount of β-glucanase added is 30~80 U / g dry matter, and the amount of xylanase added is 50~150 U / g dry matter. Based on the total volume of the gelatinized culture medium, the concentration of calcium chloride is 0.10~0.30 g / L, the concentration of magnesium sulfate heptahydrate is 0.05~0.20 g / L, the concentration of glycerol is 1~3 g / L, the concentration of soybean lecithin is 0.10~0.30 g / L, the concentration of ectoine is 0.5~2.0 g / L, and the concentration of hydroxypropyl cellulose is 0.2~0.8 g / L. The viscosity of the 2wt% aqueous solution of the hydroxypropyl cellulose is 2~10 mPa·s, and the cloud point of the hydroxypropyl cellulose in the gelatinized medium is 39~44℃.

4. The liquid culture method for Daqu (a type of starter culture) according to claim 1, characterized in that, The culture additive is prepared by the following steps: A1. Hydroxypropyl cellulose is added to water for dispersion and hydration to obtain a hydroxypropyl cellulose dispersion. A2. Add ectoine, glycerol, calcium chloride, magnesium sulfate heptahydrate and water-dispersed soybean lecithin to the hydroxypropyl cellulose dispersion obtained in step A1 to obtain a mixture. A3. Add β-glucanase and xylanase to the mixture obtained in step A2, and mix to obtain the culture additive.

5. The liquid culture method for Daqu (a type of Chinese liquor) according to claim 1, characterized in that: In step S2, the gelatinized culture medium is cooled to 30-32°C and then the culture additive is added, and the medium is treated at 30-32°C for 30-60 minutes. In step S3, the inoculation amount of the Daqu liquid seed source is 30% to 35% of the volume of the gelatinized culture medium, and after inoculation, it is cultured at 28 to 35°C for 24 to 30 hours under stirring and aeration conditions.

6. The liquid culture method for Daqu (a type of Chinese liquor) according to claim 1, characterized in that, Step S4 includes the following steps: S41. Adjust the culture medium obtained in step S3 to 33~35℃ and culture it for 24~32 hours under stirring and ventilation conditions. S42. Adjust the culture medium obtained in step S41 to 35~39℃ and culture it for 48~56 hours under stirring and ventilation and oxygen supply conditions. S43. Add the polyvinylpyrrolidone suspension to the culture medium obtained in step S42, adjust the culture medium to 39~42℃, and culture for 24~36 hours under stirring and ventilation and oxygen supply conditions. S44. Detect the total number of bacteria in the culture medium obtained in step S43. If the total number of bacteria is less than 250 million / mL, extend the culture time of step S43. If the total number of bacteria is not less than 250 million / mL, adjust the culture medium to 42~45℃ and culture for 24~36 hours under stirring and ventilation conditions to obtain the liquid Daqu culture mash.

7. The liquid culture method for Daqu (a type of Chinese liquor) according to claim 1, characterized in that, The polyvinylpyrrolidone suspension is prepared by the following method: porous cross-linked polyvinylpyrrolidone particles are added to water, stirred, dispersed, and hydrated to obtain the polyvinylpyrrolidone suspension.

8. The liquid culture method for Daqu (a type of Chinese liquor) according to claim 7, characterized in that, The median particle size D50 of the porous cross-linked polyvinylpyrrolidone particles is 20~80μm, the D10 is not less than 5μm, and the mass proportion of particles with a particle size less than 2μm is not higher than 1%; the content of porous cross-linked polyvinylpyrrolidone particles in the polyvinylpyrrolidone suspension is 5~10wt%; and the hydration time is 15~120min.

9. The liquid culture method for Daqu (a type of Chinese liquor) according to claim 1, characterized in that, In step S43: after the culture in step S42 is completed, before adjusting the culture medium to 39~42℃, the polyvinylpyrrolidone suspension is added; the amount of polyvinylpyrrolidone suspension added is such that the concentration of porous cross-linked polyvinylpyrrolidone particles in the culture medium is 0.2~1.0 g / L; the mass ratio of the porous cross-linked polyvinylpyrrolidone particles to the hydroxypropyl cellulose added in step S2 is 0.5∶1~2∶1.

10. The liquid culture method for Daqu (a type of Chinese liquor) according to claim 1, characterized in that, In step S5, a microporous membrane with a pore size of 2 μm is used to perform cross-flow filtration on the liquid Daqu culture mash. In step S6, the filtrate obtained in step S5 is dehydrated through a reverse osmosis membrane until the water content is no more than 80%. In step S7, the encapsulating agent is dextrin, and the amount of dextrin added is 5% to 7% of the mass of the Daqu concentrate. The Daqu concentrate with added dextrin is mixed and then spray granulated.

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