Preparation method of monascus mother starter

Abstract

The invention is applicable to the technical field of monascus fermentation, and provides a preparation method and application of monascus mother koji, and the preparation method of the monascus mother koji comprises the following steps: carrying out composite stress adaptability pretreatment on monascus production strains; thermosensitive hydrogel is compounded with grains to form an intelligent carrier, and metabolism-induced nutrients are added; in the fermentation process, temperature switching is triggered by monitoring the CO2 release rate in real time, and when the temperature rises to be higher than the phase transformation point of the hydrogel, the carrier shrinks to apply physical stress to hyphae and release nutrients in a pulse mode, so that secondary metabolism is strongly activated. Through multi-level cooperation of strain pretreatment, intelligent carrier, time sequence nutrition and dynamic process, accurate and active regulation and control on growth and metabolism of monascus are realized, the prepared mother yeast is high in activity and short in fermentation period, the yield of monacolin K is remarkably increased, and the method is suitable for efficiently producing high-value monascus products.

Description

Technical Field

[0001] This invention belongs to the field of Monascus fermentation technology, and particularly relates to a method for preparing Monascus mother koji. Background Technology

[0002] Monascus purpureus is a traditional edible fungus widely used in the production of red yeast rice, red yeast wine, and other products. Its metabolites, such as monacolin K, possess physiological activities such as lowering blood lipids and antioxidation. The mother koji, as the seed koji for Monascus purpureus fermentation, directly affects the efficiency and quality of the final product. Existing methods for preparing Monascus purpureus mother koji mostly use grain substrates (such as rice and wheat) for solid-state fermentation, but these methods suffer from problems such as long cultivation cycles and low yields of metabolites. For example, conventional methods typically employ a single temperature for cultivation (such as a constant temperature of 30°C), resulting in a simple culture medium that leads to slow mycelial growth and unstable monacolin K yields. Furthermore, current technologies lack optimization of culture medium structure and nutrient fortification, making it difficult to achieve efficient and high-quality production.

[0003] Therefore, the present invention aims to design a method for preparing Monascus purpureus mother koji through multiple innovations in formulation and process, thereby improving the activity and application value of the mother koji. Summary of the Invention

[0004] This invention provides a method for preparing Monascus purpureus mother koji, aiming to solve the above-mentioned problems.

[0005] This invention is achieved as follows: a method for preparing Monascus purpureus mother koji includes the following steps: Pretreatment of the strain: The Monascus purpureus production strain was subjected to compound stress adaptation treatment; Culture medium preparation: The thermosensitive hydrogel was mixed with the grain matrix and a metabolically inducible nutrient component containing mulberry leaf extract and selenium yeast was added, followed by sterilization. Inoculation and dynamic fermentation: The pretreated strains are inoculated into the culture medium and subjected to segmented variable-temperature solid-state fermentation; the switching from the first growth temperature to the second metabolic temperature is triggered by in-situ monitoring of the CO2 release rate, wherein the first growth temperature is 25-27℃ and the second metabolic temperature is 33-35℃. Carrier response and metabolic activation: When the temperature switches to the secondary metabolic temperature, the thermosensitive hydrogel undergoes phase transition shrinkage, exerts physical stress on the hyphal network, and releases its loaded nutrients in a pulsed manner. Post-processing: After fermentation, the starter culture is dried to obtain highly active Monascus purpureus starter culture.

[0006] Preferably, an infrared CO2 sensor is used to monitor the fermentation exhaust gas in situ, with a sampling frequency of 1 time per hour; when the CO2 release rate (CER) reaches the first peak, if the CER value fluctuates by ≤5% for 2 consecutive hours, it is determined to be stable, and the trigger temperature is switched from the first growth temperature to the second metabolic temperature.

[0007] The fermentation cycle of this invention refers to the total time from the inoculation of the strain when the CO2 release rate drops to half of its peak value and tends to stabilize.

[0008] Preferably, the stress agent used in the composite stress adaptation treatment includes a nonionic permeation stress agent (such as polyethylene glycol PEG-4000) and a trace amount of Monacoline K.

[0009] Preferably, the method for the combined stress adaptation treatment includes: spreading spores of Monascus purpureus strains on stress plates containing a non-ionic osmotic stress agent and Monacolin K for culture and screening, and culturing at 26-30°C until the cells grow robustly; the non-ionic osmotic stress agent is polyethylene glycol PEG-4000 with a mass-volume concentration of 3%-6%, and the trace amount of Monacolin K has a mass-volume concentration of 0.003%-0.008%.

[0010] The combined stress breeding method of "non-ionic osmotic stress (PEG)" and "product feedback inhibition (low-dose monacolin K)" has a clear directional selectivity compared with conventional ultraviolet or chemical mutagenesis, and can be used to activate / enhance the strain.

[0011] Preferably, the thermosensitive hydrogel is a poly(N-isopropylacrylamide) (PNIPAM)-based hydrogel with a minimum critical dissolution temperature (LCST) in the range of 28-32°C.

[0012] Preferably, poly(N-isopropylacrylamide)-based hydrogels are synthesized using a free radical polymerization method. The free radical polymerization method includes: mixing N-isopropylacrylamide monomer (concentration 10%-15%, w / v) with initiator ammonium persulfate (0.5%-1% of monomer mass) and crosslinking agent N,N'-methylenebisacrylamide (0.1%-0.3% of monomer mass), reacting at 60℃-65℃ for 4-6 hours, cooling, crushing, and sieving to obtain the hydrogel. The LCST of the hydrogel is determined by differential scanning calorimetry to be 28-32℃.

[0013] Preferably, the thermosensitive hydrogel is preloaded with sodium propionate and GABA at low temperature before use. Specifically, the precursor inducer, sodium propionate, is a direct precursor of the monacolin K synthesis pathway and can "unblock" and enhance the target metabolic flux. The signaling molecule inducer is γ-aminobutyric acid (GABA). Studies have shown that GABA can act as a stress signaling molecule in fungi, activating their secondary metabolic defense system, thereby promoting the synthesis of secondary metabolites such as antibiotics and pigments.

[0014] At low temperatures (e.g., 26-28°C): This temperature is below the LCST of the hydrogel, which is in a swollen state with loose, wide internal pores and a huge specific surface area. This structure is extremely conducive to the germination of Monascus purpureus spores and the rapid, uniform extension and colonization of mycelia, providing optimal physical space for mycelial growth.

[0015] At high temperatures (e.g., 33-35°C): When fermentation enters the secondary metabolic stage and the system temperature switches to 33-35°C, this temperature is higher than the LCST of the hydrogel. The hydrogel undergoes a volume phase transition, changing from a swollen state to a dehydrated and shrinking state. This process leads to: The contraction and compression of the pore structure exerts a mild physical pressure on the embedded hyphal network, simulating the stress state of microorganisms in nature when resources are limited and space is compressed. This mild stress has been shown to efficiently activate secondary metabolic pathways (i.e., "adversity produces secondary metabolites"), strongly promoting the synthesis of monacolin K and pigments.

[0016] Simultaneously, during the shrinkage process, the absorbed and loaded water and nutrients (such as pre-loaded GABA and sodium propionate) are "squeezed out" to the vicinity of the mycelium. The nutrient solution squeezed out during shrinkage achieves "in-situ precise feeding" of the mycelium, resulting in extremely high nutrient utilization efficiency.

[0017] Preferably, the culture medium in step b) comprises the following components by weight: 70-80 parts of cereal matrix, 20-30 parts of the thermosensitive hydrogel, 1-2 parts of mulberry leaf extract (70% ethanol reflux extraction for 2 hours, concentrated to a solid content ≥50%, total flavonoids ≥8%, 1-deoxynojirimycin ≥0.2%), and 0.1-0.5 parts of selenium yeast (food-grade selenium yeast, each gram of selenium yeast contains 2000μg-3000μg of selenium). The cereal matrix is ​​composed of rice flour and wheat bran mixed at a mass ratio of 1.5-2.5:1.

[0018] Preferably, the specific method for preparing the culture medium in step b) is as follows: Preloading: The thermosensitive hydrogel particles are immersed in a solution containing 0.05%-0.15% sodium propionate and 0.02%-0.08% γ-aminobutyric acid and swelled at 2-10℃ for 12-36 hours to complete the preloading. Mixing: Dry mix the preloaded hydrogel particles with the cereal matrix, mulberry leaf extract and selenium yeast in a certain proportion; Humidification and sterilization: Add water to the mixed materials to adjust the moisture content to 45%-50%, and then perform high-pressure sterilization.

[0019] Compared with the prior art, the embodiments of this application have the following main advantages: through composite stress adaptation treatment, the stress resistance and metabolic activity of Monascus purpureus strains in the subsequent fermentation process are significantly improved; the thermosensitive hydrogel achieves precise phase transition under temperature induction, and synergistically releases sodium propionate and GABA in pulses, further promoting the accumulation of secondary metabolites; combined with real-time regulation of fermentation temperature conversion by CO2 release rate, dynamic response and optimized control of process parameters are realized, effectively improving the yield of monacolin K and selenoamino acids. Attached Figure Description

[0020] Figure 1 This is a process flow diagram of a method for preparing Monascus purpureus mother koji provided by the present invention. Detailed Implementation

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0022] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0023] Example 1 This invention provides a method for preparing Monascus purpureus mother koji, such as... Figure 1 As shown, it includes the following steps: I. Compound stress adaptation treatment of Monascus purpureus production strains Preparation of stress culture medium: Prepare PDA medium, autoclave and cool to approximately 50°C. Under aseptic conditions, add filtered and sterilized polyethylene glycol PEG-4000 and monacolin K standard solutions to a final concentration of 3% (w / v) and 0.003% (w / v), respectively. Pour into plates for later use.

[0024] Pretreatment: Prepare a spore suspension of Monascus purpureus CICC 5012 (concentration approximately 1×10⁻⁶). 6 (CFU / mL) was evenly spread onto a stress culture medium plate using a sterile spreader.

[0025] Stress culture: Place the plates in a 26℃ constant temperature incubator and incubate in the dark for 5 days until robust colonies with vigorous pigment secretion are formed.

[0026] Obtaining pretreated inoculum: Pick a piece of mycelium (about 5 mm in diameter) from the edge of the colony. This can be used as a pretreated inoculum and directly used for inoculation in the subsequent preparation of the mother culture.

[0027] II. Preparation of Dynamic Adaptive Carriers and Culture Medium Preparation and preloading of thermosensitive hydrogels: a. Hydrogel synthesis: Poly(N-isopropylacrylamide) hydrogel was synthesized using conventional free radical polymerization. After synthesis, it was mechanically crushed and sieved to obtain particles in the range of 40-60 mesh.

[0028] b. Nutrient Preloading: The above hydrogel particles were immersed in a sterile aqueous solution containing 0.05% (w / v) sodium propionate and 0.02% (w / v) γ-aminobutyric acid. The mixture was then allowed to stand at 2°C (below its LCST) for 24 hours to allow the hydrogel to fully swell and adsorb the inducer. Afterwards, excess liquid was filtered off to obtain "thermosensitive hydrogel particles loaded with the key inducer".

[0029] Culture medium mixing: The components are dry-mixed according to the following weight parts: 70 parts of cereal matrix, 20 parts of the temperature-sensitive hydrogel, 1 part of mulberry leaf extract, and 0.1 parts of selenium yeast. The cereal matrix is ​​made by mixing rice flour and wheat bran at a mass ratio of 1.5:1.

[0030] Humidity conditioning and sterilization: Slowly add sterile water to the above-mentioned uniformly mixed dry materials while stirring, until the moisture content of the culture medium reaches 45%-50%. Then, autoclave at 120℃ for 25 minutes.

[0031] III. Dynamic biomimetic fermentation for preparing mother koji Inoculation: The obtained pretreated strains (mycelial blocks) are inoculated into the culture medium at an inoculation rate of 5% of the dry weight of the sterilized culture medium under aseptic conditions.

[0032] Dynamic fermentation: The inoculated culture medium is transferred into a solid-state fermenter equipped with an in-situ CO2 sensor and a precise temperature control system.

[0033] Phase 1 (cell growth, 25℃): The temperature was set at 25℃, and the ventilation rate was 0.5 L / (min·kg). At this stage, the hydrogel was in a swollen state with large internal pores, providing optimal physical space for rapid mycelial growth and colonization. The system monitored the CO2 release rate (CER) in real time.

[0034] Phase Two (Metabolic Transformation and Activation): When the CER curve reaches its first peak and tends to stabilize (approximately 40-48 hours after fermentation), the control system automatically executes the following: a. Change the fermentation temperature from 25℃ to 33℃.

[0035] b. When the temperature is raised to 33℃ (above the LCST of the hydrogel), the hydrogel undergoes a significant volume phase transition within 1-2 hours, changing from a swollen state to a shrinking state. This process generates uniform physical compression on the hyphal network that has grown into it; at the same time, it concentrates and "extrudes" the pre-loaded GABA and sodium propionate around the hyphae.

[0036] Phase 3 (efficient product synthesis, 33°C): Incubate at 33°C for approximately 72 hours. During this phase, physical stress signals and high concentrations of biochemical induction signals work synergistically to strongly activate the biosynthetic pathway of monacolin K.

[0037] Termination and Drying: After fermentation is complete, the material is removed and placed in a 40℃ hot air drying oven to dry until the moisture content is ≤10%. Finally, it is pulverized and passed through an 80-mesh sieve to obtain the highly active Monascus purpureus mother koji described in this invention.

[0038] Example 2 This invention provides a method for preparing Monascus purpureus mother koji, such as... Figure 1 As shown, it includes the following steps: I. Compound stress adaptation treatment of Monascus purpureus production strains Preparation of stress culture medium: Prepare PDA medium, autoclave and cool to approximately 50°C. Under aseptic conditions, add filtered sterilized polyethylene glycol PEG-4000 and monacolin K standard solutions to a final concentration of 4% (w / v) and 0.004% (w / v), respectively. Pour into plates for later use.

[0039] Pretreatment: Prepare a spore suspension of Monascus purpureus CICC 5012 (concentration approximately 1×10⁻⁶). 6 (CFU / mL) was evenly spread onto a stress culture medium plate using a sterile spreader.

[0040] Stress culture: Place the plates in a 27°C incubator and incubate in the dark for 5 days until robust colonies with vigorous pigment secretion are formed.

[0041] Obtaining pretreated inoculum: Pick a piece of mycelium (about 5 mm in diameter) from the edge of the colony. This can be used as a pretreated inoculum and directly used for inoculation in the subsequent preparation of the mother culture.

[0042] II. Preparation of Dynamic Adaptive Carriers and Culture Medium Preparation and preloading of thermosensitive hydrogels: a. Hydrogel synthesis: Poly(N-isopropylacrylamide) hydrogel was synthesized using conventional free radical polymerization. After synthesis, it was mechanically crushed and sieved to obtain particles in the range of 40-60 mesh.

[0043] b. Nutrient Preloading: The above hydrogel particles were immersed in a sterile aqueous solution containing 0.08% (w / v) sodium propionate and 0.03% (w / v) γ-aminobutyric acid. The mixture was then allowed to stand at 4°C (below its LCST) for 24 hours to allow the hydrogel to fully swell and adsorb the inducer. Afterwards, excess liquid was filtered off to obtain "thermosensitive hydrogel particles loaded with the key inducer".

[0044] Culture medium mixing: The components are dry-mixed according to the following weight parts: 72 parts of cereal matrix, 23 parts of the thermosensitive hydrogel, 1.2 parts of mulberry leaf extract, and 0.2 parts of selenium yeast. The cereal matrix is ​​made by mixing rice flour and wheat bran at a mass ratio of 1.8:1.

[0045] Humidity conditioning and sterilization: Slowly add sterile water to the above-mentioned uniformly mixed dry materials while stirring, until the moisture content of the culture medium reaches 45%-50%. Then, autoclave at 121°C for 28 minutes.

[0046] III. Dynamic biomimetic fermentation for preparing mother koji Inoculation: The obtained pretreated strains (mycelial blocks) are inoculated into the culture medium at an inoculation rate of 5% of the dry weight of the sterilized culture medium under aseptic conditions.

[0047] Dynamic fermentation: The inoculated culture medium is transferred into a solid-state fermenter equipped with an in-situ CO2 sensor and a precise temperature control system.

[0048] Phase 1 (cell growth, 25℃): The temperature was set at 25℃, and the ventilation rate was 0.5 L / (min·kg). At this stage, the hydrogel was in a swollen state with large internal pores, providing optimal physical space for rapid mycelial growth and colonization. The system monitored the CO2 release rate (CER) in real time.

[0049] Phase Two (Metabolic Transformation and Activation): When the CER curve reaches its first peak and tends to stabilize (approximately 40-48 hours after fermentation), the control system automatically executes the following: a. Change the fermentation temperature from 25℃ to 33℃.

[0050] b. When the temperature is raised to 33℃ (above the LCST of the hydrogel), the hydrogel undergoes a significant volume phase transition within 1-2 hours, changing from a swollen state to a shrinking state. This process generates uniform physical compression on the hyphal network that has grown into it; at the same time, it concentrates and "extrudes" the pre-loaded GABA and sodium propionate around the hyphae.

[0051] Phase 3 (efficient product synthesis, 33°C): Incubate at 33°C for approximately 72 hours. During this phase, physical stress signals and high concentrations of biochemical induction signals work synergistically to strongly activate the biosynthetic pathway of monacolin K.

[0052] Termination and Drying: After fermentation is complete, the material is removed and placed in a 40℃ hot air drying oven to dry until the moisture content is ≤10%. Finally, it is pulverized and passed through an 80-mesh sieve to obtain the highly active Monascus purpureus mother koji described in this invention.

[0053] Example 3 This invention provides a method for preparing Monascus purpureus mother koji, such as... Figure 1 As shown, it includes the following steps: I. Compound stress adaptation treatment of Monascus purpureus production strains Preparation of stress culture medium: Prepare PDA medium, autoclave and cool to approximately 50°C. Under aseptic conditions, add filtered sterilized polyethylene glycol PEG-4000 and monacolin K standard solutions to a final concentration of 4.5% (w / v) and 0.005% (w / v), respectively. Pour into plates for later use.

[0054] Pretreatment: Prepare a spore suspension of Monascus purpureus CICC 5012 (concentration approximately 1×10⁻⁶). 6 (CFU / mL) was evenly spread onto a stress culture medium plate using a sterile spreader.

[0055] Stress culture: Place the plates in a 28°C incubator and incubate in the dark for 6 days until robust colonies with vigorous pigment secretion are formed.

[0056] Obtaining pretreated inoculum: Pick a piece of mycelium (about 5 mm in diameter) from the edge of the colony. This can be used as a pretreated inoculum and directly used for inoculation in the subsequent preparation of the mother culture.

[0057] II. Preparation of Dynamic Adaptive Carriers and Culture Medium Preparation and preloading of thermosensitive hydrogels: a. Hydrogel synthesis: Poly(N-isopropylacrylamide) hydrogel was synthesized using conventional free radical polymerization. After synthesis, it was mechanically crushed and sieved to obtain particles in the range of 40-60 mesh.

[0058] b. Nutrient Preloading: The above hydrogel particles were immersed in a sterile aqueous solution containing 0.1% (w / v) sodium propionate and 0.05% (w / v) γ-aminobutyric acid. The mixture was then allowed to stand at 6°C (below its LCST) for 24 hours to allow the hydrogel to fully swell and adsorb the inducer. Afterwards, excess liquid was filtered off to obtain "thermosensitive hydrogel particles loaded with the key inducer".

[0059] Culture medium mixing: The components are dry-mixed according to the following weight parts: 75 parts of cereal matrix, 25 parts of the thermosensitive hydrogel, 1.5 parts of mulberry leaf extract, and 0.3 parts of selenium yeast. The cereal matrix is ​​made by mixing rice flour and wheat bran in a mass ratio of 2:1.

[0060] Humidity control and sterilization: Slowly add sterile water to the above-mentioned uniformly mixed dry materials while stirring, until the moisture content of the culture medium reaches 45%-50%. Then, autoclave at 120-125℃ for 25-35 minutes.

[0061] III. Dynamic biomimetic fermentation for preparing mother koji Inoculation: The obtained pretreated strains (mycelial blocks) are inoculated into the culture medium at an inoculation rate of 5% of the dry weight of the sterilized culture medium under aseptic conditions.

[0062] Dynamic fermentation: The inoculated culture medium is transferred into a solid-state fermenter equipped with an in-situ CO2 sensor and a precise temperature control system.

[0063] Phase 1 (cell growth, 26℃): The temperature was set at 26℃, and the ventilation rate was 0.5 L / (min·kg). At this stage, the hydrogel was in a swollen state with large internal pores, providing optimal physical space for rapid mycelial growth and colonization. The system monitored the CO2 release rate (CER) in real time.

[0064] Phase Two (Metabolic Transformation and Activation): When the CER curve reaches its first peak and tends to stabilize (approximately 40-48 hours after fermentation), the control system automatically executes the following: a. Change the fermentation temperature from 26℃ to 34℃.

[0065] b. When the temperature is raised to 34℃ (above the LCST of the hydrogel), the hydrogel undergoes a significant volume phase transition within 1-2 hours, changing from a swollen state to a shrinking state. This process generates uniform physical compression on the hyphal network that has grown into it; at the same time, it concentrates and "extrudes" the pre-loaded GABA and sodium propionate around the hyphae.

[0066] Phase 3 (efficient product synthesis, 34°C): Incubate at 34°C for approximately 72 hours. During this phase, physical stress signals and high concentrations of biochemical induction signals work synergistically to strongly activate the biosynthetic pathway of monacolin K.

[0067] Termination and Drying: After fermentation is complete, the material is removed and placed in a 40℃ hot air drying oven to dry until the moisture content is ≤10%. Finally, it is pulverized and passed through an 80-mesh sieve to obtain the highly active Monascus purpureus mother koji described in this invention.

[0068] Example 4 This invention provides a method for preparing Monascus purpureus mother koji, such as... Figure 1 As shown, it includes the following steps: I. Compound stress adaptation treatment of Monascus purpureus production strains Preparation of stress culture medium: Prepare PDA medium, autoclave and cool to approximately 50°C. Under aseptic conditions, add filtered and sterilized polyethylene glycol PEG-4000 and Monacoline K standard solutions to achieve final concentrations of 5% (w / v) and 0.006% (w / v), respectively. Pour into plates for later use.

[0069] Pretreatment: Prepare a spore suspension of Monascus purpureus CICC 5012 (concentration approximately 1×10⁻⁶). 6 (CFU / mL) was evenly spread onto a stress culture medium plate using a sterile spreader.

[0070] Stress culture: Place the plates in a 29°C incubator and incubate in the dark for 7 days until robust colonies with vigorous pigment secretion are formed.

[0071] Obtaining pretreated inoculum: Pick a piece of mycelium (about 5 mm in diameter) from the edge of the colony. This can be used as a pretreated inoculum and directly used for inoculation in the subsequent preparation of the mother culture.

[0072] II. Preparation of Dynamic Adaptive Carriers and Culture Medium Preparation and preloading of thermosensitive hydrogels: a. Hydrogel synthesis: Poly(N-isopropylacrylamide) hydrogel was synthesized using conventional free radical polymerization. After synthesis, it was mechanically crushed and sieved to obtain particles in the range of 40-60 mesh.

[0073] b. Nutrient Preloading: The above hydrogel particles were immersed in a sterile aqueous solution containing 0.12% (w / v) sodium propionate and 0.07% (w / v) γ-aminobutyric acid. The mixture was then allowed to stand at 8°C (below its LCST) for 24 hours to allow the hydrogel to fully swell and adsorb the inducer. Afterwards, excess liquid was filtered off to obtain "thermosensitive hydrogel particles loaded with the key inducer".

[0074] Culture medium mixing: The components are dry-mixed according to the following weight parts: 78 parts of cereal matrix, 27 parts of the thermosensitive hydrogel, 1.8 parts of mulberry leaf extract, and 0.4 parts of selenium yeast. The cereal matrix is ​​made by mixing rice flour and wheat bran at a mass ratio of 2.3:1.

[0075] Humidity control and sterilization: Slowly add sterile water to the above-mentioned uniformly mixed dry materials while stirring, until the moisture content of the culture medium reaches 45%-50%. Then, autoclave at 124℃ for 32 minutes.

[0076] III. Dynamic biomimetic fermentation for preparing mother koji Inoculation: The obtained pretreated strains (mycelial blocks) are inoculated into the culture medium at an inoculation rate of 5% of the dry weight of the sterilized culture medium under aseptic conditions.

[0077] Dynamic fermentation: The inoculated culture medium is transferred into a solid-state fermenter equipped with an in-situ CO2 sensor and a precise temperature control system.

[0078] Phase 1 (cell growth, 27℃): The temperature was set at 27℃, and the ventilation rate was 0.5 L / (min·kg). At this stage, the hydrogel was in a swollen state with large internal pores, providing optimal physical space for rapid mycelial growth and colonization. The system monitored the CO2 release rate (CER) in real time.

[0079] Phase Two (Metabolic Transformation and Activation): When the CER curve reaches its first peak and tends to stabilize (approximately 40-48 hours after fermentation), the control system automatically executes the following: a. Change the fermentation temperature from 27℃ to 35℃.

[0080] b. When the temperature is raised to 35℃ (above the LCST of the hydrogel), the hydrogel undergoes a significant volume phase transition within 1-2 hours, changing from a swollen state to a shrinking state. This process generates uniform physical compression on the hyphal network that has grown into it; at the same time, it concentrates and "extrudes" the pre-loaded GABA and sodium propionate around the hyphae.

[0081] Phase 3 (efficient product synthesis, 35°C): Incubate at 35°C for approximately 72 hours. During this phase, physical stress signals and high concentrations of biochemical induction signals work synergistically to strongly activate the biosynthetic pathway of monacolin K.

[0082] Termination and Drying: After fermentation is complete, the material is removed and placed in a 40℃ hot air drying oven to dry until the moisture content is ≤10%. Finally, it is pulverized and passed through an 80-mesh sieve to obtain the highly active Monascus purpureus mother koji described in this invention.

[0083] Example 5 This invention provides a method for preparing Monascus purpureus mother koji, such as... Figure 1 As shown, it includes the following steps: I. Compound stress adaptation treatment of Monascus purpureus production strains Preparation of stress culture medium: Prepare PDA medium, autoclave and cool to approximately 50°C. Under aseptic conditions, add filtered sterilized polyethylene glycol PEG-4000 and monacolin K standard solutions to a final concentration of 6% (w / v) and 0.008% (w / v), respectively. Pour into plates for later use.

[0084] Pretreatment: Prepare a spore suspension of Monascus purpureus CICC 5012 (concentration approximately 1×10⁻⁶). 6 (CFU / mL) was evenly spread onto a stress culture medium plate using a sterile spreader.

[0085] Stress culture: Place the plates in a 30℃ constant temperature incubator and incubate in the dark for 7 days until robust colonies with vigorous pigment secretion are formed.

[0086] Obtaining pretreated inoculum: Pick a piece of mycelium (about 5 mm in diameter) from the edge of the colony. This can be used as a pretreated inoculum and directly used for inoculation in the subsequent preparation of the mother culture.

[0087] II. Preparation of Dynamic Adaptive Carriers and Culture Medium Preparation and preloading of thermosensitive hydrogels: a. Hydrogel synthesis: Poly(N-isopropylacrylamide) hydrogel was synthesized using conventional free radical polymerization. After synthesis, it was mechanically crushed and sieved to obtain particles in the range of 40-60 mesh.

[0088] b. Nutrient Preloading: The above hydrogel particles were immersed in a sterile aqueous solution containing 0.15% (w / v) sodium propionate and 0.08% (w / v) γ-aminobutyric acid. The mixture was then allowed to stand at 10°C (below its LCST) for 24 hours to allow the hydrogel to fully swell and adsorb the inducer. Afterwards, excess liquid was filtered off to obtain "thermosensitive hydrogel particles loaded with the key inducer".

[0089] Culture medium mixing: The components are dry-mixed according to the following weight parts: 80 parts of cereal matrix, 30 parts of the temperature-sensitive hydrogel, 2 parts of mulberry leaf extract, and 0.5 parts of selenium yeast. The cereal matrix is ​​made by mixing rice flour and wheat bran at a mass ratio of 2.5:1.

[0090] Humidity control and sterilization: Slowly add sterile water to the above-mentioned uniformly mixed dry materials while stirring, until the moisture content of the culture medium reaches 45%-50%. Then, autoclave at 125℃ for 35 minutes.

[0091] III. Dynamic biomimetic fermentation for preparing mother koji Inoculation: The obtained pretreated strains (mycelial blocks) are inoculated into the culture medium at an inoculation rate of 5% of the dry weight of the sterilized culture medium under aseptic conditions.

[0092] Dynamic fermentation: The inoculated culture medium is transferred into a solid-state fermenter equipped with an in-situ CO2 sensor and a precise temperature control system.

[0093] Phase 1 (cell growth, 27℃): The temperature was set at 27℃, and the ventilation rate was 0.5 L / (min·kg). At this stage, the hydrogel was in a swollen state with large internal pores, providing optimal physical space for rapid mycelial growth and colonization. The system monitored the CO2 release rate (CER) in real time.

[0094] Phase Two (Metabolic Transformation and Activation): When the CER curve reaches its first peak and tends to stabilize (approximately 40-48 hours after fermentation), the control system automatically executes the following: a. Change the fermentation temperature from 27℃ to 35℃.

[0095] b. When the temperature is raised to 35℃ (above the LCST of the hydrogel), the hydrogel undergoes a significant volume phase transition within 1-2 hours, changing from a swollen state to a shrinking state. This process generates uniform physical compression on the hyphal network that has grown into it; at the same time, it concentrates and "extrudes" the pre-loaded GABA and sodium propionate around the hyphae.

[0096] Phase 3 (efficient product synthesis, 35°C): Incubate at 35°C for approximately 72 hours. During this phase, physical stress signals and high concentrations of biochemical induction signals work synergistically to strongly activate the biosynthetic pathway of monacolin K.

[0097] Termination and Drying: After fermentation is complete, the material is removed and placed in a 40℃ hot air drying oven to dry until the moisture content is ≤10%. Finally, it is pulverized and passed through an 80-mesh sieve to obtain the highly active Monascus purpureus mother koji described in this invention.

[0098] Comparative Example 1 (Traditional Method): Using a matrix similar to that of the traditional method, without stress treatment, hydrogels, or specific inducers, the original strain was cultured at a constant temperature of 30°C for 8 days as a baseline control.

[0099] Comparative Example 2 (No Stress Treatment): The difference from Example 3 is that the original bacterial strain was used.

[0100] Comparative Example 3 (hydrogel-free carrier): The difference from Example 3 is that an equal amount of rice flour was used instead of hydrogel, and GABA and sodium propionate were directly mixed into the culture medium. The same segmented temperature variation was used (but based on fixed-time switching).

[0101] Comparative Example 4 (without dynamic triggering): The difference from Example 3 is that a fixed time control (48 hours to 34°C) is used instead of CO2 signal triggering.

[0102] Comparative Example 5 (without specific inducers): The difference from Example 3 is that GABA and sodium propionate were not preloaded in the hydrogel.

[0103] Comparative Example 6 (Single Stress): The difference from Example 3 is that the strain was pretreated only in a medium containing 5% PEG-4000 (monacolin K-free).

[0104] Comparative Example 7 (Carrier Replacement): The difference from Example 3 is that static, non-thermosensitive porous starch-based microspheres were used instead of thermosensitive hydrogels, and GABA and sodium propionate were directly mixed into the culture medium.

[0105] Comparative Example 8 (Reverse Logic Test): The difference from Example 3 is that the temperature program was reversed (incubated at 34°C first, then reduced to 26°C).

[0106] The methods of Examples 1-5 and Comparative Examples 1-8 were tested as follows: Monacoline K detection: High performance liquid chromatography was used with a C18 column (250 mm × 4.6 mm, 5 μm), a mobile phase of methanol-0.1% phosphoric acid aqueous solution (85:15, v / v), a detection wavelength of 238 nm, a flow rate of 1.0 mL / min, and a column temperature of 30 °C.

[0107] Spore count detection: The hemocytometer method was used. The dried mother koji sample was serially diluted with sterile physiological saline and counted under a microscope to calculate the number of spores per gram of mother koji.

[0108] The results are shown in Table 1 below: Table 1 Test results for each preparation method This invention achieves precise and active regulation of Monascus growth and metabolism by organically combining strains pretreated under stress, intelligent carriers with dynamic response capabilities, precise metabolic inducers, and physiological state-based triggering processes. The resulting mother koji has high activity, a short fermentation cycle, and a significantly increased Monacoline K yield.

[0109] The mother koji prepared in Example 3 of this invention and the mother koji prepared in Comparative Example 1 were inoculated into steamed rice at an inoculation rate of 3% to prepare red yeast rice (fermented at 30°C for 7 days). The results showed that the red yeast rice prepared using the mother koji of this invention had a monacolin K content of 8.5 mg / g and a color value of 3800 U / g, both of which were significantly better than the product prepared using the mother koji of Comparative Example 1 (monacolin K 5.1 mg / g, color value 2100 U / g).

[0110] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

Claims

1. A method for preparing a red koji mold, characterized by, The method comprises the following steps: a) strain pretreatment: the Monascus production strain is subjected to compound stress adaptive treatment with non-ionic osmotic stress agent and monacolin K; b) preparation of culture medium: the temperature-sensitive hydrogel loaded with sodium propionate and γ-aminobutyric acid is mixed with grain substrate, mulberry leaf extract and selenium yeast, and sterilized; c) inoculation and fermentation: the strain of step a) is inoculated into the culture medium of step b), and subjected to staged temperature solid-state fermentation; the staged temperature comprises a first growth temperature and a second secondary metabolism temperature; during the fermentation, the switching from the first growth temperature to the second secondary metabolism temperature is triggered by in-situ monitoring of the CO2 release rate in the fermentation tail gas; d) when the temperature is switched to the second secondary metabolism temperature, the temperature-sensitive hydrogel undergoes phase transition and shrinkage, thereby producing physical stress on the mycelium and releasing at least part of the loaded nutritional components; e) after the fermentation, the Monascus mother koji is obtained by drying.

2. The preparation method of the red koji of Monascus according to claim 1, characterized in that, The method of compound stress adaptive treatment in step a) comprises the following steps: spores of the Monascus strain are inoculated on a stress plate containing non-ionic osmotic stress agent and monacolin K for culture and screening, and the culture is carried out at 26-30°C until the strain grows robustly.

3. The preparation method of the Monascus mother rice according to claim 2, characterized in that, The non-ionic osmotic stress agent is polyethylene glycol PEG-4000 with a mass-volume concentration of 3%-6%, and the trace monacolin K has a mass-volume concentration of 0.003%-0.008%.

4. The preparation method of the Monascus mother rice according to claim 1, characterized in that, The temperature-sensitive hydrogel loaded with sodium propionate and γ-aminobutyric acid is prepared by the following method: poly(N-isopropyl acrylamide) based hydrogel is synthesized by free radical polymerization, and after synthesis, the hydrogel is crushed and sieved to select particles with a size of 40-60 mesh; before use, the hydrogel is soaked in a solution containing 0.05%-0.15% sodium propionate and 0.02%-0.08% γ-aminobutyric acid under conditions below the lower critical solution temperature, and swelled at 2-10°C for 12-36 hours to complete the loading.

5. The preparation method of the Monascus mother rice according to claim 4, characterized in that, The lower critical solution temperature of the temperature-sensitive hydrogel is in the range of 28-32°C.

6. The preparation method of the red koji of Monascus according to claim 1, characterized in that, The culture medium in step b) comprises the following components by weight: grain substrate 70-80 parts, temperature-sensitive hydrogel 20-30 parts, mulberry leaf extract 1-2 parts, and selenium yeast 0.1-0.5 parts; the grain substrate is prepared by mixing rice flour and wheat bran at a mass ratio of 1.5-2.5:

1.

7. The preparation method of the Monascus mother rice according to claim 6, characterized in that, The method for preparing the culture medium comprises the following steps: preparing temperature-sensitive hydrogel loaded with sodium propionate and γ-aminobutyric acid; dry mixing the loaded hydrogel particles with grain substrate, mulberry leaf extract and selenium yeast in proportion; adding water to the mixed material to adjust the moisture content to 45%-50%, and then performing high-pressure sterilization.

8. The preparation method of the Monascus mother rice according to claim 6, characterized in that, In step c), the first growth temperature is 25-27°C, and the second secondary metabolism temperature is 33-35°C.

9. Monascus mother koji prepared by the method of any one of claims 1-8.

10. Use of the Monascus mother koji of claim 9 in the preparation of red kojic rice, red kojic wine or monacolin K extract.

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