Method for preparing traditional Chinese medicinal material compound preparation by utilizing monascus fermentation

Abstract

The invention relates to the technical field of traditional Chinese medicinal material fermentation, in particular to a method for preparing a traditional Chinese medicinal material compound preparation through monascus fermentation. The method for preparing the traditional Chinese medicinal material compound preparation by utilizing the monascus fermentation comprises the following steps: uniformly mixing sticky rice and black rice, adding water to soak for 10-20 hours, fishing out and steaming, then adding traditional Chinese medicinal materials to uniformly mix, and cooling to room temperature to obtain a premixed matrix; adjusting the water content of the premixed matrix, standing for 5-10 days in a ventilated environment, sterilizing, adding the lactobacillus fermentation supernatant, uniformly mixing, adjusting the pH value of the system to 4.8-5.2, and performing pasteurization to obtain a matrix; adding monascus liquid or composite monascus microspheres into the matrix, uniformly mixing, then adding neutral protease, fully mixing, and culturing at 30-35 DEG C; when the water content of the system is lower than 30%, the water content is adjusted to be 60-70%, and glucose and vinegar residues are added; and when the water content of the system is lower than 20%, ending the culture, uniformly turning and spreading, and performing vacuum drying.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine fermentation, and in particular to a method for preparing a compound preparation of traditional Chinese medicine by fermenting red yeast rice. Background Art

[0002] Red yeast rice is a characteristic microbial fermentation product with a history of thousands of years in China. It is prepared by solid-state fermentation of rice using Monascus spp. as the functional strain. Its applications span both the food and pharmaceutical fields.

[0003] In the food industry, it is used as a natural colorant (producing the red pigment Monascorubramine) and a saccharifying fermenting agent for the brewing of yellow rice wine; while in ancient books, it is recorded that it has the effect of promoting blood circulation and removing blood stasis. Modern pharmacology has confirmed that it contains Monacolin K (the precursor of lovastatin), which can significantly reduce total serum cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL) by inhibiting HMG-CoA reductase, and at the same time increase high-density lipoprotein (HDL), becoming a representative of natural lipid-lowering drugs.

[0004] Although China dominates the global production of red yeast rice, the production process still mainly follows the traditional model: using rice as the single matrix, relying on artificial inoculation and staged fermentation with segmented water replenishment and humidity control (60 - 80% RH). However, the content of secondary metabolites such as Monacolin K is restricted by factors such as strain degradation and contamination by miscellaneous bacteria in open fermentation, resulting in the problem that the biological activity cannot meet the requirements. If traditional Chinese medicine is added to the production of red yeast rice to prepare a preparation of medicinal red yeast rice and improve the biological activity of the preparation, it has excellent research prospects. Summary of the Invention

[0005] The purpose of the present invention is to solve the disadvantages existing in the prior art, and to propose a method for preparing a compound preparation of traditional Chinese medicine by fermenting red yeast rice.

[0006] A method for preparing a compound preparation of traditional Chinese medicine by fermenting red yeast rice, which is characterized by comprising the following steps:

[0007] S1. Mix glutinous rice and black rice evenly, soak them in water for 10 - 20 h, fish them out and steam them, then add traditional Chinese medicine and mix evenly, and cool to room temperature to obtain a premixed matrix;

[0008] Adjust the water content of the premixed matrix to 60 - 80%, let it stand for 5 - 10 days under a ventilation environment, the standing temperature is 25 - 30 °C, sterilize it, add the supernatant of lactic acid bacteria fermentation and mix evenly, adjust the pH value of the system to 4.8 - 5.2, and perform pasteurization to obtain the matrix;

[0009] S2. Add Monascus liquid or composite Monascus microspheres to the substrate, mix evenly, then add neutral protease and mix well. Incubate at 30 - 35°C with a humidity of 80 - 90%. When the water content in the system is lower than 30%, adjust the water content to 60 - 70% and add glucose and vinegar residue. When the water content in the system is lower than 20%, end the incubation, turn it evenly and spread it flat, and then dry it under vacuum.

[0010] Preferably, in S1, the mass ratio of glutinous rice, black rice, Chinese medicinal materials, and lactic acid bacteria fermentation supernatant is 5 - 15:1 - 5:10 - 30:1 - 2.

[0011] Preferably, the lactic acid bacteria fermentation supernatant is prepared by the following specific operation: Inoculate lactic acid bacteria into MRS liquid medium with an inoculation amount of 3%, incubate at 37°C for 24 h, centrifuge at 6000 rpm for 10 min, and the centrifugation temperature is 4°C.

[0012] Preferably, in S1, the Chinese medicinal materials include: licorice, poria, eucommia leaves, ganoderma lucidum; the mass ratio of licorice, poria, eucommia leaves, and ganoderma lucidum is 5 - 9:4 - 6:3 - 7:1 - 3.

[0013] Preferably, in S2, the viable bacteria concentration of the Monascus liquid is 1 - 2×10 8 CFU / mL.

[0014] Preferably, the composite Monascus microspheres used in S2 are prepared by the following steps:

[0015] a. Mix Monascus liquid, trehalose, and glutamine, incubate at 28 - 32°C with shaking for 1 - 2 h, add chitosan oligosaccharide and continue stirring for 40 - 60 min to obtain a pretreated bacterial solution;

[0016] b. Add hydroxypropyl-β-cyclodextrin and sodium carboxymethylcellulose to the citric acid - disodium hydrogen phosphate buffer system and stir evenly. Add methacrylated carboxymethylcellulose sodium and a photo - crosslinking agent to obtain a pretreated sol;

[0017] c. Form microspheres from the pretreated bacterial solution and the pretreated sol through a coaxial nozzle. The microspheres include the pretreated bacterial solution and a pretreated sol layer covering the outside of the pretreated bacterial solution. Crosslink by visible light irradiation and then freeze - dry under vacuum.

[0018] Among them, methacrylated carboxymethylcellulose sodium and the photo - crosslinking agent can be referred to in CN114230678A (A photo - crosslinkable hydrogel embolization system for intravascular treatment and its use method, application number CN202111521041.1, application date December 13, 2021, publication date March 25, 2022).

[0019] The photo-crosslinking agent is composed of eosin-Y, triethanolamine, and N-vinylcaprolactam, and the mass ratio of the three is 3:188:125.

[0020] More preferably, the mass ratio of Monascus bacteria solution, trehalose, glutamine, chitosan oligosaccharide, hydroxypropyl-β-cyclodextrin, sodium carboxymethylcellulose, methacrylated sodium carboxymethylcellulose, and photo-crosslinking agent is 5-10:1-2:0.01-0.1:0.01-0.1:1-2:0.1-1:0.15-0.2:0.001-0.01; the viable bacteria concentration of the Monascus bacteria solution is 1-2×10 8 CFU / mL, and the molecular weight of chitosan oligosaccharide is 2-3 kDa.

[0021] More preferably, the particle size of the microspheres is 100-300 μm.

[0022] More preferably, the visible light wavelength is 400-600 nm, and the irradiation time is 1-3 min.

[0023] Preferably, in S2, the mass ratio of the substrate, Monascus bacteria solution, neutral protease, glucose, and vinegar residue is 20-50:5-10:1-2:1-2:1-2; or, the mass ratio of the substrate, composite Monascus microspheres, neutral protease, glucose, and vinegar residue is 20-50:7.5-15:1-2:1-2:1-2.

[0024] Preferably, in S2, intermittent negative pressure ventilation is carried out during the cultivation at 30-35 °C after adding neutral protease, specifically: negative pressure ventilation at -0.05 MPa for 1-3 min, and then stopping negative pressure ventilation for 57-59 min.

[0025] Beneficial effects:

[0026] The present invention uses traditional Chinese medicines in combination with glutinous rice and black rice. Among them, Eucommia ulmoides leaves can effectively inhibit miscellaneous bacteria and effectively maintain the purity of the bacterial community, while glycyrrhizin can be compounded with the exopolysaccharide in the supernatant of lactic acid bacteria fermentation to effectively protect the fermentation system.

[0027] The present invention forms microspheres through the combination of hydroxypropyl-β-cyclodextrin / chitosan oligosaccharide composite carriers and photocuring to achieve the slow release and protection of Monascus bacteria. When compounded with the substrate, it improves the survival rate and metabolic activity of the bacteria in the middle and late stages of fermentation. Not only is the fermentation process stable, but it also promotes the proliferation of bacteria, effectively enhances secondary metabolism, improves the generation of effective active ingredients, and at the same time has strong glucoamylase activity and protease activity, shortening the fermentation cycle by more than 20%.

[0028] The present invention can provide a good medium for the growth of microorganisms, enhance the adaptability of microorganisms to Chinese medicinal materials, effectively maintain the vigorous growth state of the microbial flora during the fermentation cycle, enable the rapid fermentation of Chinese medicinal materials, enhance the oxygen diffusion efficiency through intermittent negative pressure ventilation, and promote the metabolic conversion of Monascus from the mycelial growth stage to the spore formation stage in combination with the regulation of the moisture gradient, greatly improving the biological activity of the product and being applicable to large-scale production.

[0029] The present invention can effectively improve the performance of medicated red yeast rice, with excellent glucoamylase activity, protease activity, and esterase activity. It not only effectively retains the active ingredients of the used Chinese medicinal materials, but also has excellent health care functions, can achieve a central sedative effect, and effectively enhance the anti-fatigue effect of the body and the myocardial hypoxia tolerance.

[0030] The present invention can avoid the structural damage and loss of active ingredients caused by high temperature during the extraction of Chinese medicine components by the traditional decoction method, improve the extraction efficiency of Chinese medicine active ingredients, maximize the medicinal properties of the Chinese medicine formula, enhance the body's immune function, and is suitable for large-scale popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a comparison chart of the total saponin content and crude polysaccharide content of the products obtained by the methods of Example 5 and Comparative Examples 1-3.

[0032] Figure 2 It is a comparison chart of the glucoamylase activity and protease activity of the products obtained by the methods of Example 5 and Comparative Examples 1-3.

[0033] Figure 3 It is a comparison chart of the number of spontaneous activities and pole climbing time of mice in the control group, Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group.

[0034] Figure 4 It is a comparison chart of the survival time of mice in the control group, Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group after intraperitoneal injection of 2% sodium nitrite solution. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention will be further explained below in conjunction with specific embodiments.

[0036] Example 1

[0037] A method for preparing a compound preparation of Chinese medicinal materials by Monascus fermentation includes the following steps:

[0038] S1. Mix 500 g of glutinous rice and 100 g of black rice evenly, soak them in water for 10 h, fish them out and steam them on a steaming bed. After steaming, add 1000 g of Chinese medicinal materials and mix evenly, and cool to room temperature to obtain a premixed matrix;

[0039] The Chinese medicinal materials include: licorice, poria cocos, eucommia leaves, and ganoderma lucidum. The mass ratio of licorice, poria cocos, eucommia leaves, and ganoderma lucidum is 5:4:3:1;

[0040] Adjust the water content of the premixed matrix to 60%, let it stand for 5 days under a ventilated environment, the standing temperature is 25°C, sterilize it, add 100 g of lactic acid bacteria fermentation supernatant and mix evenly, adjust the pH value of the system to 4.8 - 5.2 with citric acid, and perform pasteurization to obtain the matrix;

[0041] S2. Add 500 g of monascus bacteria solution with a viable bacteria concentration of 1×10 8 CFU / mL to 2000 g of the matrix and mix evenly, then add 100 g of neutral protease and stir well. Incubate at a temperature of 30°C, and the incubation humidity is 80%. Intermittent negative pressure ventilation is carried out during this incubation stage, specifically: perform negative pressure ventilation at -0.05 MPa for 1 min, and then stop negative pressure ventilation for 59 min; when the water content of the system is lower than 30%, adjust the water content to 60%, and add 100 g of glucose and 100 g of vinegar residue; when the water content of the system is lower than 20%, end the incubation, mix evenly and spread flat, and perform vacuum drying.

[0042] Example 2

[0043] A method for preparing a compound preparation of Chinese medicinal materials by Monascus fermentation, comprising the following steps:

[0044] S1. Mix 1500 g of glutinous rice and 500 g of black rice evenly, soak them in water for 20 h, take them out and steam them on a steaming bed. After steaming, add 3000 g of Chinese medicinal materials and mix evenly, and let it cool to room temperature to obtain a premixed matrix;

[0045] The Chinese medicinal materials include: licorice, poria cocos, eucommia leaves, and ganoderma lucidum. The mass ratio of licorice, poria cocos, eucommia leaves, and ganoderma lucidum is 9:6:7:3;

[0046] Adjust the water content of the premixed matrix to 80%, let it stand for 10 days under a ventilated environment, the standing temperature is 30°C, sterilize it, add 200 g of lactic acid bacteria fermentation supernatant and mix evenly, adjust the pH value of the system to 4.8 - 5.2 with citric acid, and perform pasteurization to obtain the matrix;

[0047] S2. Add 1000 g of monascus bacteria solution with a viable bacteria concentration of 2×10 8 CFU / mL to 5000 g of the matrix and mix evenly, then add 200 g of neutral protease and stir well. Incubate at a temperature of 35°C, and the incubation humidity is 90%. Intermittent negative pressure ventilation is carried out during this incubation stage, specifically: perform negative pressure ventilation at -0.05 MPa for 3 min, and then stop negative pressure ventilation for 57 min; when the water content of the system is lower than 30%, adjust the water content to 70%, and add 200 g of glucose and 200 g of vinegar residue; when the water content of the system is lower than 20%, end the incubation, mix evenly and spread flat, and perform vacuum drying.

[0048] Example 3

[0049] A method for preparing a compound preparation of traditional Chinese medicine by Monascus fermentation, comprising the following steps:

[0050] S1. Mix 800 g of glutinous rice and 400 g of black rice evenly, soak them in water for 12 h, fish them out and steam them on a steaming bed. After steaming, add 2500 g of traditional Chinese medicine and mix evenly, and cool to room temperature to obtain a premixed matrix;

[0051] The traditional Chinese medicine includes: licorice, poria cocos, eucommia ulmoides leaves, and ganoderma lucidum. The mass ratio of licorice, poria cocos, eucommia ulmoides leaves, and ganoderma lucidum is 6:5.5:4:2.5;

[0052] Adjust the water content of the premixed matrix to 65%, let it stand for 9 days in a ventilation environment, the standing temperature is 26 °C, sterilize, add 180 g of lactic acid bacteria fermentation supernatant and mix evenly, adjust the pH value of the system to 4.8 - 5.2 with citric acid, and perform pasteurization to obtain the matrix;

[0053] S2. Add 1500 g of compound Monascus microspheres to 3000 g of the matrix and mix evenly, then add 130 g of neutral protease and stir well. Cultivate at a temperature of 34 °C, and the cultivation humidity is 82%. Intermittent negative pressure ventilation is carried out in this cultivation stage, specifically: carry out negative pressure ventilation at -0.05 MPa for 2 min, and then stop negative pressure ventilation for 58 min; when the water content of the system is lower than 30%, adjust the water content to 68%, and add 130 g of glucose and 180 g of vinegar residue; when the water content of the system is lower than 20%, end the cultivation, turn it evenly and spread it flat, and perform vacuum drying.

[0054] Among them, the compound Monascus microspheres are prepared by the following steps:

[0055] a. Mix 500 g of Monascus liquid with a viable bacteria concentration of 2×10 8 CFU / mL, 100 g of trehalose, and 10 g of glutamine, oscillate and cultivate at a temperature of 28 °C for 2 h, add 1 g of chitosan oligosaccharide with a molecular weight of 3 kDa to it and continue to stir for 40 min to obtain a pretreated bacterial body;

[0056] b. Add 200 g of hydroxypropyl-β-cyclodextrin and 10 g of sodium carboxymethylcellulose to 3000 g of a citric acid - disodium hydrogen phosphate buffer system with a pH of 5 - 6 and a concentration of 0.1 mol / L, stir evenly, add 20 g of methacrylated carboxymethylcellulose sodium and 0.1 g of photo-crosslinking agent to it to obtain a pretreated sol;

[0057] c. Form microspheres with an average particle size of 250 μm from the pretreated bacterial solution and the pretreated sol through a coaxial nozzle. The microspheres include the pretreated bacterial solution and a pretreated sol layer coating the outside of the pretreated bacterial solution; irradiate with 400 nm visible light for 3 min and then perform vacuum freeze-drying.

[0058] Example 4

[0059] A method for preparing a traditional Chinese medicine compound preparation by Monascus fermentation, comprising the following steps:

[0060] S1. Mix 1200 g of glutinous rice and 200 g of black rice evenly, soak them in water for 18 h, take them out and steam them on a steaming bed. After steaming, add 1500 g of traditional Chinese medicine and mix evenly, and let it cool to room temperature to obtain a premixed matrix;

[0061] The traditional Chinese medicine includes: licorice, poria cocos, eucommia ulmoides leaves, ganoderma lucidum, and the mass ratio of licorice, poria cocos, eucommia ulmoides leaves, ganoderma lucidum is 8:4.5:6:1.5;

[0062] Adjust the water content of the premixed matrix to 75%, let it stand for 7 days in a ventilated environment, the standing temperature is 28 °C, sterilize it, add 120 g of lactic acid bacteria fermentation supernatant and mix evenly, adjust the pH value of the system to 4.8 - 5.2 with citric acid, and perform pasteurization to obtain the matrix;

[0063] S2. Add 750 g of compound Monascus microspheres to 4000 g of the matrix and mix evenly, then add 170 g of neutral protease and stir well. Culture at a temperature of 32 °C, and the culture humidity is 88%. Intermittent negative pressure ventilation is carried out during this culture stage, specifically: carry out negative pressure ventilation at -0.05 MPa for 2 min, and then stop negative pressure ventilation for 58 min; when the water content of the system is lower than 30%, adjust the water content to 62%, and add 170 g of glucose and 120 g of vinegar residue; when the water content of the system is lower than 20%, end the culture, mix evenly and level it, and perform vacuum drying.

[0064] Among them, the compound Monascus microspheres are prepared by the following steps:

[0065] a. Mix 1000 g of Monascus bacteria solution with a viable bacteria concentration of 1×10 8 CFU / mL, 200 g of trehalose, and 1 g of glutamine, oscillate and culture at a temperature of 32 °C for 1 h, add 10 g of chitosan oligosaccharide with a molecular weight of 2 kDa to it and continue to stir for 60 min to obtain pretreated bacteria;

[0066] b. Add 100 g of hydroxypropyl-β-cyclodextrin and 100 g of sodium carboxymethylcellulose to 1000 g of a citric acid - disodium hydrogen phosphate buffer system with pH = 5 - 6 and a concentration of 0.3 mol / L, stir evenly, add 15 g of methacrylated carboxymethylcellulose sodium and 1 g of photo-crosslinking agent to it to obtain pretreated sol;

[0067] c. Form microspheres with an average particle size of 150 μm from the pretreated bacterial solution and the pretreated sol through a coaxial nozzle. The microspheres include the pretreated bacterial solution and a layer of pretreated sol coating the outside of the pretreated bacterial solution; irradiate with 600 nm visible light for 1 min and then perform vacuum freeze-drying.

[0068] Example 5

[0069] A method for preparing a traditional Chinese medicine compound preparation by Monascus fermentation, comprising the following steps:

[0070] S1. Mix 1000 g of glutinous rice and 300 g of black rice evenly, soak them in water for 15 h, take them out and steam them on a steaming bed. After steaming, add 2000 g of traditional Chinese medicine and mix evenly, and let it cool to room temperature to obtain a premixed matrix;

[0071] The traditional Chinese medicine includes: licorice, poria, eucommia ulmoides leaves, and ganoderma lucidum. The mass ratio of licorice, poria, eucommia ulmoides leaves, and ganoderma lucidum is 7:5:5:2;

[0072] Adjust the water content of the premixed matrix to 70%, let it stand for 8 days in a ventilated environment, the standing temperature is 27 °C, sterilize it, add 150 g of lactic acid bacteria fermentation supernatant and mix evenly, adjust the pH value of the system to 4.8 - 5.2 with citric acid, and perform pasteurization to obtain a matrix;

[0073] S2. Add 1200 g of compound Monascus microspheres to 3500 g of the matrix and mix evenly, then add 150 g of neutral protease and stir thoroughly. Cultivate at a temperature of 33 °C, and the cultivation humidity is 85%. Intermittent negative pressure ventilation is carried out during this cultivation stage, specifically: carry out negative pressure ventilation at -0.05 MPa for 2 min, and then stop negative pressure ventilation for 58 min; when the water content of the system is lower than 30%, adjust the water content to 65%, and add 150 g of glucose and 150 g of vinegar residue; when the water content of the system is lower than 20%, end the cultivation, mix evenly and spread it flat, and perform vacuum drying.

[0074] Among them, the compound Monascus microspheres are prepared by the following steps:

[0075] a. Mix 800 g of Monascus bacterial solution with a viable cell concentration of 1.5×10 8 CFU / mL, 150 g of trehalose, and 5 g of glutamine, oscillate and cultivate at a temperature of 30 °C for 1.5 h, add 5 g of chitosan oligosaccharide with a molecular weight of 2.5 kDa to it and continue to stir for 50 min to obtain pretreated bacteria;

[0076] b. Add 150 g of hydroxypropyl-β-cyclodextrin and 50 g of sodium carboxymethylcellulose to 2000 g of a citric acid - disodium hydrogen phosphate buffer system with a pH of 5 - 6 and a concentration of 0.2 mol / L, stir evenly, add 18 g of methacrylated carboxymethylcellulose sodium and 0.5 g of photo-crosslinking agent to it to obtain a pretreated sol;

[0077] c. Form microspheres with an average particle size of 200 μm from the pretreated bacterial solution and the pretreated sol through a coaxial nozzle. The microspheres include the pretreated bacterial solution and a pretreated sol layer coating the outside of the pretreated bacterial solution; irradiate with 500-nm visible light for 2 min and then perform vacuum freeze-drying.

[0078] Comparative Example 1

[0079] A method for preparing a traditional Chinese medicine compound preparation by Monascus fermentation, comprising the following steps:

[0080] S1. Mix 1000 g of glutinous rice and 300 g of black rice evenly, soak in water for 15 h, take out and steam on a steaming bed until cooked. After cooking, add 2000 g of traditional Chinese medicine and mix evenly, and let it cool to room temperature to obtain a premixed matrix.

[0081] The traditional Chinese medicine includes: licorice, poria, ganoderma lucidum. The mass ratio of licorice, poria, eucommia ulmoides leaves, and ganoderma lucidum is 12:5:2.

[0082] Adjust the water content of the premixed matrix to 70%, let it stand for 8 days in a ventilated environment at a standing temperature of 27 °C, sterilize, add 150 g of lactic acid bacteria fermentation supernatant and mix evenly, adjust the pH value of the system to 4.8 - 5.2 with citric acid, and perform pasteurization to obtain the matrix.

[0083] S2. Add 1200 g of compound Monascus microspheres to 3500 g of the matrix and mix evenly, then add 150 g of neutral protease and stir well. Incubate at a temperature of 33 °C with a cultivation humidity of 85%. Intermittent negative pressure ventilation is carried out during this cultivation stage, specifically: perform negative pressure ventilation at -0.05 MPa for 2 min, then stop negative pressure ventilation for 58 min; when the water content of the system is lower than 30%, adjust the water content to 65% and add 150 g of glucose and 150 g of vinegar residue; when the water content of the system is lower than 20%, end the cultivation, mix evenly and spread flat, and perform vacuum drying.

[0084] Among them, the compound Monascus microspheres are prepared by the following steps:

[0085] a. Mix 800 g of Monascus liquid with a viable bacteria concentration of 1.5×10 8 CFU / mL, 150 g of trehalose, and 5 g of glutamine, incubate with shaking at a temperature of 30 °C for 1.5 h, add 5 g of chitosan oligosaccharide with a molecular weight of 2.5 kDa and continue stirring for 50 min to obtain pretreated bacteria.

[0086] b. Add 150 g of hydroxypropyl-β-cyclodextrin and 50 g of sodium carboxymethylcellulose to 2000 g of a citric acid-disodium hydrogen phosphate buffer system with a pH of 5 - 6 and a concentration of 0.2 mol / L, stir evenly, add 18 g of methacrylated carboxymethylcellulose sodium and 0.5 g of photo-crosslinking agent thereto to obtain a pretreated sol;

[0087] c. Form microspheres with an average particle size of 200 μm from the pretreated bacterial solution and the pretreated sol through a coaxial nozzle. The microspheres include the pretreated bacterial solution and a pretreated sol layer coating the outside of the pretreated bacterial solution; irradiate with 500 nm visible light for 2 min and then perform vacuum freeze-drying.

[0088] Comparative Example 2

[0089] A method for preparing a Chinese medicinal material composite preparation by Monascus fermentation includes the following steps:

[0090] S1. Mix 1000 g of glutinous rice and 300 g of black rice evenly, soak them in water for 15 h, fish them out and steam them on a steaming bed. After steaming, add 2000 g of Chinese medicinal materials and mix evenly, and let it cool to room temperature to obtain a premixed matrix;

[0091] The Chinese medicinal materials include: licorice, poria cocos, eucommia ulmoides leaves, and ganoderma lucidum. The mass ratio of licorice, poria cocos, eucommia ulmoides leaves, and ganoderma lucidum is 7:5:5:2;

[0092] Adjust the water content of the premixed matrix to 70%, let it stand for 8 days in a ventilated environment at a standing temperature of 27°C, sterilize it, add 150 g of lactic acid bacteria fermentation supernatant and mix evenly, adjust the pH value of the system to 4.8 - 5.2 with citric acid, and perform pasteurization to obtain a matrix;

[0093] S2. Add 1200 g of composite Monascus microspheres to 3500 g of the matrix and mix evenly, then add 150 g of neutral protease and stir thoroughly. Cultivate at a temperature of 33°C with a cultivation humidity of 85%. Intermittent negative pressure ventilation is carried out during this cultivation stage, specifically: perform negative pressure ventilation at -0.05 MPa for 2 min, and then stop negative pressure ventilation for 58 min; when the water content of the system is lower than 30%, adjust the water content to 65% and add 150 g of glucose and 150 g of vinegar residue; when the water content of the system is lower than 20%, end the cultivation, turn it over and level it, and perform vacuum drying.

[0094] Among them, the composite Monascus complex is prepared by the following steps:

[0095] a. Mix 800 g of Monascus liquid with a viable bacteria concentration of 1.5×108 CFU / mL, 150 g of trehalose, and 5 g of glutamine, cultivate with shaking at a temperature of 30°C for 1.5 h, add 5 g of chitosan oligosaccharide with a molecular weight of 2.5 kDa thereto, and continue stirring for 50 min to obtain a pretreated bacterial body;

[0096] b. Add the pretreated bacterial cells, 150 g of hydroxypropyl-β-cyclodextrin, and 50 g of sodium carboxymethylcellulose to 2028.5 g of a citric acid-disodium hydrogen phosphate buffer system with a pH of 5 - 6 and a concentration of 0.2 mol / L, stir evenly, and perform vacuum freeze-drying to obtain the compound Monascus complex.

[0097] Comparative Example 3

[0098] A method for preparing a traditional Chinese medicine compound preparation by Monascus fermentation, comprising the following steps:

[0099] S1. Mix 1000 g of glutinous rice and 300 g of black rice evenly, soak them in water for 15 h, take them out and steam them on a steaming bed. After steaming, add 2000 g of traditional Chinese medicine and mix evenly, and cool to room temperature to obtain a premixed matrix;

[0100] The traditional Chinese medicine includes: licorice, Poria cocos, Eucommia ulmoides leaves, Ganoderma lucidum, and the mass ratio of licorice, Poria cocos, Eucommia ulmoides leaves, and Ganoderma lucidum is 7:5:5:2;

[0101] Adjust the water content of the premixed matrix to 70%, let it stand for 8 days in a ventilated environment at a standing temperature of 27°C, sterilize, add 150 g of lactic acid bacteria fermentation supernatant and mix evenly, adjust the pH value of the system to 4.8 - 5.2 with citric acid, and perform pasteurization to obtain the matrix;

[0102] S2. Add 1200 g of compound Monascus microspheres to 3500 g of the matrix and mix evenly, then add 150 g of neutral protease and stir thoroughly, and culture at a temperature of 33°C with a culture humidity of 85%; when the water content of the system is lower than 30%, adjust the water content to 65%, and add 150 g of glucose and 150 g of vinegar residue. Intermittent negative pressure ventilation is carried out during this culture stage, specifically: perform negative pressure ventilation at -0.05 MPa for 2 min, and then stop negative pressure ventilation for 58 min; when the water content of the system is lower than 20%, end the culture, turn it over and level it, and perform vacuum drying.

[0103] Among them, the compound Monascus microspheres are prepared by the following steps:

[0104] a. Mix 800 g of Monascus liquid with a viable bacteria concentration of 1.5×10 8 CFU / mL, 150 g of trehalose, and 5 g of glutamine, oscillate and culture at a temperature of 30°C for 1.5 h, add 5 g of chitosan oligosaccharide with a molecular weight of 2.5 kDa to it and continue to stir for 50 min to obtain the pretreated bacterial cells;

[0105] b. Add 150 g of hydroxypropyl-β-cyclodextrin and 50 g of sodium carboxymethylcellulose to 2000 g of a citric acid-disodium hydrogen phosphate buffer system with a pH of 5 - 6 and a concentration of 0.2 mol / L, stir evenly, add 18 g of methacrylated carboxymethylcellulose sodium and 0.5 g of photo-crosslinking agent to it to obtain the pretreated sol;

[0106] c. The pretreated bacterial liquid and the pretreated sol are formed into microspheres with an average particle size of 200 μm through a coaxial nozzle. The microspheres include the pretreated bacterial liquid and a pretreated sol layer coating the outside of the pretreated bacterial liquid; they are irradiated with 500-nm visible light for 2 min and then vacuum freeze-dried.

[0107] Samples were taken at the end of S2 culture in Example 5 and Comparative Examples 1-3. The total saponin and crude polysaccharide contents of each group of samples were determined. Subsequently, each group of samples was freeze-dried to detect their glucoamylase activity and protease activity.

[0108] As Figure 1 - Figure 2 shown, the total saponin and crude polysaccharide contents of the product fermented by the method of Example 5 are the highest, and the glucoamylase activity and protease activity are also the highest, all superior to those of Comparative Examples 1-3 (P < 0.05).

[0109] 100 healthy Kunming mice (50 males and 50 females, 20 ± 2 g) were randomly divided into 5 groups, namely the control group, the Example 5 group, the Comparative Example 1 group, the Comparative Example 2 group, and the Comparative Example 3 group, with 20 mice in each group. The mice in the Example 5 group were intragastrically administered the product fermented by the method of Example 5, the mice in the Comparative Example 1 group were intragastrically administered the product fermented by the method of Comparative Example 1, the mice in the Comparative Example 2 group were intragastrically administered the product fermented by the method of Comparative Example 2, the mice in the Comparative Example 3 group were intragastrically administered the product fermented by the method of Comparative Example 3, and the control group was given an equal amount of physiological saline by gavage. The daily dose of each group was 10 g / kg bw. After continuous gavage for 7 days, 1 h after the last administration, the mice were placed in a mouse activity counter. After adapting for 4 min, the number of spontaneous activities of each mouse within 10 min was recorded.

[0110] 100 healthy Kunming mice (50 males and 50 females, 20 ± 2 g) were randomly divided into 5 groups, namely the control group, the Example 5 group, the Comparative Example 1 group, the Comparative Example 2 group, and the Comparative Example 3 group, with 20 mice in each group. The mice in the Example 5 group were intragastrically administered the product fermented by the method of Example 5, the mice in the Comparative Example 1 group were intragastrically administered the product fermented by the method of Comparative Example 1, the mice in the Comparative Example 2 group were intragastrically administered the product fermented by the method of Comparative Example 2, the mice in the Comparative Example 3 group were intragastrically administered the product fermented by the method of Comparative Example 3, and the control group was given an equal amount of physiological saline by gavage. The daily dose of each group was 10 g / kg bw. After continuous gavage for 7 days, 1 h after the last administration, the mice were placed on a smooth glass rod with a length of 110 cm and a diameter of 0.7 cm and timed, and the time when the mice slipped off the glass rod due to muscle fatigue was observed.

[0111] As Figure 3 shown, the number of spontaneous activities of the mice in the Example 5 group is the least, and the pole-climbing time is the longest, superior to those of the Comparative Examples 1-3 groups and the control group (P < 0.05).

[0112] 100 healthy Kunming mice (50 males and 50 females, 20±2 g) were randomly divided into 5 groups, namely the control group, Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group, with 20 mice in each group. The mice in the Example 5 group were intragastrically administered with the product fermented by the method of Example 5, the mice in the Comparative Example 1 group were intragastrically administered with the product fermented by the method of Comparative Example 1, the mice in the Comparative Example 2 group were intragastrically administered with the product fermented by the method of Comparative Example 2, the mice in the Comparative Example 3 group were intragastrically administered with the product fermented by the method of Comparative Example 3, and the control group was given an equal amount of normal saline by gavage. The daily dose of each group was 10 g / kg bw. After continuous gavage for 14 days, 1 h after the last administration, each mouse was intraperitoneally injected with 2% sodium nitrite solution (10 mL / kg bw), and the timing was started immediately to compare the survival time of the mice in each group.

[0113] As Figure 4 shown, the survival time of the mice in the Example 5 group was the longest, superior to that of the Comparative Example 1-3 groups and the control group (P < 0.05).

[0114] Through Figure 3 and Figure 4 verification, the product fermented by the method of Example 5 was more capable of reducing the number of spontaneous activities of mice, prolonging the time of pole climbing and hypoxia tolerance of mice, achieving a central sedative effect, effectively enhancing the anti-fatigue effect of the body and the hypoxia tolerance ability of the myocardium.

[0115] The applicant believes that: this is because the present invention uses traditional Chinese medicinal materials in combination with glutinous rice and black rice. Among them, Eucommia ulmoides leaves can effectively inhibit miscellaneous bacteria and effectively maintain the purity of the bacterial flora, while glycyrrhizin can be combined with the exopolysaccharide in the fermentation supernatant of lactic acid bacteria to effectively protect the fermentation system; and the present invention forms microspheres through the combination of hydroxypropyl-β-cyclodextrin / chitosan oligosaccharide composite carriers and photocuring to achieve the slow release and protection of Monascus purpureus, and is compounded with the matrix to improve the survival rate and metabolic activity of the bacteria in the middle and late stages of fermentation. Not only is the fermentation process stable, but it also promotes the proliferation of bacteria, effectively enhances the secondary metabolism, improves the generation of effective active ingredients, and at the same time has strong glucoamylase activity and protease activity, shortening the fermentation cycle by more than 20%. At the same time, the present invention can provide a good medium for the growth of microorganisms, enhance the adaptability of microorganisms to traditional Chinese medicinal materials, effectively maintain the vigorous growth state of the microbial flora during the fermentation cycle, enable the rapid fermentation of traditional Chinese medicinal materials, enhance the oxygen diffusion efficiency through intermittent negative pressure ventilation, and combine with the regulation of the moisture gradient to promote the metabolic conversion of Monascus purpureus from the mycelial growth stage to the spore formation stage, greatly improving the biological activity of the product and being suitable for large-scale production.

[0116] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution of the present invention and its inventive concept, makes equivalent substitutions or changes, and all should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a Chinese medicinal material compound preparation by using red yeast rice fermentation, characterized in that: The steps include: S1, glutinous rice and black rice are mixed evenly, soaked in water for 10-20 hours, taken out and steamed, and then Chinese medicinal materials are added and mixed evenly, and cooled to room temperature to obtain a premixed matrix; The water content of the premixed matrix is ​​adjusted to 60-80%, and the premixed matrix is ​​allowed to stand for 5-10 days in a ventilated environment at a temperature of 25-30° C., sterilized, and lactic acid bacteria fermentation supernatant is added and mixed evenly, and the pH value of the system is adjusted to 4.8-5.2, and the matrix is ​​pasteurized to obtain the matrix; S2. Add Monascus liquid or composite Monascus microspheres to the matrix and mix evenly, then add neutral protease and mix thoroughly, culture at 30-35° C., and the culture humidity is 80-90%; when the water content of the system is lower than 30%, adjust the water content to 60-70%, and add glucose and vinegar dregs; when the water content of the system is lower than 20%, end the culture, turn over and flatten, and vacuum dry.

2. The method for preparing a composite Chinese medicinal material preparation by using red yeast rice fermentation according to claim 1, characterized in that: In S1, the mass ratio of glutinous rice, black rice, Chinese medicinal materials, and lactic acid bacteria fermentation supernatant is 5-15:1-5:10-30:1-2; the lactic acid bacteria fermentation supernatant is prepared by the following specific operations: inoculating lactic acid bacteria in MRS liquid culture medium, culturing at 37°C for 24h, centrifuging at 6000rpm for 10min, and the centrifugation temperature is 4°C.

3. The method for preparing a composite Chinese medicinal material preparation by using red yeast rice fermentation according to claim 1, characterized in that: In S1, the Chinese medicinal materials include: licorice, Poria cocos, Eucommia ulmoides leaves, and Ganoderma lucidum; the mass ratio of licorice, Poria cocos, Eucommia ulmoides leaves, and Ganoderma lucidum is 5-9:4-6:3-7:1-3.

4. The method for preparing a composite Chinese medicinal material preparation by using red yeast rice fermentation according to claim 1, characterized in that: In S2, the concentration of live bacteria in the Monascus liquid is 1-2×10 8 CFU / mL.

5. The method for preparing a composite Chinese medicinal material preparation by using red yeast rice fermentation according to claim 1, characterized in that: The composite Monascus microspheres used in S2 are prepared by the following steps: a. Mix the Monascus liquid, trehalose and glutamine, and culture them under shaking at 28-32°C for 1-2 hours, add chitosan oligosaccharide and continue stirring for 40-60 minutes to obtain a pretreated bacterial liquid; b. Add hydroxypropyl-β-cyclodextrin and sodium carboxymethyl cellulose to a citric acid-disodium hydrogen phosphate buffer system and stir evenly, and add methacrylylated sodium carboxymethyl cellulose and a photocrosslinking agent thereto to obtain a pretreated sol; c. The pretreated bacterial solution and the pretreated sol are formed into microspheres through a coaxial nozzle. The microspheres include the pretreated bacterial solution and the pretreated sol layer covering the outer side of the pretreated bacterial solution. The microspheres are cross-linked by visible light irradiation and vacuum freeze-dried.

6. The method for preparing a composite Chinese medicinal material preparation by using red yeast rice fermentation according to claim 5, characterized in that: The mass ratio of Monascus liquid, trehalose, glutamine, chitosan oligosaccharide, hydroxypropyl-β-cyclodextrin, sodium carboxymethyl cellulose, sodium methacryloyl carboxymethyl cellulose, and photocrosslinking agent is 5-10:1-2:0.01-0.1:0.01-0.1:1-2:0.1-1:0.15-0.2:0.001-0.01; the concentration of live bacteria in Monascus liquid is 1-2×10 8 CFU / mL, the molecular weight of chitosan oligosaccharide is 2-3kDa.

7. The method for preparing a composite Chinese medicinal material preparation by using red yeast rice fermentation according to claim 5, characterized in that: The particle size of the microspheres is 100-300 μm.

8. The method for preparing a composite Chinese medicinal material preparation by using red yeast rice fermentation according to claim 5, characterized in that: The wavelength of visible light is 400-600nm, and the irradiation time is 1-3min.

9. The method for preparing a composite Chinese medicinal material preparation by using red yeast rice fermentation according to claim 1, characterized in that: In S2, the mass ratio of the substrate, Monascus liquid, neutral protease, glucose, and vinegar residue is 20-50:5-10:1-2:1-2:1-2; or, the mass ratio of the substrate, composite Monascus microspheres, neutral protease, glucose, and vinegar residue is 20-50:5-10:1-2:1-2:1-2. 7.5-15：1-2：1-2：1-2。 10. The method for preparing a composite Chinese medicinal material preparation by using red yeast rice fermentation according to claim 1, characterized in that: In S2, after adding neutral protease, intermittent negative pressure ventilation was performed during the culture at 30-35°C, specifically: ventilation at a negative pressure of -0.05 MPa for 1-3 minutes, and then negative pressure ventilation was stopped for 57-59 minutes.

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