Method for solid state fermentation of poria cocos by purple monascus csu-m183 and enzyme coordination

Abstract

The application provides a method for solid-state fermentation of poria cocos by purple monascus CSU-M183 and enzymes, and the method is characterized in that: the method is a biological transformation method, and the traditional Chinese medicine poria cocos is used as raw material, and the poria cocos is subjected to bidirectional solid-state fermentation by enzyme pretreatment and purple monascus CSU-M183 to improve the yield of Monacolin K, water-soluble polysaccharides and triterpenoid compounds, and no citrinin is generated. The method improves the efficient utilization of poria cocos resources, increases the added value of the poria cocos, provides a new processing technology guidance for poria cocos products, and provides a method for saving resources, reducing cost and high safety for high-value comprehensive utilization of the traditional Chinese medicine poria cocos.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological conversion, and particularly relates to a method for solid-state fermentation of poria cocos by using purple red koji CSU-M183 and enzymes. BACKGROUND

[0002] Poria cocos is a common traditional Chinese medicinal material in China, has the effects of promoting water excretion and dampness, calming the heart and invigorating the spleen, and the chemical components mainly include terpenes and polysaccharides, has the pharmacological effects of anti-tumor, blood lipid regulation, anti-virus and anti-oxidation, and is widely used in clinical application. Poria cocos is usually used as a resource for both medicine and food, has good health care function, but more is used in the form of powder or block after drying as medicine or food, the high-value deep processing conversion rate is low, and the efficient use of poria cocos resources is limited. The main components in poria cocos are poria cocos polysaccharide and poria acid, most of the polysaccharide is alkali-soluble polysaccharide, the main component is beta-1, 3 glucan, is difficult to dissolve in water, and has low biological activity, while the content of water-soluble poria cocos polysaccharide (beta-glucan) with high biological activity is low, and the active free terpenes are few due to the combination of terpenes and polysaccharides through chemical bonds, which leads to low bioavailability of poria cocos in the traditional medicine and food mode. The traditional processing methods such as decocting, boiling and simmering can only extract 1%-2% of the active components, and the remaining part including a large amount of alkali-soluble polysaccharide and triterpenoids is discarded as dregs, which causes a large amount of waste of poria cocos resources and limits the development of poria cocos industry.

[0003] At present, through the biotransformation of beneficial microorganisms, probiotics can produce powerful enzymes such as amylase, cellulase, protease, esterase and the like in the fermentation process of Chinese herbal medicine. These enzymes are the material basis for chemical reactions in the fermentation process of Chinese herbal medicine, which can decompose and transform Chinese herbal medicine into new components, and promote the release and transformation of bioactive components in Chinese herbal medicine. At the same time, it can further improve the synthesis of beneficial secondary metabolites of beneficial microorganisms, greatly improve the bioavailability and utilization rate of Chinese herbal medicine, save resources, reduce costs and protect the environment. As a fungus for both medicine and food, Monascus can produce enzymes such as saccharifying enzyme, protease, glucoamylase, pectinase and acetic acid enzyme, and can also produce a large amount of secondary metabolites such as Monacolin K, red pigment, red yeast polysaccharide and gamma-aminobutyric acid, which have the effects of enhancing immunity, antioxidant, anti-tumor, reducing cholesterol, reducing blood lipids, reducing blood pressure and reducing blood sugar. Therefore, the fermentation of Chinese herbal medicine by Monascus can not only produce enzymes to modify and transform the components of Chinese herbal medicine to improve the content of active substances, but also can utilize the nutritional components of Chinese herbal medicine to produce metabolites with biological activity during the growth and metabolism of Monascus. This provides the nutrients required for fungal growth and also decomposes, transforms and even produces new components from the components and organization of the medicinal matrix, so that the final product of fermentation has the effects of both Monascus and Chinese herbal medicine, making the fermentation effect bidirectional. For example, the fermentation product of bidirectional fermentation of Monascus and ginseng has the main active ingredients and functions of medicinal fungus Monascus and ginseng, and has a certain transformation ability for ginsenosides; the content of beta-glucan in the fermentation system of bidirectional fermentation of Monascus and white birch fungus is increased, so that the fermentation product has good blood lipid-lowering activity. Therefore, the use of medicinal and edible fungi such as Monascus for biotransformation of Chinese herbal medicine has good application prospect in the field of Chinese herbal medicine fermentation. However, when using bidirectional fermentation technology of Monascus to biotransform active components of Chinese herbal medicine, it is only directed to a certain active component, and mycotoxin citrinin is produced in the fermentation product, thereby limiting the application of the technology. SUMMARY

[0004] In view of the above problems, the present application provides a method for solid-state fermentation of Poria cocos by purple Monascus CSU-M183 and enzymes, which utilizes biotransformation method to pretreat the enzymes and then cooperates with purple Monascus CSU-M183 for bidirectional solid-state fermentation of Poria cocos to improve the yield of Monacolin K, water-soluble polysaccharides and triterpenoids, and does not produce citrinin. The present application improves the efficient utilization of Poria cocos resources, increases the added value, provides a new processing technology guidance for Poria cocos products, and provides a resource-saving, cost-reducing and safe method for high-value comprehensive utilization of Poria cocos.

[0005] The present application is realized by the following technical solutions.

[0006] In one aspect, the present application provides a purple monascus CSU-M183, characterized in that the monascus M183 is preserved in China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 2018224, located at No. 299, Baoyi Road, Wuchang District, Wuhan City, Hubei Province, China, and a preservation date of April 19, 2018; and a classification name of Monascus purpureus CSU-M183. The purple monascus CSU-M183 as a fermentation strain can simultaneously increase the yields of Monacolin K, water-soluble polysaccharides and triterpenoids in the fermented product of Poria cocos, and does not produce citrinin.

[0007] Preferably, the purple monascus CSU-M183 as a fermentation strain can simultaneously increase the yields of Monacolin K, water-soluble polysaccharides and triterpenoids in the fermented product of Poria cocos to 19 mg / kg, 130 mg / g and 2.6 mg / g or more, respectively, and does not produce citrinin.

[0008] In another aspect, the present application provides a method for simultaneously increasing the yields of Monacolin K, water-soluble polysaccharides and triterpenoids by enzyme-assisted monascus fermentation of Poria cocos, characterized by comprising the following steps:

[0009] (1) Enzyme pretreatment

[0010] After the Poria cocos block is crushed and sieved, Poria cocos powder is obtained, and then enzymes are added to the Poria cocos powder for enzymatic hydrolysis;

[0011] (2) Activation culture of fermentation strain

[0012] The purple monascus CSU-M183 with a preservation number of CCTCC NO: M 2018224 is used as a fermentation strain for PDA plate activation culture, and then a spore suspension is prepared with sterile water or physiological saline and inoculated into a seed culture medium for shaking culture, so as to obtain a monascus seed liquid;

[0013] (3) Preparation of solid-state fermentation medium

[0014] Carbon source, nitrogen source and pure water are added to the Poria cocos powder treated in step (1), and after high-pressure sterilization and cooling, a Poria cocos solid-state fermentation medium is obtained;

[0015] (4) Solid-state fermentation

[0016] The monascus seed liquid of step (2) is inoculated into the Poria cocos solid-state fermentation medium of step (3) for solid-state fermentation, and after the fermentation is completed, the fermentation product is sequentially dried, crushed and sieved, so as to obtain a Poria cocos fermentation product containing Monacolin K, water-soluble polysaccharides and triterpenoids, but not containing citrinin.

[0017] Preferably, in the step (1), the enzyme added is one or more of xylanase, cellulase and beta-glucanase; the enzyme addition amount is 45-75 U / g based on the mass of the Poria cocos powder; the enzymolysis temperature is 30-50 DEG C; and the enzymolysis time is 2-3 h.

[0018] Preferably, in the step (2), the PDA plate activation culture time is 5-7 days.

[0019] Preferably, in the step (2), the concentration of the Monascus spore suspension is controlled to be 1x105-1x107 CFU.

[0020] Preferably, in the step (2), the culture temperature of the Monascus seed liquid is 28-32 DEG C, the culture time is 2-3 days, and the shaking bed rotation speed is 150-200 rpm.

[0021] Preferably, in the step (3), the carbon source is one or more of glucose, starch and sucrose, and the nitrogen source is one or more of yeast powder, beef extract, peptone, sodium nitrate, ammonium chloride, ammonium nitrate, ammonium dihydrogen phosphate and diammonium hydrogen phosphate; the addition amount of the carbon source and the nitrogen source is 1%-5% based on the mass of the Poria cocos powder; and the solid-state fermentation medium is controlled to have a liquid-to-solid ratio of 1:0.5-2 w / v.

[0022] Preferably, in the step (4), the inoculation amount of the Monascus seed liquid to the solid-state substrate is controlled to be 0.4-0.6:1 v / w.

[0023] Preferably, in the step (4), the solid-state fermentation conditions are as follows: static fermentation at 28-32 DEG C for 5-8 days, turning the material every 2-3 days during the fermentation process, and supplementing water to keep the water content of the material at 30%-50% on the 3rd-5th day.

[0024] The present application has the following advantages:

[0025] (1) The present application uses Poria cocos as raw material, and uses biological transformation method to prepare Monacolin K while increasing the yield of water-soluble beta-glucan and triterpenoids such as pachymic acid in Poria cocos by using the related proteases metabolized by Monascus, thereby improving the utilization rate of Poria cocos. The technology makes it possible for the medicinal bacteria produced by the bidirectional solid-state fermentation system of Monascus and enzyme pretreated Poria cocos to have the effect of advantage complementation when they are taken as medicine respectively, thereby improving the utilization of Poria cocos resources.

[0026] (2) In the prior art, the content of active ingredients of Poria cocos resources is improved by conventional enzyme treatment or microbial fermentation method, only the content of water-soluble polysaccharide of Poria cocos is improved, and the yield is low. In addition, the development of Monascus products is limited due to the detection of mycotoxin-citrinin which is harmful to humans and animals in the related products fermented by Monascus. In the present application, Poria cocos is treated by enzyme and cooperated with Monascus purpureus CSU-M183 bidirectional solid-state fermentation, the content of various active ingredients in the fermentation product is significantly increased, through detection, the yield of Monacolin K, water-soluble polysaccharide and triterpenoid compounds in the Poria cocos fermentation product is increased to 19 mg / kg, 130 mg / g and 2.6 mg / g or more respectively by the method of the present application, and no citrinin is produced. In conclusion, the method of the present application has low cost, high safety and good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 HPLC chromatogram of citrinin in different Monascus fermentation products. DETAILED DESCRIPTION

[0028] The present application will be further described in conjunction with specific embodiments. It should be pointed out that the following embodiments are only illustrative explanations of the present application by way of example, but the protection scope of the present application is not limited thereto, and all equivalent substitutions of the present application made by those skilled in the art within the spirit of the present application fall within the protection scope of the present application.

[0029] Example 1

[0030] Monascus purpureus CSU-M183 is obtained by mutagenesis of Monascus purpureus (CICC 5013, purchased from China Industrial Microbial Culture Collection Center) using carbon ion beam (80 MeV / u, 250 Gy) irradiation of Lanzhou Heavy Ion Research Accelerator National Laboratory (Institute of Modern Physics, Chinese Academy of Sciences). 12 C 6 ) irradiation (80 MeV / u, 250 Gy). CSU-M183 colony grows slowly, is pelliculose, surface is dry, has many wrinkles, is raised, sometimes top is cracked, edge is fine and round-toothed, colony color changes from earth red to deep purple red, back is purple red or chestnut color. Conidia are single, double or catenulate, spherical or pear-shaped, surface is smooth, contains particles, sometimes contains oil droplets, orange red or colorless; perithecium is spherical, orange red, a few are colorless, surface is irregular; ascospores are oval or ovoid, surface is smooth, colored or colorless. Saccharifying enzyme is produced, and the activity is high.

[0031] Example 2

[0032] The embodiment provides a method for synchronously improving the production of Monacolin K, water-soluble polysaccharide and triterpenoid compounds by enzyme cooperation and Monascus fermentation of Poria cocos, and the specific steps are as follows:

[0033] (1) Enzyme pretreatment

[0034] The Poria cocos is crushed to pass through a 40-60 mesh sieve to obtain Poria cocos powder; 10 mL of an enzyme solution is prepared by adding xylanase at an addition amount of 45 U / g of the substrate Poria cocos powder into a sodium citrate buffer solution with a pH of 4.5-5.5, and then 20 g of the Poria cocos powder is added to react at 40-50 DEG C for 2-3 h;

[0035] (2) Activation culture of the fermentation strain

[0036] The purple Monascus CSU-M183 is taken out from a test tube stored at 4 DEG C, and is activated and cultured on a PDA plate for 5-7 days; a spore suspension with a concentration of 1x10 5 ~ 1x10 7 CFU is prepared by using sterile water or normal saline, 2 mL of which is inoculated into a seed culture medium, and the seed culture is carried out under the conditions that the culture temperature is 28-32 DEG C, the culture time is 2-3 days, and the rotation speed of a shaking table is 150-200 rpm, so that a Monascus seed liquid is obtained; wherein the seed culture medium formula is corn starch 30 g / L, sodium nitrate 3 g / L, FeSO4.7H2O 0.01 g / L, KH2PO4 4 g / L, and the pH is natural;

[0037] (3) Preparation of a solid-state fermentation medium

[0038] The carbon source, the nitrogen source and pure water are added to the Poria cocos powder treated in the step (1), and the solid-state fermentation medium is controlled to have a ratio of solid to liquid of 1:0.5 w / v; wherein the solid-state fermentation medium uses glucose as the carbon source and uses beef extract as the nitrogen source, and the addition amount of the carbon source and the nitrogen source is 1% based on the mass of the Poria cocos powder; after high-pressure sterilization at 121 DEG C for 30 min, the solid-state fermentation medium is cooled to obtain the solid-state fermentation medium;

[0039] (4) Solid-state fermentation

[0040] The solid-state fermentation medium is inoculated with the Monascus seed liquid at a ratio of 0.4:1 v / w, and is statically fermented at 28-32 DEG C for 6-8 days, and is turned every 2-3 days; water is supplemented on the 3rd to 5th day to keep the water content of the material at 30%-50%; and after the fermentation is completed, the product is dried at 50-60 DEG C for 12-24 h, is crushed to pass through a 40-60 mesh sieve, and Poria cocos fermentation products are obtained.

[0041] Example 3

[0042] The embodiment provides a method for synchronously improving the production of Monacolin K, water-soluble polysaccharide and triterpenoid compounds by enzyme cooperation and Monascus fermentation of Poria cocos, and the specific steps are as follows:

[0043] (1) Enzyme pretreatment

[0044] The Poria cocos is crushed to pass through a 40-60 mesh sieve to obtain Poria cocos powder; 10 mL of enzyme solution is prepared by adding the cellulase at an addition amount of 60 U / g to a sodium citrate buffer solution with a pH of 4.5-5.5, and then 20 g of the Poria cocos powder is added to the enzyme solution for reaction at 40-50 DEG C for 2-3 h, with the mass of the substrate Poria cocos powder being taken as the basis;

[0045] (2) Activation culture of the fermentation strain

[0046] The Monascus purpureus CSU-M183 is taken out from a test tube stored at 4 DEG C and activated on a PDA plate for 5-7 days, and the Monascus purpureus CSU-M183 is taken out from a test tube stored at 4 DEG C and activated on a PDA plate for 5-7 days, a spore suspension with a concentration of 1x10 5 ~1x10 7 CFU is prepared with sterile water or normal saline, 2 mL of which is inoculated into a seed culture medium, and the seed culture is carried out under the conditions that the culture temperature is 28-32 DEG C, the culture time is 2-3 days, and the shaking speed is 150-200 rpm, so as to obtain a Monascus seed liquid; the seed culture medium has the same formula as that in Embodiment 2.

[0047] (3) Preparation of a solid-state fermentation medium

[0048] The carbon source, the nitrogen source and pure water are added to the Poria cocos powder treated in step (1), and the solid-state fermentation medium is prepared by controlling the ratio of the solid-state fermentation medium to the liquid to be 1:2 w / v, wherein the solid-state fermentation medium uses starch as the carbon source and uses yeast powder as the nitrogen source, and the addition amount of the carbon source and the nitrogen source is 5% based on the mass of the Poria cocos powder; after high-pressure sterilization at 121 DEG C for 30 min, the solid-state fermentation medium is cooled to obtain the solid-state fermentation medium;

[0049] (4) Solid-state fermentation

[0050] The solid-state fermentation medium is inoculated with the Monascus seed liquid at a ratio of 0.6:1 v / w, and is statically fermented at 28-32 DEG C for 6-8 days, and is turned over every 2-3 days, and water is supplemented to keep the water content of the material to be 30%-50% on the 3rd to 5th day. After the fermentation is completed, the product is dried at 50-60 DEG C for 12-24 h, and is crushed to pass through a 40-60 mesh sieve to obtain a Poria cocos fermentation product.

[0051] Embodiment 4

[0052] The embodiment provides a method for synchronously improving the production of Monacolin K, water-soluble polysaccharide and triterpenoid compounds by enzyme cooperation and Monascus fermentation of Poria cocos, and the specific steps are as follows:

[0053] (1) Enzyme pretreatment

[0054] The Poria cocos is crushed to pass through a 40-60 mesh sieve to obtain Poria cocos powder; 10 mL of enzyme solution is prepared by adding cellulase at an addition amount of 75 U / g of the mass of the substrate Poria cocos powder into a sodium citrate buffer solution with a pH of 4.5-5.5, and then 20 g of the Poria cocos powder is added to react at 40-50 DEG C for 2-3 h;

[0055] (2) Activation culture of the fermentation strain

[0056] The purple Monascus CSU-M183 is taken out from a test tube stored at 4 DEG C and activated on a PDA plate for 5-7 days, a spore suspension with a concentration of 1x10 5 ~ 1x10 7 CFU is prepared with sterile water or normal saline, 2 mL of which is inoculated into a seed culture medium, and seed culture is carried out under the conditions that the culture temperature is 28-32 DEG C, the culture time is 2-3 days, and the shaking speed is 150-200 rpm, so that a Monascus seed liquid is obtained; the seed culture medium formula is the same as that in Embodiment 2;

[0057] (3) Preparation of a solid-state fermentation medium

[0058] The carbon source, the nitrogen source and pure water are added to the Poria cocos powder treated in step (1), and the solid-state fermentation medium is prepared by controlling the ratio of the solid-state fermentation medium to the liquid to be 0.75:2 w / v, wherein sucrose is used as the carbon source and peptone is used as the nitrogen source, and the addition amount of the carbon source and the nitrogen source is 3% based on the mass of the Poria cocos powder; after high-pressure sterilization at 121 DEG C for 30 min, the solid-state fermentation medium is obtained after cooling;

[0059] (4) Solid-state fermentation

[0060] The solid-state fermentation medium is inoculated with the Monascus seed liquid at a ratio of 0.5:1 v / w, and is placed for fermentation at 28-32 DEG C for 6-8 days, and is turned over every 2-3 days; water is supplemented on the 3rd-5th day to keep the water content of the material to be 30%-50%. After the fermentation is completed, the product is dried at 50-60 DEG C for 12-24 h, and is crushed to pass through a 40-60 mesh sieve to obtain a Poria cocos fermentation product.

[0061] Comparative Example 1

[0062] The present comparative example provides a conventional Monascus fermentation method of Poria cocos, which adopts the method of Example 2, but does not perform enzyme pretreatment, and uses Monascus anka mutant MYM2 (CGMCC 2275) as the fermentation strain. The specific steps are as follows:

[0063] (1) Activation culture of the fermentation strain

[0064] The Monascus anka mutant MYM2 (CGMCC 2275) preserved in the laboratory was taken out from the test tube stored at 4°C and activated on a PDA plate for 5-7 days. A spore suspension with a concentration of 1 x 10 5 ~ 1 x 10 7 CFU was prepared with sterile water or normal saline, 2 mL of which was inoculated into the seed culture medium. The seed culture was performed in a shaking incubator under the conditions of a culture temperature of 28-32°C, a culture time of 2-3 days, and a shaking speed of 150-200 rpm, to obtain the Monascus seed liquid. The seed culture medium formula is the same as that of Example 2.

[0065] (2) Preparation of the solid-state fermentation medium

[0066] Poria cocos was ground through a 40-60 mesh sieve to obtain Poria cocos powder. Carbon source, nitrogen source, and pure water were added to the Poria cocos powder, and the solid-state fermentation medium was prepared by controlling the solid-liquid ratio to be 1:0.5 w / v. Glucose was used as the carbon source, and beef extract was used as the nitrogen source. The addition amount of the carbon source and the nitrogen source was 1% based on the mass of the Poria cocos powder. After high-pressure sterilization at 121°C for 30 min, the solid-state fermentation medium was cooled to obtain the solid-state fermentation medium.

[0067] (3) Solid-state fermentation

[0068] The solid-state fermentation medium was inoculated with the Monascus seed liquid at a ratio of 0.4:1 v / w, and was statically fermented at 28-32°C for 6-8 days. The material was turned every 2-3 days, and water was supplemented on the 3rd-5th day to maintain the water content of the material at 30%-50%. After the fermentation was completed, the product was dried at 50-60°C for 12-24 h, ground through a 40-60 mesh sieve, to obtain the Poria cocos fermentation product.

[0069] Comparative Example 2

[0070] The present comparative example provides a conventional Monascus fermentation method of Poria cocos, which adopts the method of Example 2, but does not perform enzyme pretreatment, and uses Monascus anka mutant MYM2 (CGMCC 2275) as the fermentation strain. The specific steps are as follows:

[0071] (1) Activation culture of the fermentation strain

[0072] Monascus purpureus LQ-6 (CCTCC: NO: M2018600) preserved in our laboratory was taken from the test tube stored at 4°C and activated on PDA plates for 5-7 days. A spore suspension with a concentration of 1 x 10 5 ~ 1 x 10 7 CFU was prepared with sterile water or normal saline, 2 mL of which was inoculated into the seed culture medium. The seed culture was obtained by shaking culture under the conditions of a culture temperature of 28-32°C, a culture time of 2-3 days, and a shaking speed of 150-200 rpm. The seed culture medium was prepared according to Example 2.

[0073] (2) Preparation of the solid-state fermentation medium

[0074] Poria cocos was ground through a 40-60 mesh sieve to obtain Poria cocos powder. Carbon and nitrogen sources and pure water were added to the Poria cocos powder. The solid-state fermentation medium was prepared by adding glucose as the carbon source and beef extract as the nitrogen source, with the addition amount of each being 1% based on the mass of the Poria cocos powder. The solid-state fermentation medium was sterilized at 121°C for 30 min and then cooled to obtain the solid-state fermentation medium.

[0075] (3) Solid-state fermentation

[0076] The solid-state fermentation medium was inoculated with the seed culture of Monascus purpureus at a ratio of 0.4:1 v / w, and then fermented at 28-32°C for 6-8 days. The medium was stirred every 2-3 days, and water was added on the 3rd-5th day to maintain the water content of the medium at 30%-50%. The product was dried at 50-60°C for 12-24 h, ground through a 40-60 mesh sieve, and then obtained as the fermentation product of Poria cocos.

[0077] Comparative Example 3

[0078] This comparative example provides a method for fermenting Poria cocos with conventional Monascus purpureus, which is prepared according to the method of Example 2, but without enzyme pretreatment. The specific steps are as follows:

[0079] (1) Activation culture of the fermentation strain

[0080] Monascus purpureus CSU-M183 was taken from the test tube stored at 4°C and activated on PDA plates for 5-7 days. A spore suspension with a concentration of 1 x 10 5 ~ 1 x 10 7 CFU was prepared with sterile water or normal saline, 2 mL of which was inoculated into the seed culture medium. The seed culture was obtained by shaking culture under the conditions of a culture temperature of 28-32°C, a culture time of 2-3 days, and a shaking speed of 150-200 rpm. The seed culture medium was prepared according to Example 2.

[0081] (2) Preparation of solid-state fermentation medium

[0082] The poria cocos is crushed to pass through a 40-60 mesh sieve to obtain poria cocos powder; a carbon source, a nitrogen source, and pure water are added to the poria cocos powder, and the solid-state fermentation medium is prepared by controlling the solid-liquid ratio to be 1:0.5 w / v, wherein the solid-state fermentation medium uses glucose as the carbon source and beef extract as the nitrogen source, and the addition amount of the carbon source and the nitrogen source is 1% based on the mass of the poria cocos powder; the solid-state fermentation medium is sterilized at 121°C for 30 min under high pressure and then cooled to obtain the solid-state fermentation medium.

[0083] (3) Solid-state fermentation

[0084] The solid-state fermentation medium is inoculated with the red yeast seed liquid at a ratio of 0.4:1 v / w, and then fermented at 28-32°C for 6-8 days, and the material is turned over every 2-3 days; water is added to maintain the water content of the material at 30%-50% during the 3rd to 5th day; and the product is dried at 50-60°C for 12-24 h, crushed to pass through a 40-60 mesh sieve, and obtained as the poria cocos fermentation product.

[0085] Comparative Example 4

[0086] The present comparative example provides a method for conventional enzymatic poria cocos, and the specific steps are as follows: the poria cocos is crushed to pass through a 40-60 mesh sieve to obtain poria cocos powder; 10 mL of enzyme solution is prepared by adding xylanase at an addition amount of 60 U / g to a pH 4.5-5.5 sodium citrate buffer, and then added to 20 g of poria cocos powder to react at 40-50°C for 2-3 h; and then the enzyme is inactivated in a 100°C water bath for 1-10 min, and finally dried to obtain the enzymatic poria cocos powder.

[0087] Example 4

[0088] The content of active ingredients and citrinin in the poria cocos powder treated by different methods.

[0089] 1. Experimental method and results

[0090] The poria cocos powder without any treatment is used as the Ck group, and the products treated by Examples 2-4 and Comparative Examples 1-3 are detected for the content of water-soluble polysaccharides, total triterpenes, poricoic acid, Monacolin K, and citrinin, respectively.

[0091] The water-soluble polysaccharides are detected as follows: 0.5 g of sample is added to 20 mL of pure water, heated in a 80°C water bath for 2 h, centrifuged at 8000 r / min for 10 min at 4°C, and the supernatant is taken as the water-soluble polysaccharide detection solution; and the polysaccharides are determined by the phenol-sulfuric acid method at 490 nm.

[0092] Detection of total triterpenoids and pachymic acid: 0.5 g of sample was added into anhydrous methanol at 1:30 (w / v) and placed in a 60°C water bath for 1 h, then centrifuged at 8000 r / min for 10 min at 4°C, and the supernatant was rotary evaporated at 45°C. The sample was dissolved in methanol to obtain the test solution. The total triterpenoids were determined by vanillin-perchloric acid colorimetry at 548 nm. The pachymic acid was determined by high performance liquid chromatography (HPLC). The test solution was filtered through a 0.22 μm organic membrane and analyzed. The specific analysis conditions were as follows: a HPLC system with a UV detector, an absorption wavelength of 208 nm, a column temperature of 30°C, and a C18 chromatographic column. The mobile phase was A: acetonitrile and B: 0.1% phosphoric acid aqueous solution, and the flow rate was 1.0 mL / min. Gradient elution was performed as follows: 0-5 min, 60% A-70% A; 5-10 min, 70% A-80% A; 10-15 min, 80% A-85%; and 15-25 min, 85% A (all by volume).

[0093] Detection of monacolin K and citrinin: 0.3 g of sample was added into 75% ethanol at 1:30 (w / v) and ultrasonicated at room temperature for 30 min, then oscillated at 200 rpm for 30 min, centrifuged at 8000 r / min for 10 min at 4°C, and the supernatant was filtered through a 0.22 μm organic membrane and analyzed by HPLC. The analysis conditions for monacolin K were as follows: a HPLC system with a UV detector, an absorption wavelength of 238 nm, a column temperature of 30°C, a C18 chromatographic column, and a mobile phase of acetonitrile: pure water: phosphoric acid = 650:350:0.6 (v / v) at a flow rate of 1.0 mL / min. Isocratic elution was performed as follows: 0-15 min, 100% mobile phase. The analysis conditions for citrinin were as follows: a HPLC system with a fluorescence detector, an excitation wavelength of 350 nm, an emission wavelength of 500 nm, a column temperature of 30°C, a C18 chromatographic column, and a mobile phase of acetonitrile: isopropyl alcohol: phosphoric acid solution = 350:100:550 (v / v) at a flow rate of 1.0 mL / min. Isocratic elution was performed as follows: 0-20 min, 100% mobile phase.

[0094] 2. Result analysis

[0095] The results of determination of active ingredients and citrinin content in Poria cocos powder after treatment of Examples 2-4 and Comparative Examples 1-4 are shown in Table 1. The HPLC chromatograms of citrinin in different Monascus fermentation products are shown in Figure 1 , Figure 1 MYM2 is Comparative Example 1, LQ-6 is Comparative Example 2, M183 is Comparative Example 3, and enzyme+M183 is Example 1.

[0096] Table 1 Determination of active ingredients and citrinin content in Poria cocos powder after treatment of Examples 2-4 and Comparative Examples 1-4

[0097]

[0098]

[0099] From Table 1 and Figure 1 It can be seen that, in the comparative examples 1 and 2, the water-soluble polysaccharide and the pachymic acid content are reduced after the fermentation of Poria cocos by the Monascus MYM2 and the Monascus LQ-6 respectively, the total triterpenoid content changes insignificantly, but the citrinin content in the fermentation product is detected, which is far more than the national limited standard by 79 times and 35.5 times respectively. In the comparative example 3, the water-soluble polysaccharide, the total triterpenoid and the pachymic acid content are significantly improved after the fermentation of Poria cocos by the Monascus M183 without the enzyme treatment, and the citrinin content is not detected, which indicates that the fermentation of Poria cocos by the self-made Monascus in the application has a significant effect, the product has high safety, and has a significant advantage compared with other Monascus.

[0100] From Table 1, compared with the comparative example 3 using only the Monascus M183 as the fermentation strain or the comparative example 4 using only the enzyme treatment, the method (the examples 2-4) of the application uses the enzyme to ferment Poria cocos in cooperation with the Monascus M183, the enzyme pretreatment of Poria cocos has a promoting effect on the growth of the Monascus M183 and the accumulation of the secondary metabolite Monacolin K, the Monacolin K content can reach 17.62-25.45 mg / kg, at the same time, the water-soluble polysaccharide content in the product is increased by about 443%-456% compared with that before the fermentation, the total triterpenoid content is increased by about 191%-287% compared with that before the fermentation, and the pachymic acid is increased by about 30%-83% compared with that before the fermentation, so as to achieve the purpose of the enzyme cooperating with the Monascus M183 to ferment Poria cocos in two directions to synchronously increase the active ingredient content of the fermentation product, and the mycotoxin citrinin is not detected, which meets the quantitative limit of 80 μg / kg of the Monascus and its products in the “Determination of Citrinin in Foods” (GB 5009.222-2016).

[0101] In summary, the Monascus M183 created in the laboratory has a significant advantage in the solid-state fermentation of Poria cocos compared with other Monascus, the active ingredient content of the fermentation product is significantly increased and has high safety, the enzyme is further used to pretreat the Poria cocos powder, which damages the cell wall of Poria cocos to a certain extent, promotes the dissolution of polysaccharide, and further improves the content of the active substance in Poria cocos in cooperation with the Monascus M183 fermentation, so that the prepared Monascus Poria cocos powder has stronger pharmacological activity and higher application value. It is indicated that the method can effectively promote the high-value utilization of Poria cocos resources, and the method has the advantages of simple operation, low cost, high safety, good color of the product, and great application potential in the food or health care field.

Claims

1. A method for simultaneously increasing the yields of Monacolin K, water-soluble polysaccharides, and triterpenoids in Poria cocos fermentation using enzyme-synergistic fermentation with Monascus purpureus, characterized in that, Includes the following steps: (1) Enzyme pretreatment After crushing the Poria cocos pieces and sieving them, Poria cocos powder is obtained. Then, xylanase or cellulase is added to the Poria cocos powder for enzymatic hydrolysis. (2) Activation culture of fermentation strains The purple Monascus purpureus with accession number CCTCC NO: M 2018224 ( Monascus purpureus CSU-M183 was used as the fermentation strain for PDA plate activation culture, and then a spore suspension was prepared with sterile water or physiological saline and inoculated into the seed culture medium for shake culture to obtain purple Monascus seed liquid. (3) Preparation of solid-state fermentation culture medium Add carbon source, nitrogen source and pure water to the Poria cocos powder after step (1), and then sterilize and cool it under high pressure to obtain the Poria cocos solid fermentation culture medium. (4) Solid-state fermentation The purple Monascus seed liquid from step (2) was inoculated into the solid fermentation medium of Poria cocos in step (3) for solid fermentation. After the fermentation was completed, the fermentation product was dried, crushed and sieved in sequence to obtain the Poria cocos fermentation product containing Monacolin K, water-soluble polysaccharides and triterpenoids, but without citrinin.

2. The method for simultaneously increasing the yields of Monacolin K, water-soluble polysaccharides, and triterpenoids in Poria cocos fermentation with enzyme synergistic effect as described in claim 1, characterized in that, In step (1), the amount of enzyme added is 45-75 U / g based on the mass of the substrate Poria cocos powder; the enzymatic hydrolysis temperature is 30-50℃; and the enzymatic hydrolysis time is 2-3h.

3. The method for simultaneously increasing the yield of Monacolin K, water-soluble polysaccharides, and triterpenoids in Poria cocos fermentation with enzyme synergistic with Monascus purpureus as described in claim 1, characterized in that, In step (2), the PDA plate activation culture time is 5 to 7 days.

4. The method for simultaneously increasing the yield of Monacolin K, water-soluble polysaccharides, and triterpenoids in Poria cocos fermentation with enzyme synergistic effect as described in claim 1, characterized in that, In step (2), the concentration of the purple Monascus spore suspension is controlled to be 1×10⁻⁶. 5 ~1×10 7 CFU.

5. The method for simultaneously increasing the yield of Monacolin K, water-soluble polysaccharides, and triterpenoids in Poria cocos fermentation with enzyme synergistic effect as described in claim 1, characterized in that, In step (2), the culture temperature of the purple Monascus seed liquid is 28-32℃, the culture time is 2-3 days, and the shaking speed is 150-200 rpm.

6. The method for simultaneously increasing the yield of Monacolin K, water-soluble polysaccharides, and triterpenoids in Poria cocos fermentation with enzyme synergistic with Monascus purpureus as described in claim 1, characterized in that, In step (3), the carbon source is one or more of glucose, starch, and sucrose, and the nitrogen source is one or more of yeast powder, beef extract, peptone, sodium nitrate, ammonium chloride, ammonium nitrate, ammonium dihydrogen phosphate, ammonium sulfate, and diammonium hydrogen phosphate; the amount of carbon source and nitrogen source added is 1% to 5% based on the mass of Poria cocos powder; and the material-to-liquid ratio in the solid fermentation medium is controlled to be 1:0.5 to 2 w / v.

7. The method for simultaneously increasing the yield of Monacolin K, water-soluble polysaccharides, and triterpenoids in Poria cocos fermentation with enzyme synergistic effect as described in claim 1, characterized in that, In step (4), the ratio of the inoculum amount of purple Monascus seed liquid to the solid substrate is controlled to be 0.4-0.6:1v / w.

8. The method for simultaneously increasing the yields of Monacolin K, water-soluble polysaccharides, and triterpenoids in Poria cocos fermentation with enzyme synergistic with Monascus purpureus as described in claim 1, characterized in that, In step (4), the solid-state fermentation conditions are: static fermentation at 28-32℃ for 5-8 days, turning the material every 2-3 days during the fermentation process, and adding water on the 3rd-5th day to keep the moisture content of the material at 30%-50%.

Images