CN104388322A - Solid state fermentation method of lipid-lowering Monascus purpureus Zhang-MP - Google Patents
Solid state fermentation method of lipid-lowering Monascus purpureus Zhang-MP Download PDFInfo
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- CN104388322A CN104388322A CN201410743264.6A CN201410743264A CN104388322A CN 104388322 A CN104388322 A CN 104388322A CN 201410743264 A CN201410743264 A CN 201410743264A CN 104388322 A CN104388322 A CN 104388322A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- C12R2001/645—Fungi ; Processes using fungi
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
Abstract
The invention relates to a solid state fermentation method of lipid-lowering Monascus purpureus Zhang-MP, belonging to the technical field of microbes. Monascus purpureus Zhang-MP CGMCC No.9221 is activated, and subjected to amplification culture, solid state fermentation and other steps to prepare red yeast rice containing rich Monacolin K. By using a nonglutinous rice-tartary buckwheat mixture as a basic fermentation culture medium, optimizing the solid state fermentation culture medium formula and adding the glucose, 501 rapeseed peptone and ZnSO4 as a carbon source, a nitrogen source and an inorganic salt for high-yield production of Monacolin K by Monascus purpureus P-MP fermentation, the synthesis yield of the Monacolin K from Monascus purpureus Zhang-M is enhanced by 2.56 times as compared with the Monacolin K yield before optimization; and the effects of the buckwheat and red yeast rice are combined, No notalin is detected in the red yeast rice fermented from Monascus purpureus Zhang-MP.
Description
Technical field
The present invention relates to the method for the red colouring agent for food, also used as a Chinese medicine solid state fermentation of microbial technology field, utilize solid state substrate cheap and easy to get for raw material, produce red colouring agent for food, also used as a Chinese medicine physiologically active substance by biological fermentation process---Monacolin K, thus realize high value added product conversion.
Background technology
Red colouring agent for food, also used as a Chinese medicine is mainly used in brewageing the aspects such as red wine, leavened food, anti-corrosive fresh-keeping, food color and medicine.Red colouring agent for food, also used as a Chinese medicine is referred to as red bent ancient times, is a kind of medicine-food two-purpose traditional Chinese medicine, is inoculated in rice is prepared from through fermentation by Monascus fungus.In ancient times in many pharmacopeia, red colouring agent for food, also used as a Chinese medicine has the effects such as promoting blood circulation and removing blood stasis, strengthening the spleen to promote digestion, is used for the treatment of that dyspepsia is glutted, the disease such as post partum longtime lochia, stasis stomachache and wound.From ancient times a lot of to the applied research of red colouring agent for food, also used as a Chinese medicine, and the research of pharmacology, medicineization is less.Until Japanese scholars Teng Zhang doctor Endo far away in 1976 has found trace from the meta-bolites of 1 strain from the monascus purpureus (Monascus purpureus) of China, after the material Monacolin K that can reduce human serum cholesterol, just extensively cause World Focusing, and start the in-depth research of red colouring agent for food, also used as a Chinese medicine.Modern science proves: have the natural statin substance that can reduce serum cholesterol in the meta-bolites of red colouring agent for food, also used as a Chinese medicine, particularly containing lovastatin (a kind of active substance with remarkable blood lipid regulation ability, i.e. Monacolin K), many compositions useful to human body can also be produced, as indispensable amino acid, unsaturated fatty acids etc. simultaneously.Large quantity research finds the remarkable comprehensive therapeutic effect that it has very powerful reduction total cholesterol, reduces low density lipoprotein cholesterol, reduces serum triglyceride, reduces atherogenic index, high density lipoprotein increasing cholesterol, and take that security is high, side effect is little, and effectively can treat the cardiovascular and cerebrovascular diseases such as coronary heart disease, cerebral apoplexy and the disease relevant to hyperlipidemia, as diabetes, nephrotic syndrome and fatty liver.At present, China is that the treatment cardiovascular and cerebrovascular diseases medicament that main raw material makes has become the best seller of European & American Market with red colouring agent for food, also used as a Chinese medicine.
Radix Et Rhizoma Fagopyri Tatarici is buckwheat dicotyledons, formal name used at school hull buckwheat, and be a kind of exclusive ancient crop of China, buckwheat originates from China, and cultivation history is long.Radix Et Rhizoma Fagopyri Tatarici is a herb, has the effects such as the treatment cold of insufficiency type, hypertension, coronary heart disease.Radix Et Rhizoma Fagopyri Tatarici, containing a large amount of flavonoid compounds, is especially rich in rutin, has the tension force maintaining capillary vessel, reduce its permeability and fragility, promote hyperplasia and prevent the aggegation of hemocyte, reducing blood-fat in addition, coronary artery dilator, strengthens the effects such as coronary artery blood flow.In buckwheat flour, contained abundant supporting one's family have the effect reducing human body blood fat and cholesterol, is treatment hypertension, the important ancillary drug of cardiovascular diseases.In addition, containing some trace elements in buckwheat flour, as magnesium, iron, copper, potassium etc. have provide protection for cardiovascular.
At present, the domestic report having a lot of patent to relate to red colouring agent for food, also used as a Chinese medicine solid state fermentation production Monacolin K, mainly studies production technique aspect.There is a small amount of patent to optimize the composition of solid fermentation culture medium, thus determine that red colouring agent for food, also used as a Chinese medicine produces Monacolin K optimum medium matter.Such as Southern Yangtze University is permitted the method for carrier Monascus anka Nakazawa et sato fermentative production Mo Nakelin K " a kind of be with solid state substrate " (CN102911977 A) of flourish, the thin rich application of gentle of Lu Li, its fermentation difficult water-soluble inert support of base matrix or granular cereals material, and with nutritive medium by certain mass than mixing, cultivate after inoculation; " a kind of preparation method of coix seed red yeast rice " (CN102429928 A) of people's applications such as Chengdu Ge Lanyang biological medicine Science and Technology Ltd. Zou Kun and Chen Shufeng is using the seed of Job's tears as raw material, and the access red colouring agent for food, also used as a Chinese medicine sort of quyi, through preparing red colouring agent for food, also used as a Chinese medicine process and obtaining; " a kind of making method of tartary buckwheat monascus and application thereof " (CN 102429163 A) fermented substrate based on bitter buckwheat of Hangzhou Twin-Horse Biological Engineering Co., Ltd. Gan Feng, week vertical gentle Feng Jianping application, add a large amount of carbon sources, nitrogenous source and trace element, to improve the content of active substance Monacolin K in product.But about utilizing long-grained nonglutinous rice and buckwheat mixed-matrix as fermention medium, and more additional nutritive ingredients, comprising a kind of novel nitrogen source (rapeseed protein peptone), there is not yet Patents and bibliographical information.This kind of fermention medium not only increases the output of Monacolin K, and containing distinctive functional ingredient in buckwheat.
Summary of the invention
The object of the present invention is to provide the process for solid state fermentation of the monascus purpureus of a kind of high yield Monacolin K.
The present invention is achieved by the following technical solutions:
(1) monascus purpureus Zhang-MP bacterial classification
Monascus purpureus (Monascus purpureus) Zhang-MP applied in described method is Laboratories Accession bacterial classification, and being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 12nd, 2014, preserving number is: CGMCC No.9221.In MEA substratum, cultivate 7d, colony diameter 30 ~ 32mm for 25 DEG C, color swallow chin is red, and bacterium colony quality is velvet shape, and the irregular branch of mycelia, has tabula, wall has orange crystal, and width is 4 ~ 5.5 μm; The single raw or chain life of conidium, raw on mycelia top or side shoot top, falls pyriform to spherical; Thecaspore ovalize.
(2) monascus purpureus Zhang-MP fermention medium is prepared
In described method, monascus purpureus Zhang-MP solid-state fermentation culture medium formula is: substratum based on long-grained nonglutinous rice 70%+ buckwheat 30%, and adds 0.1% glucose, 0.3%501 rapeseed protein peptone and 0.04%ZnSO
4.
Long-grained nonglutinous rice and Radix Et Rhizoma Fagopyri Tatarici are cleaned, drains after soaking 24h; Take 35g long-grained nonglutinous rice and 15g Radix Et Rhizoma Fagopyri Tatarici is mixed in 250mL triangular flask, add 5g glucose, 1.5g 501 rapeseed protein peptone and 2g ZnSO
4, the moisture content to 50% of adjustment substratum, puts into high-pressure steam sterilizing pan 0.1MPa, sterilizing 20min with gauze after sealing.
(3) fermentation culture
In described method, the technological condition for fermentation of monascus purpureus is: after red koji fermentation medium sterilization, sterile glass rod is utilized to be broken up by culture medium, after cooling, by spore suspension in the ratio access solid-state fermentation culture medium of 6% (v/v), 12 ~ 15d is cultivated in 28 DEG C, every 3d turns once therebetween, treats that solid medium is that namely red-purple obtains cultivating ripe Red kojic rice.
(5) dry
In described method, the sample after fermentation ends is dried at 55 DEG C, and pulverizes 80 mesh sieves, namely obtains the red colouring agent for food, also used as a Chinese medicine powder-product being rich in Monacolin K.
The process for solid state fermentation of the monascus purpureus of the high yield Monacolin K that aforesaid method obtains, and patent bacterial classification belongs to scope.
Embodiment
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Percentage composition in following embodiment, if no special instructions, is the percentage composition of weight to weight.
The best process for solid state fermentation of embodiment 1, monascus purpureus Zhang-MP high yield Monacolin K
1, the preparation of purplish red aspergillus spore suspension
(1) separation of Monascus anka Nakazawa et sato
Adopt dilution spread flat band method.Take Red kojic rice 1g, put into 9ml sterile saline, vibrate about 10 minutes, make spore be scattered in water, make 10
-1bacteria suspension.Therefrom get 1mL to add in 9mL sterile saline, fully vibrate, make 10
-2bacteria suspension.Do 2 dilutions with 10 times of extension rates successively again, totally 4 extent of dilution, are respectively 10,100,1000,10000 times.Each extent of dilution gets the bacteria suspension of 0.1mL, adds in wort agar substratum, even spread, is inverted in by culture dish in 28 DEG C of incubators, cultivates 2 ~ 6d, until produce early stage bacterium colony for white, the later stage is red, in fine hair felted single bacterium colony.
(2) cultivation of Monascus anka Nakazawa et sato
Choose the mono-bacterium colony of purplish red bent Zhang-MP that slat chain conveyor obtains, aseptic inoculation, on wort agar slant medium, is cultivated 7d, is put 4 DEG C of Refrigerator stores for 28 DEG C, for subsequent use.
(3) preparation of Monascus anka Nakazawa et sato spore suspension
Choose the inclined-plane grown fine, by stroke-physiological saline solution by spore wash-out gently, double-layer sterile filtered through gauze, fully vibrates, and is prepared into homogeneous spore suspension, stand-by.Spore suspension concentration is measured with blood counting chamber.
(4) seed liquor is prepared
Spore suspension liquid is linked into seed culture medium (seed culture based formulas: glucose 60g, peptone 30g, NaNO in the ratio of 10% (v/v)
32g, MgSO
41g, water 1000mL) in, in 28 DEG C, 250r/min aerlbic culture 3d, obtains seed liquor.
2, the preparation of monascus purpureus Zhang-MP fermention medium
Long-grained nonglutinous rice and Radix Et Rhizoma Fagopyri Tatarici are cleaned, drains after soaking 24h; Take 35g long-grained nonglutinous rice and 15g Radix Et Rhizoma Fagopyri Tatarici is mixed in 250mL triangular flask, add 5g glucose, 1.5g 501 rapeseed protein peptone and 2g ZnSO
4, the moisture content to 50% of adjustment substratum, puts into high-pressure steam sterilizing pan 0.1MPa, sterilizing 20min with gauze after sealing.
3, purplish red koji fermentation processing condition
After red koji fermentation medium sterilization, sterile glass rod is utilized to be broken up by culture medium, after cooling, by spore suspension in the ratio access solid-state fermentation culture medium of 6% (v/v), 12 ~ 15d is cultivated in 28 DEG C, every 3d turns once therebetween, treats that solid medium is that namely red-purple obtains cultivating ripe Red kojic rice.
4, the mensuration of Monacolin K content
Get the Red kojic rice of oven dry, precision takes 1g, puts in 200mL volumetric flask, adds ethanolic soln and is diluted to scale, shakes up, and soak 8h, the centrifugal 20min of 3000r/min, gets supernatant liquor, suitably dilutes, be placed in 10mL colorimetric cylinder, adopt the outer spectrophotometer of 752 types to measure absorbancy under 238nm ultraviolet wavelength, look into the content that typical curve calculates Monacolin K, test parallel three times.It is appropriate that another precision takes standard substance (Monacolin K standard substance are purchased from SIGMA company), adds dissolve with ethanol and be quantitatively diluted to the solution containing 10 μ g in every 1mL, as standard solution.
5, single factor test multilevel test optimizes the formula of solid-state fermentation culture medium
(1) determination of monascus purpureus Zhang-MP solid state fermentation basic medium
For determining the suitableeest basic medium, select long-grained nonglutinous rice, Radix Et Rhizoma Fagopyri Tatarici, both are mixed into different basic medium, carry out fermenting experiment under certain condition.
The different basic medium of table 1 is on the impact of Monacolin K output
As shown in Table 1, when choosing substratum based on long-grained nonglutinous rice 70%+ buckwheat 30%, monascus purpureus Zhang-MP produces Monacolin K ability the most by force, and output can reach 3.9mg/g, and matrix is optimized further based on this.
(2) carbon source and addition thereof are on the impact of Monacolin K output
Apply the basic medium after above-mentioned optimization, adding massfraction is respectively that 1% glucose, 1% sucrose, 1% Zulkovsky starch are as additional carbon source, observe its impact on Monacolin K output, and then choose optimum interpolation carbon source, select optimum to add concentration further.
Table 2 different carbon source kind is on the impact of Monacolin K output
Table 3 carbon source concentration is on the impact of Monacolin K output
From table 2, table 3, additional glucose in basic medium, and when addition is 1%, its Monacolin K output is the highest; When glucose addition is too high, Monacolin K production declining.Its reason may form glucose metabolism and check and cause.Therefore determine that the glucose of 1% is for better additional carbon source.
(3) nitrogenous source and addition thereof are on the impact of Monacolin K output
Apply the basic medium after above-mentioned optimization, wherein respectively add massfraction be 0.1% peptone, 0.1% yeast leaching powder, 0.1% 501 rapeseed protein peptones, 0.1% 502 rapeseed protein peptones, 0.1% 502 rapeseed protein peptones as additional nitrogenous source, observe its impact on Monacolin K output, and then choose optimum interpolation nitrogenous source, select optimum to add concentration further.
Table 4 different nitrogen sources kind is on the impact of Monacolin K output
Table 5 nitrogenous source content is on the impact of Monacolin K output
From table 4, table 5, additional 501 rapeseed protein peptones in basic medium, and when addition is 0.3%, its Monacolin K output is the highest, therefore determine that the 501 rapeseed protein peptones of 0.3% are for better additional nitrogenous source.
(4) inorganic salt are on the impact of Monacolin K output
Apply the basic medium after above-mentioned optimization, adding massfraction respectively is wherein 0.01%MgSO
4, 0.01%ZnSO
4and 0.01%KH
2pO
4as additional inorganic salt, observe its impact on Monacolin K output.
The different Inorganic Salts of table 6 is on the impact of Monacolin K output
Table 7 inorganic salt content is on the impact of Monacolin K output
From table 6, table 7, additional ZnSO in basic medium
4, and when addition is 0.04% and 0.05%, its Monacolin K output is the highest, therefore determines that the ZnSO of 0.04% is better additional inorganic salt.
(5) proof test
Carry out the test of fermentation Red kojic rice under condition before solid-state fermentation culture medium optimization and after optimizing, the results are shown in Table 8.
Table 8 proof test
As shown in Table 8, the rear monascus purpureus Zhang-MP of optimization produces Monacolin K content and can reach 6.9mg/g, than optimizing front Monacolin K output increased 2.56 times.
Present method is fermention medium based on long-grained nonglutinous rice and Radix Et Rhizoma Fagopyri Tatarici mixture, by auxiliary interpolation 0.1% glucose, 0.3% 501 rapeseed protein peptone and 0.04%ZnSO
4to ferment the carbon source of high yield Monacolin K, nitrogenous source and inorganic salt as monascus purpureus Zhang-MP, not only increase the output that monascus purpureus Zhang-MP synthesizes Monacolin K, but also buckwheat is combined with effect of Red kojic rice, produce Monacolin K content in Red kojic rice and can reach 6.9mg/g, Monacolin K output increased 2.56 times before optimizing than fermention medium, does not detect Citrinin simultaneously in monascus purpureus Zhang-MP to ferment the Red kojic rice that obtains.
Project 1 belonging to this patent: the sub-problem of the Department of Science and Technology " 12 " national high-tech research evolutionary operation(EVOP) (863) project " livestock product pathogenic bacteria safety control technology ".
Item number: 2012AA101606-05
The project beginning and ending time: 2012.01-2015.12
Project leader: Zhang Hongxing
Project 2 belonging to this patent: Beijing institution of higher education directly under the jurisdiction of a municipal government high-level personnel Introduction and cultivation planning item " natural lactobacillin is to the antimicrobial mechanism of important pathogenic bacteria in livestock product and Effect study ".
Item number: CIT & TCD20140315
The project beginning and ending time: 2014.01-2016.12
Project leader: Zhang Hongxing
Project 3 belonging to this patent: Department of Science and Technology's national science and technology key special subjects " disease-resistant transgenic sheep rearing new variety " sub-problem " foundation that disease-resistant transgenic sheep expansion traditional font is/disease-resistant transgenic goat-anti disease, production performance and safety evaluation "
Item number: 2013ZX08008-005
The project beginning and ending time: 2013.01-2013.12
Project leader: Liu Hui.
Claims (2)
1. monascus purpureus (Monascus purpureus) Zhang-MP CGMCC No.9221.
2. the process for solid state fermentation of a lipopenicillinase monascus purpureus Zhang-MP, it is characterized in that: consisting of of monascus purpureus (Monascus purpureus) solid-state fermentation culture medium: fermention medium based on long-grained nonglutinous rice 70%+ buckwheat 30%, auxiliary interpolation 0.1% glucose, 0.3% 501 rapeseed protein peptone and 0.04%ZnSO
4; Take 35g long-grained nonglutinous rice and 15g Radix Et Rhizoma Fagopyri Tatarici is mixed in 250mL triangular flask, add 5g glucose, 1.5g 501 rapeseed protein peptone and 2g ZnSO
4the moisture content to 50% of adjustment substratum, after sealing with gauze, after 0.1MPa high pressure steam sterilization 20min, sterile glass rod is utilized to be broken up by culture medium, after cooling, by monascus purpureus Zhang-MP spore suspension by the inoculum size immigration solid-state fermentation culture medium of 6%, in 28 DEG C of quiescent culture 12 ~ 15d, every 3d turns once therebetween, treats that solid-state fermentation culture medium is that namely red-purple obtains cultivating ripe Red kojic rice; Dry under Red kojic rice 55 DEG C of conditions, pulverized 80 mesh sieve, namely obtain the red colouring agent for food, also used as a Chinese medicine powder-product being rich in Monacolin K.
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CN104560790A (en) * | 2014-12-15 | 2015-04-29 | 北京农学院 | Functional lactobacillus plantarum and preparation method of compound bacterial powder of functional lactobacillus plantarum |
CN104962483A (en) * | 2015-07-20 | 2015-10-07 | 光明乳业股份有限公司 | Monascus purpureus strain and application thereof |
CN105062894A (en) * | 2015-07-20 | 2015-11-18 | 光明乳业股份有限公司 | Monascus purpureus strain and application thereof |
CN105105110A (en) * | 2015-08-04 | 2015-12-02 | 江苏大学 | Making method of monascus and tartary buckwheat functional food and application thereof |
CN105361136A (en) * | 2015-08-04 | 2016-03-02 | 江苏大学 | Preparation method and application of monascus transformed tartary buckwheat fermentation liquor |
CN105755048A (en) * | 2016-04-28 | 2016-07-13 | 福建农林大学 | Mixed-medium two-stage liquid fermentation method for preparing functional red yeast rice product |
CN107058433A (en) * | 2017-05-16 | 2017-08-18 | 昆明理工大学 | A kind of preparation method of the bitter buckwheat active peptide with antibacterial and anti-oxidant double activated |
CN108157355A (en) * | 2018-03-30 | 2018-06-15 | 潍坊学院 | Cut Carnation Flower liquid for immersing in bottle |
CN108707555A (en) * | 2018-06-05 | 2018-10-26 | 成都医学院 | Monascus strain and its application of the high yield rich in Monacolin K |
CN109022293A (en) * | 2018-08-24 | 2018-12-18 | 北京农学院 | A kind of monascus purpureus bacterial strain and its fermented product and fermentation process |
CN109554301A (en) * | 2018-11-22 | 2019-04-02 | 浙江三禾生物工程股份有限公司 | A kind of monascus purpureus, fermentation process and tunning reduce the application in interior fat product in preparation |
CN110551636A (en) * | 2019-08-14 | 2019-12-10 | 南京工业大学 | Monascus purpureus MY-21 strain and application thereof |
CN110592156A (en) * | 2019-10-21 | 2019-12-20 | 北京工商大学 | Application of nicotinamide to improvement of monacolin K produced by monascus purpureus |
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CN108157355A (en) * | 2018-03-30 | 2018-06-15 | 潍坊学院 | Cut Carnation Flower liquid for immersing in bottle |
CN108707555A (en) * | 2018-06-05 | 2018-10-26 | 成都医学院 | Monascus strain and its application of the high yield rich in Monacolin K |
CN109022293B (en) * | 2018-08-24 | 2019-12-06 | 北京农学院 | Monascus purpureus strain, and fermentation product and fermentation method thereof |
CN109022293A (en) * | 2018-08-24 | 2018-12-18 | 北京农学院 | A kind of monascus purpureus bacterial strain and its fermented product and fermentation process |
CN109554301A (en) * | 2018-11-22 | 2019-04-02 | 浙江三禾生物工程股份有限公司 | A kind of monascus purpureus, fermentation process and tunning reduce the application in interior fat product in preparation |
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CN110592156A (en) * | 2019-10-21 | 2019-12-20 | 北京工商大学 | Application of nicotinamide to improvement of monacolin K produced by monascus purpureus |
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