CN105112305A - Red yeast rice rich in Monacolin K and preparation method of red yeast rice - Google Patents
Red yeast rice rich in Monacolin K and preparation method of red yeast rice Download PDFInfo
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Abstract
The invention discloses red yeast rice rich in Monacolin K and a preparation method of the red yeast rice. The preparation method comprises the steps of taking 40 monascus strains which are obtained in a separated mode as objects, and screening out a monascus strain, namely FG-8, wherein the monascus strain produces the Monacolin K in a high-yield mode and produces no citrinin, and the Monacolin K content of a fermentation product of the monascus strain is 2.46 mg/g and 2.29 times that of a type strain ACCC 30501. Through molecular identification, the strain is determined as red monascus. The red monascus, namely the FG-8, is treated as an original strain, 26 different varieties of rice raw materials are screened, when the FG-8 is founded, polished rice of a rice variety, namely 'California red rice', is treated as substrates for conducting fermentation, and the red yeast rice rich in the Monacolin K is obtained. The red yeast rice is simple in preparation method, the Monacolin K content measured under the optimum fermentation condition is 4.64 mg/g and approximately 8.30 times that of Furuta red yeast rice on the market, and no citrinin is detected.
Description
Technical field
The present invention relates to a kind of Red kojic rice and preparation method thereof, particularly relate to a kind of Red kojic rice being rich in MonacolinK and preparation method thereof.
Background technology
Red colouring agent for food, also used as a Chinese medicine, originates from China, apart from modern existing history of more than one thousand years, is often called red song or red song in Ancient Times in China.It is a kind of is raw material with long-grained nonglutinous rice, and a kind of red-purple rice made through Fermentation Condition of Monascus spp is bent, the foodstuff additive be a kind of pure natural, security is high, being of value to HUMAN HEALTH, liking always deeply by our people.In blood of human body, cholesterol level was in high state for a long time, easily caused the cardiovascular and cerebrovascular diseases such as atherosclerosis.1979, Japanese scholars rattan chapter professor far away has found the material that can reduce human serum cholesterol---Monacolin K (MonacolinK from the secondary metabolite of monascus ruber, MK), or claim lovastatin (Lovastatin), cause the extensive concern of medical circle to Red kojic rice.MonacolinK is the inhibitor of the rate-limiting enzyme HMG-CoA reductase of body's cholesterol route of synthesis.1985, American scientist Goldstein and Brown worked out the mechanism of action that MonacolinK suppresses cholesterol biosynthesis further, and therefore obtains the Nobel prize, and red colouring agent for food, also used as a Chinese medicine also gains considerable fame thus, is still the focus of world's blood lipid-lowering medicine research field so far.
Yellow rice wine is the rarity of the Chinese nation, so far the history of existing more than 5,000 year, with beer, grape wine claim the world three great Gu wine.The yellow rice wine place of production is comparatively wide, and kind is numerous, is mainly divided into: Zhejiang wheat koji yellow rice wine, Shandong chestnut rice yellow rice wine and Fujian red rice yellow wine three major types.Fujian red rice yellow wine take glutinous rice as main raw material, and employing red colouring agent for food, also used as a Chinese medicine is saccharifying ferment, forms through traditional technology brew, and wine liquid is amber and the strong delicate fragrance of tool red colouring agent for food, also used as a Chinese medicine.Containing MonacolinK in the red rice yellow wine of Fujian, having the advantage that wheat koji, chestnut rice yellow rice wine are incomparable, is a gorgeous wonderful work in China's yellow rice wine.MonacolinK in red rice yellow wine mainly comes from the monascus ruber in red colouring agent for food, also used as a Chinese medicine, monascus ruber energy own metabolism synthesis MonacolinK.MonacolinK is the favourable agents of the decreasing cholesterol that various countries generally acknowledge, the cardiovascular disorder that the yellow rice wine being rich in MonacolinK not only can prevent hyperlipidemia to cause effectively, hypoglycemic, hypotensive, anti-cancer and the effect such as antifatigue, the most important thing is the side effect not having pharmacological agent in addition.
But, because biologically active substance MonacolinK content contained in most of red colouring agent for food, also used as a Chinese medicine is lower, its application is restricted.At present, domestic and international Most scholars all puts forth effort on the content how research improves MonacolinK in monascus product, mainly by transforming production bacterial strain, to transform or by the mode such as physics, chemomorphosis as adopted Molecular tools to its key gene.But, about different varieties rice source, the Difference of MonacolinK is produced for monascus ruber fermentation, at home and abroad rarely seen report.Therefore, the innovative point of this research is: on the basis of monascus strain separating high yield MonacolinK, with the rice of different varieties for raw material, filter out the substrate that optimum rice source ferments as monascus ruber, optimize its fermention medium and zymotechnique, develop and be rich in MonacolinK and the Red kojic rice not detecting Citrinin, can be applicable to the production of being rich in MonacolinK red rice yellow wine.
Summary of the invention
The object of the present invention is to provide a kind of Red kojic rice being rich in MonacolinK and preparation method thereof.
Technical scheme provided by the present invention is:
Described monascus strain (
monascusruber) FG-8, this bacterial strain on August 28th, 2015 in China General Microbiological culture presevation administrative center (CGMCC) preservation, address is No. 3, No. 1, Chaoyang District Beijing North Star West Road institute, and preserving number is CGMCCNO.11277.
Be rich in a Red kojic rice of MonacolinK, in described Red kojic rice, the content of MonacolinK is 4.64mg/g, is about 8.30 times of commercially available Gutian Red kojic rice, and does not detect Citrinin.
A kind of preparation method being rich in the Red kojic rice of MonacolinK, described Red kojic rice is with rice varieties " California red rice " (river and mountain etc., sooner or later season paddy rice polished rice and the Genotypic Difference of meter Pi selenium content, plant genetic resources journal, 2005,6 (4): 448-452) polished rice is substrate, with monascus FG-8 for starting strain, ferments according to its optimal conditions of fermentation.
The preparation method of described Red kojic rice comprises the following steps:
(1) the preparation of rice medium: cleaned by the polished rice of rice varieties " California red rice ", clear water soaks three hours, steams 20min after draining in food steamer, and every fermentation flask takes 50g rice, adds the glucose of 3wt%, the NaNO of 4wt% according to the per-cent of rice quality
3, 1wt% sodium acetate, after good seal, autoclaving 20min at 121 DEG C.
(2) the preparation of monascus ruber seed liquid: by the monascus strain FG-8 of high yield MonacolinK on PDA flat board 28 DEG C after activation culture 5-7 days, the punch tool that diameter is 5mm is used to get bacterium block, then respectively add 5 bacterium cakes in the 250mL triangular flask that 50mL seed liquor substratum is housed, in 30 DEG C, namely obtain seed liquor after shaking culture 72h under 150rpm condition; Described seed liquor substratum is glucose 50g, peptone 5g, yeast extract paste 1g, KH
2pO
41g, FeSO
47H
2o0.01g, MgSO
47H
2o0.5g, adds water and is settled to 1000mL.
(3) inoculation and fermentation culture: it is 1 × 10 that the seed liquor after cultivating 72h is diluted to spore concentration
7individual/mL, the seed liquor of dilution being added to by 10% inoculum size is equipped with in the plastics fermentation flask of rice medium, stirs with aseptic bamboo let, and is placed in 28 DEG C of constant incubators and cultivates 14d, at Initial stage of culture 2-3d, slightly take on a red color bottom rice fermentation substratum; When being cultured to 4d, plastics fermentation flask is taken out, rice is clapped loose, make Monascus anka Nakazawa et sato can utilize rice raw material more fully; When being cultured to 7d, Monascus anka Nakazawa et sato filament growth is vigorous; During 8d, moved to by plastics fermentation flask in 26 DEG C of constant incubators, continue to be cultured to 14d, rice fermentation is complete, finally obtains Red kojic rice.
Described Red kojic rice is specifically prepared in accordance with the following steps:
1) screening being separated the 40 strain monascus strains obtained, obtaining high yield MonacolinK, and not producing the bacterial strain of Citrinin; Again Molecular Identification is carried out to this bacterial strain, determine its specific name.
2) respectively with the polished rice of 26 parts of different varieties rice and brown rice for substrate, carry out the fermentation test of monascus ruber, filter out the rice source of high yield MonacolinK.
3) fermention medium and processing condition of preparing Red kojic rice are optimized, obtain high yield MonacolinK, and do not produce the suitableeest fermention medium and the optimal conditions of fermentation of Citrinin.
What step 1) was concrete is: from the waste water and dregs of different red rice yellow wine factory, and isolate monascus strain in the sample such as the commercially available Red kojic rice of different sources and yellow rice wine, and carry out purifying cultivation, the strain culturing after purifying, on PDA slant medium, obtains 40 strain monascus strains.Then solid state fermentation mode is adopted, using the MonacolinK content in monascus ruber tunning as index, dry to constant weight at respectively tunning Red kojic rice being placed in 50 DEG C, be ground into powder, after the pre-treatment of necessity, HPLC method is adopted to measure the content of its MonacolinK and Citrinin.The high yield MonacolinK separated and do not produce the monascus strain of Citrinin, is numbered FG-8.Adopt CTAB method to extract the genomic dna of monascus ruber, utilize ITS1/ITS4 primer pair, carry out pcr amplification and the sequencing analysis of ITS sequence.The ITS sequence of acquisition signed in on NCBI website and carry out BLASTN searching analysis, result shows: be separated the monascus strain FG-8 obtained and belong to red monascus (Monascusrubber).
Step 2) concrete be: respectively with the polished rice of 26 parts of different varieties rice and brown rice for raw material, carry out the fermentation test of monascus ruber, adopt commercially available polished rice " all sharp rice " for contrast raw material (CK1), the rice source and the commercially available rice that compare different varieties produce the impact of MonacolinK to monascus strain FG-8; Simultaneously with commercially available Gutian Red kojic rice for reference product (CK2), compare the MonacolinK content in Red kojic rice and commercially available Gutian Red kojic rice prepared by the present invention.Thus the polished rice filtering out rice variety " California red rice " is when being raw material, the MonacolinK content in tunning is the highest.
What step 3) was concrete is: the processing parameters such as research leavening temperature, initial ph value, dress rice amount, inoculum size, ventilation property, fermentation time, and add precursor substance and different carbon source, different nitrogen sources etc., tunning Red kojic rice is produced to the impact of MonacolinK and Citrinin; On this basis, response phase method is adopted to optimize the fermentation condition of high MonacolinK monascus ruber, obtain being rich in MonacolinK and do not detect the Red kojic rice of Citrinin, its suitableeest fermention medium is: in rice fermentation basic medium, add the glucose of 3%, the NaNO of 4%
3, 1% sodium acetate; Best zymotechnique is: earlier fermentation temperature is 30 DEG C, and initial pH is 5.0, and dress rice amount is 40g, and fermentation time is 17d, and inoculum size is 10%, and breather hole is adjusted to 5 holes.Wherein, the making method of rice fermentation basic medium is: cleaned by rice, and clear water soaks three hours, steams 20min after draining in food steamer, and every fermentation flask takes 50g rice, after good seal, and autoclaving 20min at 121 DEG C.
Advantage of the present invention is: on the basis of the optimum monascus strain of separation screening high yield MonacolinK and optimum rice source, optimize the zymotechnique of Red kojic rice, thus prepares and be rich in MonacolinK and the Red kojic rice not detecting Citrinin.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Embodiment is only not used in for illustration of the present invention and limits the scope of the invention.
Embodiment 1
(1) separation of monascus strain: adopt PDA plate culture medium, the waste water and dregs of different red rice yellow wine factory from Fujian Province, and isolate monascus strain in the sample such as the commercially available Red kojic rice of different sources and yellow rice wine, and after carrying out purifying cultivation, obtain 40 strain monascus strains, the strain culturing of purifying is on PDA slant medium.
(2) screening of high yield MonacolinK monascus strain: using the MonacolinK content in monascus ruber tunning and Citrinin as index, dry to constant weight at respectively the tunning Red kojic rice of 40 kinds of monascus strains being placed in 50 DEG C, be ground into powder, after the pre-treatment of necessity, HPLC method is adopted to measure the content of its MonacolinK and Citrinin.Result shows: MonacolinK output is the highest and the bacterial strain not producing Citrinin is FG-8.
(3) Molecular Identification of monascus strain: adopt CTAB method to extract the mycelial genomic dna of high yield MonacolinK monascus strain FG-8, utilize ITS1/ITS4 primer pair to carry out pcr amplification and the sequencing analysis of ITS sequence.The ITS sequence of acquisition signed in on NCBI website and carry out BLASTN searching analysis, the sequence of result and red monascus BCRC31523 (MonascusrubberstrainBCRC31523) is completely the same (Identities=100%).Therefore, bacterial strain FG-8 is defined as red monascus (Monascusrubber).
(4) biological characteristics of FG-8 bacterial strain: substratum is PDA substratum, pH nature, optimum growth temperature is 28 DEG C, aerobic.Colony growth is comparatively slow, and bacterium colony swells, and in felted, there are breach in central authorities, has obvious rill and very fine and closely woven, and neat in edge, smooth, bacterium colony color and luster be dark pink, and the back side is amaranth.The single raw or chaining of conidium, raw on conidiophore top, spherical or pyriform.
Embodiment 2
(1) preparation of monascus ruber seed liquid: by monascus strain activation culture (28 DEG C) after 5-7 days on PDA flat board, the punch tool that diameter is 5mm is used to get bacterium block, then respectively add 5 bacterium cakes and (fill little granulated glass sphere) in the 250mL triangular flask that 50mL seed liquor substratum is housed, each bacterial strain arranges three repetitions, in 30 DEG C, under 150rpm condition, after shaking culture 72h, namely obtain seed liquor.
(2) preparation of rice medium: rice is cleaned, clear water soaks three hours, steams 20min(about medium well after draining in food steamer), every fermentation flask takes 50g rice, adds the glucose of 3wt%, the NaNO of 4wt% according to the per-cent of rice quality
3, 1wt% sodium acetate, after good seal, autoclaving 20min at 121 DEG C.
(3) inoculation and fermentation culture: the seed liquor after cultivating 72h is diluted to spore concentration and is about 1 × 10
7individual/mL, to add to the seed liquor of dilution by 10% inoculum size and is equipped with in the plastics fermentation flask of rice medium, stir with aseptic bamboo let, and is placed in 28 DEG C of constant incubators and cultivates 14d.At Initial stage of culture (3d), slightly take on a red color bottom rice fermentation substratum; When being cultured to 4d, plastics fermentation flask is taken out, rice is clapped loose, make Monascus anka Nakazawa et sato can utilize rice raw material more fully; When being cultured to 7d, Monascus anka Nakazawa et sato filament growth is vigorous; During 8d, moved to by plastics fermentation flask in 26 DEG C of constant incubators, continue to be cultured to 14d, rice fermentation is complete.
(4) screening of rice source: respectively with the polished rice of 13 kinds of different varieties rice and brown rice for raw material, carry out the fermentation test of monascus ruber.Adopt commercially available high-quality " all sharp rice " for contrast raw material (CK1), compare different varieties rice source and commercially available rice produces the impact of MonacolinK to monascus strain FG-8; Simultaneously with commercially available Gutian Red kojic rice for reference product (CK2), compare the MonacolinK content in Red kojic rice and commercially available Gutian Red kojic rice prepared by the present invention.Result shows: monascus ruber FG-8 with the polished rice of rice varieties " California red rice " be raw material ferment time, the MonacolinK content in tunning is the highest.
(5) optimization of fermention medium and processing condition: the processing parameters such as research leavening temperature, initial ph value, dress rice amount, inoculum size, ventilation property, fermentation time, and add precursor substance and different carbon source, different nitrogen sources etc., tunning Red kojic rice is produced to the impact of MonacolinK and Citrinin; On this basis, response phase method is adopted to optimize the fermentation condition of high MonacolinK monascus ruber, obtain being rich in MonacolinK and do not detect the Red kojic rice of Citrinin, its suitableeest fermention medium is: in rice fermentation basic medium, add the glucose of 3wt%, the NaNO of 4wt%
3, 1wt% sodium acetate; Best zymotechnique is: earlier fermentation temperature is 30 DEG C, and initial pH is 5.0, and dress rice amount is 40g, and fermentation time is 17d, and inoculum size is 10%, and breather hole is adjusted to 5 holes.Result shows: in the Red kojic rice prepared under optimal conditions of fermentation, and MonacolinK content is 4.64mg/g, improves 28.5% than before process optimization, is about 8.30 times of commercially available Gutian Red kojic rice, and does not detect Citrinin.
In Red kojic rice, the HPLC measuring method of MonacolinK content is as follows:
Chromatographic condition: chromatographic column is VisionHTC18HL (5 μm, 250 × 4.6mm), Grace; Moving phase is V (A:B)=V (acetonitrile: 0.18% phosphate aqueous solution)=50:50; Flow velocity is 1mL/min; The wavelength of detector is 238nm; Column temperature is 28 DEG C; Sample size is 10 μ L.
The pre-treating process of testing sample: get product by solid-state fermentation and dry to constant weight in 50 DEG C, be ground to Powdered.Get 0.5g testing sample to add in the brown volumetric flask of 10mL, accurately add 5mL0.2MNaOH solution, then with methanol constant volume to 10mL, ultrasonic 1h at 50 DEG C.4 DEG C, centrifugal 10min under 8000rpm, gets supernatant liquor 5mL in the brown volumetric flask of 25mL, with methanol constant volume to 25mL.After fully shaking up, get diluent 1mL and cross 0.45 μm of filter membrane, to be checked.
Measurement result: in the Red kojic rice that the present invention is prepared under optimal conditions of fermentation, the content of MonacolinK is 4.64mg/g, is about 8.30 times of commercially available Gutian Red kojic rice.
In Red kojic rice, the HPLC measuring method of citrinin content is as follows:
Chromatographic condition: chromatographic column is Shim-PackVP-ODSC18(5 μm, 250 × 4.6nm, Japanese Shimadzu), moving phase is: acetonitrile-water (volume ratio is 35:65, adjusts pH to 2.5 with chromatographically pure phosphoric acid), and flow velocity is 1.0mL/min, column temperature 35 DEG C, determined wavelength 254nm; Sampling volume 10 μ L.
The pre-treating process of testing sample: red colouring agent for food, also used as a Chinese medicine crosses 40 mesh sieves after pulverizing, accurately take 1g as sample, slowly add 20mLTEF solution (toluene: ethyl acetate: formic acid=7:3:1 under magnetic stirring, v/v/v), stir 30min, the centrifugal 10min of 5000r/min, gets supernatant liquor through 0.22 μm of filtering with microporous membrane, to be checked.
Measurement result: the present invention adopts monascus strain FG-8 to carry out, in the standby Red kojic rice of fermentation, not detecting Citrinin.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (3)
1. produce MonacolinK and do not produce the monascus ruber of Citrinin, it is characterized in that: described bacterium be monascus strain (
monascusruber) FG-8, this bacterial strain on August 28th, 2015 in China General Microbiological culture presevation administrative center (CGMCC) preservation, preserving number is CGMCCNO.11277.
2. be rich in MonacolinK and do not produce the Red kojic rice of Citrinin, it is characterized in that: described Red kojic rice is that substrate carries out fermentation gained by monascus strain FG-8 with the polished rice of rice varieties " California red rice ".
3. be rich in the preparation method of MonacolinK Red kojic rice as claimed in claim 2, it is characterized in that: the preparation method of described Red kojic rice comprises the following steps:
(1) the preparation of rice medium: cleaned by the polished rice of rice varieties " California red rice ", clear water soaks three hours, steams 20min after draining in food steamer, and every fermentation flask takes 50g rice, adds the glucose of 3wt%, the NaNO of 4wt% according to the per-cent of rice quality
3, 1wt% sodium acetate, after good seal, autoclaving 20min at 121 DEG C;
(2) the preparation of monascus ruber seed liquid: by the monascus strain FG-8 of high yield MonacolinK on PDA flat board 28 DEG C after activation culture 5-7 days, the punch tool that diameter is 5mm is used to get bacterium block, then respectively add 5 bacterium cakes in the 250mL triangular flask that 50mL seed liquor substratum is housed, in 30 DEG C, namely obtain seed liquor after shaking culture 72h under 150rpm condition;
(3) inoculation and fermentation culture: it is 1 × 10 that the seed liquor after cultivating 72h is diluted to spore concentration
7individual/mL, the seed liquor of dilution being added to by 10% inoculum size is equipped with in the plastics fermentation flask of rice medium, stirs with aseptic bamboo let, and is placed in 28 DEG C of constant incubators and cultivates 14d, at Initial stage of culture 2-3d, slightly take on a red color bottom rice fermentation substratum; When being cultured to 4d, plastics fermentation flask is taken out, rice is clapped loose, make Monascus anka Nakazawa et sato can utilize rice raw material more fully; When being cultured to 7d, Monascus anka Nakazawa et sato filament growth is vigorous; During 8d, moved to by plastics fermentation flask in 26 DEG C of constant incubators, continue to be cultured to 14d, rice fermentation is complete, finally obtains Red kojic rice.
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