Background technology
Red colouring agent for food, also used as a Chinese medicine is the traditional medicine of China, food dual purpose product, is to form with monascus ruber fermentation culture in rice, and it uses apart from modern existing more than 1,000 year history, has a wide range of applications at aspects such as Chinese medicine, wine brewing, leavened food, food dyes.Ancient Times in China is clearly put down in writing the medicine book of red colouring agent for food, also used as a Chinese medicine medical functions, can trace back to Wu Rui in the Yuan Dynasty " daily book on Chinese herbal medicine " and chat it " red colouring agent for food, also used as a Chinese medicine wine brewing, broken capable medicine gesture of blood ".The most famous Zhu Danxi puts down in writing " red colouring agent for food, also used as a Chinese medicine, invigorating blood circulation helps digestion, the dry stomach of invigorating the spleen is controlled dysentery with red and white feces, Xia Shuigu, Chen Jiuzhe is good " in the big doctor of gold dollar four family in its " Amplification on Materia Medica addendum ".According to the theory of traditional Chinese medicine, red colouring agent for food, also used as a Chinese medicine is distinguished the flavor of sweet warm in nature, the effect of tool regulating blood fat concentration.In recent years " the Chinese medicine voluminous dictionary " of Chu Baning is defined as the major function of red colouring agent for food, also used as a Chinese medicine " promoting blood circulation and removing blood stasis, strengthening the spleen to promote digestion, the back lochiorrhea of managing property, the stasis of blood stomachache that stagnates, dyspepsia is glutted, red white diarrhea and wound ".
Along with the raising of people's living standard and the aging of population, hyperlipidaemia becomes the main killer of harm humans health as a kind of " rich man disease ".Hyperlipidaemia mainly shows as serum cholesterol TC, triglyceride level TG and raises, and a kind of whole body lipid metabolism that high density lipoprotein cholesterol HDL-C reduces, low-density lipoprotein LDL raises is unusual.In the cholesterol route of synthesis, methyl glutaryl coenzyme A (HMG-CoA) reductase enzyme is the key enzyme of control resultant velocity, just can reduce or block the synthetic of cholesterol in human body effectively so suppress the activity of this kind of enzyme, thereby prevent and treat hyperlipidaemia.Japanese scholar rattan Zhang Shouci far away in 1979 finds to have the material Monacolin K that suppresses the HMG-CoA reductase activity from the meta-bolites of red monascus.1985, U.S. scientist Goldstein and Brown further found out Monacolin K and have suppressed the cholesterol synthetic mechanism of action, and had therefore obtained the Nobel prize.In the same year, rattan chapter far away is isolated other activeconstituentss with Monacolin K structural similitude, called after respectively again from red monascus: Monacolin L, X, J, Dihydromevinolin and Dihydromonacolin L.Through clinical verification, these Monacolins active substances are effective reducing cholesterol all.
Functional Monascus claims Antilipemic monascus again, is that (lovastatin lovastatin) is Red kojic rice more than 0.4% to a kind of accent fat effective ingredient Monacolin K.Studies show that functional Monascus also contains the physiologically active substance of multiple reducing blood-fat such as a large amount of unsaturated fatty acidss, sterol, saponin, isoflavones and Moncolin K homologue, reducing cholesterol, inhibition lipogenesis, enhance immunity power.(γ-GABA) also has the effect that brings high blood pressure down to γ-An Jidingsuan in the red colouring agent for food, also used as a Chinese medicine.
Nineteen ninety-five, doctor Blanc finds that monascus can produce the Citrinin (Citrinin) with Toxicity of Kidney.Afterwards, for raising has the content of the functional factor lovastatin of using value, reduce or eliminate the Citrinin in the monascus product simultaneously, experts and scholars both domestic and external have carried out a large amount of work.Seed selection such as Bulgaria Rasheva etc. one strain do not produce Citrinin monascus parpureus Went mutant Went CBS 109.07.Screenings such as Japan NIHIRA obtain a new polyketide synthase gene, are applied to make up the monascus strain that does not produce Citrinin.Biotechnology key lab of Southern Yangtze University screening obtains monascus 9901 bacterium that Citrinin, high yield open-loop lovastatin are not produced in 1 strain, can reach 11mg/g with the content of the Red kojic rice lovastatin of this bacterium solid state fermentation production.CN1065432 discloses nineteen Monascus anka Nakazawa et sato bacterial classification and with the red colouring agent for food, also used as a Chinese medicine of its fermentative preparation; CN1206341 discloses a kind of functional Monascus bacterium quick solid cultured method, and the content of yeast culture lovastatin in the time of 28 days can reach 1.6%; CN100386425 discloses the cultural method of the functional Monascus bacterium that does not contain Citrinin; CN1908156 has screened a kind of red colouring agent for food, also used as a Chinese medicine and has belonged to new bacterial strain, lovastatin content 15-30mg/g in the red colouring agent for food, also used as a Chinese medicine, and Citrinin is smaller or equal to 100ppb.Therefore, seek the high yield lovastatin and low yield or not produce the monascus strain of Citrinin particularly necessary.
Summary of the invention
The present invention relates to a strain by separating the monascus variant obtain, the purplish red song of called after (Monascus Purpureus) 9798-0802 in the distiller's yeast through the high yield lovastatin of mutagenesis screening.The present invention also provides the method for being produced functional Monascus by this bacterial strain solid fermentation, and resulting product.
The purpose of this invention is to provide a kind of method of utilizing purplish red bent 9798-0802 fermentative production functional Monascus, make the functional meta-bolites lovastatin high yield of monascus, do not produce or low-yield citrinin.The present invention also provides a kind of functional Monascus, it is characterized in that the content 4-25mg/g of lovastatin, and citrinin content is smaller or equal to 50ppb.
Particularly, the purplish red song of monascus variant called after (MonascusPurpureus) 9798-0802 of high yield lovastatin of the present invention, this bacterial strain is preserved in China Microbial Culture Preservation Commission common micro-organisms center (Beijing) on September 16th, 2008, and preserving number is CGMCC2668.
The purplish red bent 9798-0802 of bacterial strain of the present invention (CGMCC 2668) has following Microbiological Characteristics:
1, morphologic characteristic: when examining under a microscope the bacterial strain of in solid medium, cultivating, mycelium have every, branch is multi-cellular structure, includes a small amount of light oil droplet.Diameter 4.5-6.8um, the many Dan Sheng of conidium, foreign pyriform or subsphaeroidal; Perithecium is colourless, and is transparent, subsphaeroidal, diameter 18-30um, shell wall is thicker, on appendage is arranged, 2-2.5um has pigment to take on a red color in the minority perithecium, it is not good enough that perithecium endoascus spore generates.The thecaspore oval, smooth colourless or incarnadine, 5.2-6.5 * 4-5um.
2, cultivate the characteristic of learning: this bacterial strain is 30 ℃ of cultivations on the wort agar flat board, and bacterium colony spreads growth, and it is velvet-like, more smooth to be felt, and the small amount of radiation line is arranged, white, and aerial hyphae is sparse.The mycelium initial stage is beautiful white powder, and aging back is that pomegranate is bright pink.The greenish orange redness of substrate mycelium.30 ℃ of cultivations on PDA, bacterium colony light fleece shape, baby pink, more smooth.When growing on the CYA substratum, the bacterium colony of formation is thin, and is smooth, owes rule, is beautiful pink.In above substratum, there are not secretory product and chromogenesis.
By the method for purplish red bent 9798-0802 fermentative production functional Monascus, it is characterized in that branch high temperature and low temperature fermentation cultivation monascus, thereby reach the ability that improves its metabolism lovastatin, comprise the steps:
A, spawn culture
Purplish red bent 9798-0802 use seed as producing, in wort agar substratum or PDA nutrient agar, cultivate 3~15 days under 30~40 ℃ of temperature after, through seed culture medium cultivation 3~6 days as first order seed or fermentor tank seed liquor;
B, fermention medium preparation
Fermention medium comprises by weight: starchy material 60-85%, soybean protein 1-10%, carbohydrate or contain sugar substance 5-15%, yeast 0.5-2%;
Starchy material and soybean protein are soaked in water together or directly add the water boiling, add the fermention medium remaining ingredient then, and the back high-temperature sterilization that stirs obtains fermention medium;
C, production fermentation
Described fermention medium is cooled to inoculation below 40 ℃, and inoculum size is 10-30wt%; The fermentation ware of packing into was cultivated 3-5 days in 35~41 ℃ of high-temperature chambers; Treat that mycelia is covered with whole substratum, advance freezer compartment and cultivated 8-20 days for 18~22 ℃; Tunning drying, aseptic stone roller powder obtain functional Monascus of the present invention.
As optimization, the described seed culture medium of step a contains nutrient broth 5g for every liter, bay peptone 1g, white sugar 9.5g, MgSO
40.1g, KH
2PO
42.0g, NaNO
30.15g all the other are water, regulating pH with acetic acid is 4.0.
As optimization, the inoculation below 121 ℃ of sterilization half hour postcooling to 40 ℃ of the described starchy material of step b.
As optimization, the described fermention medium composition of step b is by weight: starchy material 70-85%, soybean protein 2-8%, white sugar 5-12%, yeast 0.5-2%, water 6-10%.
As optimization, in the described fermentative medium formula of step b, starchy material can be one or more in rice, millet, barley, the corn.
As optimization, the described inoculum size of step c is 20-25wt%.
The functional Monascus of method for preparing is characterized in that: the content 4-25mg/g of lovastatin contains Citrinin smaller or equal to 50ppb, and can be directly used in preparation various tablets, capsule or liquid preparation etc. according to the pharmaceutical preparation of routine.
The functional Monascus of purplish red bent 9798-0802 fermentative production that utilizes provided by the invention has lovastatin content height simultaneously, does not produce Citrinin or the atomic characteristics of Citrinin output.Compare with the commercially available statins that is used for reducing blood-fat, the various active composition has synergy, reducing blood-fat simultaneously, hypotensive, decreasing cholesterol play the purpose of integrated control hyperlipidaemia and relevant cardiovascular and cerebrovascular diseases thereof, have good effect, safe, advantage such as price is low.
The method of purplish red bent 9798-0802 fermentative production functional Monascus of utilizing provided by the invention divides high and low temperature to cultivate monascus, can improve the ability of its metabolism lovastatin, and lovastatin reaches as high as 2.5mg/g in the prepared functional Monascus.This process cycle is short, and the efficient height is with low cost, easy handling.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment 1:
1, the present invention uses purplish red bent 9798-0802 bacterial strain, in the wort agar substratum 38 ℃ cultivate 360h after, cultivate 120h as the fermentor tank seed liquor through 35 ℃ of seed culture mediums again.
Seed culture based formulas: nutrient broth 5%, bay peptone 1%, white sugar 9.5%, MgSO
40.1%, KH
2PO
42.0%, NaNO
30.15%, all the other are water, and regulating pH with acetic acid is 4.0.
2, fermentative medium formula: rice 60%, soybean protein 10%, sucrose 15%, yeast 0.5%, extractum carnis 4%, water 10.5%.
3,105 ℃ of sterilization 90min behind sucrose, yeast, extractum carnis, the water are added in rice, the soybean protein boiling behind the 1h that is soaked in water, and are cooled to below 30 ℃ and inoculate liquid spawn, and inoculum size is 10wt%.
4, fermentation culture 5 days in 40 ℃ of high-temperature chambers is gone into 19 ℃ of freezer compartments and is continued to cultivate 10 days.
5, with the culture high-temperature sterilization, 80 ℃ of step-down oven dry obtain functional Monascus of the present invention.The content of measuring lovastatin with the HPLC method is 8.2mg/g.Citrinin content is 40.2ppb, and measuring method is seen functional red yeast rice industry standard (QB/T2847-2007), down together.
Embodiment 2:
1, the present invention uses purplish red bent 9798-0802 bacterial strain, in the PDA nutrient agar 35 ℃ cultivate 260h after, cultivate 90h as first order seed through 35 ℃ of seed culture mediums, cultivate 60h as secondary seed at 38 ℃.The seed culture based formulas is with embodiment 1.
2, fermentative medium formula: rice 25%, wheat 60%, soybean protein 1%, molasses 8%, extractum carnis 4%, water 2%.
3,121 ℃ of sterilization 30min behind molasses, extractum carnis, the water are added in rice, wheat, the soybean protein boiling behind the 2h that is soaked in water, and are cooled to below 28 ℃ and inoculate liquid spawn, and inoculum size is 20wt%.
4, fermentation culture 4 days in 38 ℃ of high-temperature chambers is gone into 20 ℃ of freezer compartments and is continued to cultivate 15 days.
5, with the culture high-temperature sterilization, 80 ℃ of step-down oven dry obtain functional Monascus of the present invention.The content of lovastatin is 20.5mg/g, and citrinin content is 48.4ppb.
Embodiment 3:
1, the present invention uses purplish red bent 9798-0802 bacterial strain, in the PDA nutrient agar 35 ℃ cultivate 320h after, cultivate 90h as first order seed through 35 ℃ of seed culture mediums, cultivate 60h as secondary seed at 38 ℃.The seed culture based formulas is with embodiment 1.
2, fermentative medium formula: rice 73%, soybean protein 6%, white sugar 10%, yeast 1%, water 10%.
3,110 ℃ of sterilization 60min behind white sugar, yeast, the water are added in rice, the soybean protein boiling behind the 1.5h that is soaked in water, and are cooled to below 28 ℃ and inoculate liquid spawn, and inoculum size is 28wt%.
4, fermentation culture 5 days in 38 ℃ of high-temperature chambers is gone into 20 ℃ of freezer compartments and is continued to cultivate 13 days.
5, with the culture high-temperature sterilization, 80 ℃ of step-down oven dry obtain functional Monascus of the present invention.The content of lovastatin is 13.6mg/g, and citrinin content is 43.1ppb.
Embodiment 4:
1, the present invention uses purplish red bent 9798-0802 bacterial strain, in the wort agar substratum 38 ℃ cultivate 260h after, cultivate 100h as the fermentor tank seed liquor through 35 ℃ of seed culture mediums.The seed culture based formulas is with embodiment 1.
2, fermentative medium formula: wheat 60%, wheat bran 10%, soybean protein 8%, white sugar 12%, yeast 0.5%, water 9.5%.
3, high-temperature steam sterilization 20min behind white sugar, yeast, the water is added in wheat, wheat bran, the soybean protein boiling behind the 1h that is soaked in water, and is cooled to below 28 ℃ and inoculates liquid spawn, and inoculum size is 30wt%.
4, fermentation culture 3 days in 41 ℃ of high-temperature chambers is gone into 19 ℃ of freezer compartments and is continued to cultivate 12 days.
5, with the culture high-temperature sterilization, 80 ℃ of step-down oven dry obtain functional Monascus of the present invention.The content of lovastatin is 11.7mg/g, and citrinin content is 47.6ppb.
Embodiment 5:
1, the present invention uses purplish red bent 9798-0802 bacterial strain, in the PDA nutrient agar 35 ℃ cultivate 260h after, cultivate 90h as first order seed through 35 ℃ of seed culture mediums, cultivate 60h as secondary seed at 38 ℃.The seed culture based formulas is with embodiment 1.
2, the fermention medium composition is by weight: rice 25%, brown rice 20%, barley 40%, soybean protein 2%, white sugar 5%, yeast 2%, water 6%.
3, rice, brown rice, barley and soybean protein are soaked in water and cook behind the 2h, add 121 ℃ of sterilization 30min behind white sugar, yeast, the water, are cooled to below 28 ℃ and inoculate liquid spawn, and inoculum size is 25wt%.
4, fermentation culture 4 days in 38 ℃ of high-temperature chambers is gone into 20 ℃ of freezer compartments and is continued to cultivate 15 days.
5, with the culture high-temperature sterilization, 80 ℃ of step-down oven dry obtain functional Monascus of the present invention.The content of lovastatin is 25.0mg/g, and citrinin content is 46.9ppb.
Embodiment 6:
1, the present invention uses purplish red bent 9798-0802 bacterial strain, in the PDA nutrient agar 35 ℃ cultivate 260h after, cultivate 90h as first order seed through 35 ℃ of seed culture mediums, cultivate 60h as secondary seed at 38 ℃.The seed culture based formulas is with embodiment 1.
2, the fermention medium composition is by weight: millet 25%, rice 50%, soybean protein 1%, maltose 5%, yeast 1%, extractum carnis 3%, peptone 7%, water 8%.
3, rice, millet and soybean protein are soaked in water and cook behind the 2h, add 121 ℃ of sterilization 30min behind maltose, yeast, extractum carnis, peptone, the water, are cooled to below 28 ℃ and inoculate liquid spawn, and inoculum size is 25wt%.
4, fermentation culture 4 days in 38 ℃ of high-temperature chambers is gone into 20 ℃ of freezer compartments and is continued to cultivate 15 days.
5, with the culture high-temperature sterilization, 80 ℃ of step-down oven dry obtain functional Monascus of the present invention.The content of lovastatin is 15.0mg/g, and citrinin content is 46.0ppb.
Embodiment 7:
1, the present invention uses purplish red bent 9798-0802 bacterial strain, in the PDA nutrient agar 35 ℃ cultivate 260h after, cultivate 90h as first order seed through 35 ℃ of seed culture mediums, cultivate 60h as secondary seed at 38 ℃.The seed culture based formulas is with embodiment 1.
2, the fermention medium composition is by weight: corn 80%, soybean protein 5%, white sugar 10%, yeast 1%, water 4%.
3, corn and soybean protein are soaked in water and cook behind the 2h, add 115 ℃ of sterilization 50min behind white sugar, yeast, the water, are cooled to below 28 ℃ and inoculate liquid spawn, and inoculum size is 20wt%.
4, fermentation culture 4 days in 38 ℃ of high-temperature chambers is gone into 20 ℃ of freezer compartments and is continued to cultivate 15 days.
5, with the culture high-temperature sterilization, 80 ℃ of step-down oven dry obtain functional Monascus of the present invention.The content of lovastatin is 16.2mg/g, and citrinin content is 43.9ppb.
Embodiment 8:
1, the present invention uses purplish red bent 9798-0802 bacterial strain, in the PDA nutrient agar 35 ℃ cultivate 260h after, cultivate 90h as first order seed through 35 ℃ of seed culture mediums, cultivate 60h as secondary seed at 38 ℃.The seed culture based formulas is with embodiment 1.
2, the fermention medium composition is by weight: millet 72%, soybean protein 8%, white sugar 10%, yeast 2%, water 8%.
3, millet and soybean protein are soaked in water and cook behind the 1.5h, add 121 ℃ of sterilization 30min behind white sugar, yeast, the water, are cooled to below 28 ℃ and inoculate liquid spawn, and inoculum size is 25wt%.
4, fermentation culture 4 days in 38 ℃ of high-temperature chambers is gone into 20 ℃ of freezer compartments and is continued to cultivate 15 days.
5, with the culture high-temperature sterilization, 80 ℃ of step-down oven dry obtain functional Monascus of the present invention.The content of lovastatin is 24.2mg/g, and citrinin content is 45.3ppb.