CN100384981C - Red rice mould mutant strain and its use in preparing fermented product with blood pressure reducing activity - Google Patents
Red rice mould mutant strain and its use in preparing fermented product with blood pressure reducing activity Download PDFInfo
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- CN100384981C CN100384981C CNB021583706A CN02158370A CN100384981C CN 100384981 C CN100384981 C CN 100384981C CN B021583706 A CNB021583706 A CN B021583706A CN 02158370 A CN02158370 A CN 02158370A CN 100384981 C CN100384981 C CN 100384981C
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- monascus
- mutant strain
- tunning
- blood pressure
- citrinin
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Abstract
The present invention relates to a mutant strain of Monascus prupureus, which is used for producing and preparing fermentation products with blood pressure reducing activity. Citrinin contents in obtained products are very low. The present invention simultaneously provide a method for preparing the fermentation products with blood pressure reducing activity by using the mutant strain of the Monascus prupureus and a purpose of the fermentation products for reducing blood pressure.
Description
Technical field
The invention provides the monascus mutant strain, and be used to prepare the purposes of hypotensive activity material.
Background technology
In recent years, cardiovascular disordeies such as hypertension have become to causing the one of the main reasons of death, and the population of suffering from this kind disease is also grown up year by year.Because hypertension is a kind of chronic disease, the patient must take Altace Ramipril for a long time.Show that according to Japan's statistical information in 1999 1 year Altace Ramipril market stability of Japan maintains 50,000,000,000 Yen, and the protective foods relevant with controlling blood pressure, its market is also by 1,400,000,000 Yen of 7,200,000,000 Yen of growing to 1999 in 1997.So the whole world for the demand of medicine with hypotensive activity or heath food, also increases day by day.
Since ancient times, monascus has been the tradition food medicine bacterial classification of China, and it also is widely used in makes various food.Contain the material that can improve hypertension in the meta-bolites of No. 61197524 case discovery Aspergillus (Aspergillus) of day disclosure laid-open patent and monascus (Monascus).(Chemical and Parmaceutical Bulletin 35:2484-2489) found that gamma amino butyric acid (GABA) and vagusstoff (Ach) in the monascus fermented liquid have hypotensive effect to people such as Kohama in 1987.No. 62298598 case of day disclosure laid-open patent then discloses by the method for collecting material for lowering blood pressure in the monascus fermented liquid.No. 03090031 case system of day disclosure laid-open patent provides the improved culture medium of monascus, to promote the output of material for lowering blood pressure.No. 2000279163 case system of day disclosure laid-open patent discloses the blood pressure reducing food that utilizes the monascus preparation, wherein contains gamma amino butyric acid and glucosamine.The announcement of WO01/31048A1 case system is fermented red rice with monascus after, by wherein preparing nitric oxide donor composition (nitricoxide donor composition), said composition has vasorelaxation and hypotensive effect.
People such as Blanc are in nineteen ninety-five (International Journal of Food Microbiology; 27,201-213) find that monascus can produce a kind of mycotoxins, Citrinin (citrinin), and the security of monascorubin is come into one's own.No. 7274978 case of Japanese Patent utilizes mutation method to reduce the generation (being lower than 1ppm) of Citrinin, precisely because product is a haematochrome, is not to be used to prepare material for lowering blood pressure.
Though red mould Pseudomonas has been widely used in the manufacturing material for lowering blood pressure, the citrinin content height of its product, causing the tunning of being produced has consideration in the security.
Summary of the invention
The invention provides a kind of new monascus (Monascus prupureus) mutant strain, it can be under need not the condition of processing treatment, with cheap natural matter, cultivate with solid or liquid mode, direct production has the tunning of hypotensive activity, and the citrinin content of this product is very low.
The present invention also provides the method for utilizing monascus strain of the present invention to prepare the tunning with hypotensive activity.
Pharmaceutical composition and foodstuff additive that the present invention provides the tunning that utilizes the new monascus mutant strain of the present invention to be produced to make in addition.
Embodiment the invention provides the monascus mutant strain of being derived and being filtered out by monascus (Monascus purpureus) sudden change.When containing in every liter, this mutant strain coming about 60 grams of ground rice, about 30 gram and the MgSO of soyflour
4.7H
2Cultivate in the substratum of about 5 grams of O, and when the concentration of gamma amino butyric acid reaches about 0.03 mg/ml in the tunning, wherein the content of Citrinin is approximately less than 1.0ppm, is preferably approximately less than 0.5ppm, is more preferred from approximately less than 0.15ppm.
According to the present invention, the maternal plant of monascus mutant strain is the monascus strain of the gamma amino butyric acid that can produce arbitrarily.For example: can (it is equivalent to ATCC 16375 available from the monascus CCRC 31497 of Hsin-chu Foodstuff Industrial and Development Inst., CBS 280.34, IFO 4489), (it is equivalent to ATCC 16358 to CCRC31498, CBS 281.34, IFO 4486), (it is equivalent to purplish red song (Monascus anka) ATCC 16360 of early stage name to CCRC31499, CBS283.34, IFO 4478, KFCC 11832), (it is equivalent to ATCC 16362 to CCRC 31501, CBS 285.34, IFO 4485), (it is equivalent to ATCC 16367 to CCRC 31504, CBS288.34, IFO 4484), (it is equivalent to ATCC 16365 for CCRC 31541 (it is equivalent to ATCC 16379, and IFO 5965) or CCRC 31542, CBS 109.07, and IFO 4513).
According to the present invention, mutant strain wherein refers to its gene order corresponding to maternal plant, have a Nucleotide difference at least, and the nucleotide sequence that is changed can change its cell physiological.The present invention's mutant strain can obtain by many methods, it comprises, and (for example: chemical mutagen, transposon or radioactive rays) mode is handled maternal plant with random mutagenesis, or with in the nucleotide sequence of nucleic acid recombinant technology with the maternal plant gene, replace, insert or delete one or more Nucleotide and (see also as Sambrook, J.Cold Spring Harbor Press, Plainview N.Y.; Ausubel, people such as R.M. (1995) Current protocols in Molecular Biology, John Wiley ﹠amp; Sons, New York N.Y.).Select gamma amino butyric acid output than the maternal plant height in the mode of screening again, and the Citrinin output mutant strain low than maternal plant.
The preferable embodiment of monascus mutant strain according to the present invention, this monascus mutant strain has and monascus M022 and the identical character of M1033, wherein monascus M022 and monascus M1033 strain on June 21st, 2002 regulation according to budapest treaty be preserved in American type culture collection, its preserving number is respectively PTA-4486 and PTA-4485.
According to the present invention, utilize the monascus mutant strain to prepare the method for tunning, can in solid or liquid nutrient medium, ferment and carry out.
According to the present invention, carbon source in the wherein above-mentioned substratum and nitrogenous source can be any known raw material.It is preferably natural matter, and wherein carbon source comprises but is not defined as and coming ground rice, W-Gum, rice starch, wheat starch, glucose, maltose, sucrose and glycerine, or its combination; And nitrogenous source comprises but be not defined as soyflour, soya bean protein, digestible protein, yeast extract, corn impregnation liquid, Vetsin, ammonium chloride and saltpetre, or its combination.
The anti-case of preferable enforcement of method produced according to the present invention, wherein the pH value of this substratum is preferably approximately between 5 to 7 approximately between 3 to 9.
According to the present invention, has the hypotensive activity material in the obtained tunning, for example gamma amino butyric acid, glucosamine and vagusstoff.Wherein gamma amino butyric acid is the main material that suppresses nerve conduction in the central nervous system, and the acceptor relevant with it has GABA
AAnd GABA
B, in experimentation on animals, find GABA
AActivity can follow the appearance of physiological phenomenons such as hypotensive, spasmolytic and anti-worry, many experiments also prove the activity of gamma amino butyric acid tool treatment hypertension, and many Altace Ramiprils all are the amount by the control gamma amino butyric acid, to reach hypotensive effect.So gamma amino butyric acid content can be used as the important pointer of measuring hypotensive activity.
The content of Citrinin is less than about 1.0ppm in the obtained tunning of monascus mutant strain of the present invention, is preferably approximately less than 0.5ppm, is more preferred from approximately less than 0.15ppm.
According to the present invention, obtained hypotensive tunning can be directly as the activeconstituents or the foodstuff additive of pharmaceutical composition.Hypotensive activity material in the tunning can further (for example: people such as Kohama (Chemical andParmaceutical Bulletin give purifying by various known technologies, 1987,35:2484-2489) and day disclosure laid-open patent purifying extracting process that No. 62298598 case disclosed).
Following embodiment is used for illustration the present invention, but not in order to limit the present invention.
Embodiment 1: the general analysis method
The analysis of gamma amino butyric acid content
After 0.6 milliliter of bacterial strain fermentation liquor and 0.6 milliliter of Lanthanum trichloride (140mM) mixed,, that this mixture is centrifugal in 60 ℃ of water-baths reaction 30 minutes down.Clear liquid on taking out 0.1 milliliter reacts the water of itself and 50 microlitre KOH (1M) and 850 microlitres, carries out centrifugal after 5 minutes and to keep its supernatant liquor standby.
This supernatant samples of 550 microlitres is added the NADP (4mM) of 150 microlitres, the phosphoric acid buffer (pH8.6) of 200 microlitres and the GABASE (2 units per ml) of 50 microlitres, after mixing, utilize its OD of spectrophotometer measurement immediately
340Light absorption value, and write down its result; In the α-Yang Daiwuersuan that wherein adds 50 microlitres, after reacting 60 minutes, measure its OD once more again
340Light absorption value, and calculate OD before and after the reaction
340The difference of light absorption value.With this difference and GABA standard substance relatively after, calculate its concentration.
Citrinin content is analyzed
Bacterial strain fermentation liquor is measured with HPLC.Its step comprises that elder generation is adjusted to 3.5 with the pH value of 7 milliliters of fermented liquids, and question response adds 3 milliliters of ethyl acetate after 1 hour again, collects this ethyl acetate layer after 30 minutes, and repeats this and add ethyl acetate and the step twice of the collection.This is collected the liquid drying.
Behind the collection sample with this drying of 1 milliliter dissolve with methanol, behind the membrane filtration of this methanol solution, get 10 microlitres and carry out the HPLC analysis with 0.2 micron pore size.The condition of its analysis is as follows:
Post: μ Bondapak C
18(10 microns, Waters, source place)
Flow velocity: 1.0 milliliters of per minutes
Detector: (Waters photodiary assay 996 analyzes ripple to the UV detector
Long 225-345 millimicron)
Mobile phase (gradient): 0.8% phosphoric acid: acetonitrile: the 2-propyl alcohol be 60: 35: 5 to 25:
70∶5
The time of carrying out: 20 minutes
Residence time: 11 minutes
Numerical value through recording and Citrinin standard substance (Sigma) calculate its concentration after comparing.
Embodiment 2: the bringing out and screen of mutant strain of the present invention
Monascus CCRC 31499 (being ATCC 16360) is inoculated on PDA (merging shape potato 20%, glucose (Bacto Dextrose) 2%, the agar 2%) inclined-plane, after cultivating 7 days under 30 ℃, spore is washed with sterilized water.Contain 10 with every milliliter
7The collection liquid of above spore is with UV rayed 2 minutes, through being applied in after the serial dilution on the PDA flat board.After cultivating 2 to 3 days under 30 ℃, colony inoculation wherein (is contained in its every liter and coming ground rice 60 grams, soyflour 30 gram and MgSO in substratum
4.7H
2O
5Gram), the stability of testing this gained mutant strain, and the output of gamma amino butyric acid and Citrinin.
Separate at last and select a stable mutant strain, called after monascus M022.This mutant strain is inoculated on the PDA inclined-plane, after cultivating 7 days under 30 ℃, spore is washed with sterilized water.With this spore inoculating (5 * 10
5Individual spore) (contains in every liter and coming ground rice 60 grams, soyflour 30 gram and MgSO in containing 50 milliliters of substratum
4.7H
2O
5Gram) triangle shakes in the bottle, collect its fermented liquid in 30 ℃, 150rpm after concussion is cultivated 5 to 7 days down, and measuring the content of gamma amino butyric acid and Citrinin in this fermented liquid, the result is 0.039 milligram for the gamma amino butyric acid content of every milliliter of fermented liquid, and the content of Citrinin is less than 0.15ppm.Under the same terms, gamma amino butyric acid content is 0.031 milligram every milliliter in maternal plant monascus CCRC 31499 fermented liquids, and the content of Citrinin is 2.1ppm.
Again monascus M022 is carried out screen mutation according to aforesaid method, the result obtains another plant mutant strain, called after monascus M1033, it (contains flour 80 grams in substratum in every liter, yeast extract 10 gram and Vetsin 10 grams) cultivate after, gamma amino butyric acid content is 2.07 milligrams every milliliter in the fermented liquid, and the content of Citrinin is less than 0.15ppm.Under the same terms, gamma amino butyric acid content is 0.834 milligram every milliliter in the monascus M022 fermented liquid, and the content of Citrinin is less than 0.15ppm.
The feature of monascus M022 in substratum is as follows:
The CYA substratum (contains sucrose 30 grams, NaNO in every liter
3
3 grams, K
2
HPO
4
1.0 gram,
MgSO
4
0.5 gram, KCl0.5 gram, FeSO
4
0.01 gram, yeast extract 1 gram, agar 15
Gram)
Cultivate after 7 days, bacterium colony is safran, and size is 11 to 14 millimeters, and aerial hyphae is white in color;
Cultivate after 10 days, bacterium colony is safran, and size is 12 to 16 millimeters, and aerial hyphae is white in color;
It is colourless that conidinphore is, and branch is irregular;
Conidium resembles a pear in shape, and size is 3-4 * 9.5-12.5 micron, and is level and smooth, about 2 microns of wall thickness;
In the CYA substratum, cultivated 21 days, do not find sexual generation yet.
(every liter contains Fructus Hordei Germinatus extract 20 grams to the MEA substratum, peptone 1 gram, glucose 20 grams, fine jade
Fat 15 grams)
Cultivate after 7 days, bacterium colony is orange, and size is 28 to 30 millimeters, and aerial hyphae is white in color;
Cultivate after 10 days, bacterium colony is orange, and size is 34 to 37 millimeters, and aerial hyphae is white in color;
Conidinphore takes on a red color, and branch is irregular;
Cultivated 21 days in the MEA substratum, ascoma is fully matured not yet, and thecaspore is avette (4.5-5 * 5-6 micron).
The feature of monascus M1033 in substratum is as follows:
The CYA substratum (contains sucrose 30 grams, NaNO in every liter
3
3 grams, K
2
HPO
4
1.0 gram,
MgSO
4
0.5 gram, KCl 0.5 gram, FeSO
4
0.01 gram, yeast extract 1 gram)
Cultivate after 7 days, bacterium colony is safran, and size is 13 to 14 millimeters, and aerial hyphae is short and small and very rare;
Cultivate after 10 days, bacterium colony is safran, and size is 17 to 18 millimeters, and aerial hyphae is short and small and very rare;
It is colourless that conidinphore is, and irregular component is " it " font branch, and outer wall is level and smooth;
Conidium resembles a pear in shape or is oval, and size is 6-12 * 8.5-13 micron;
The ascoma size is the 30-35 micron, and it is avette that thecaspore is, and size is 4.5-5 * 5.5-6.
The MEA substratum (every liter contains Fructus Hordei Germinatus extract 20 grams, peptone 1 gram, and glucose 20 grams,
Agar 15 grams)
Cultivate after 7 days, bacterium colony is orange, and size is 29 to 30 millimeters, and aerial hyphae is short and small and very rare;
Cultivate after 10 days, bacterium colony is orange, and size is 41 to 42 millimeters, and aerial hyphae is short and small and very rare;
It is colourless that conidinphore is, and branch is irregular;
Cultivated 21 days in the MEA substratum, ascoma is fully matured not yet.
Relatively be shown in following table 1 about monascus M022 of the present invention and M1033 mutant strain and its maternal plant CCRC 31499.
Table 1
| Maternal plant CCRC 31499 | Mutant strain M022 | Mutant strain M1033 | |
| The conidium size a | 8-12 * 10-13 millimeter | 3-4 * 9.5-12.46 millimeter | 6-12 * 8.5-13 millimeter |
| The ascoma size a | The 37-45 millimeter | Do not have | The 30-35 millimeter |
| The thecaspore size a | 4-5 * 5-6 millimeter | Do not have | 4-5 * 5.5-6 millimeter |
| GABA (mg/ml) | 0.031 b | 0.039 b；0.834 c | 2.07 c |
| Citrinin (ppm) | 2.1 b | ＜0.15 b；＜0.15 c | ＜0.15 c |
aIn the CYA substratum, cultivate.
bSubstratum: contain in every liter and coming ground rice 60 grams, soyflour 30 gram and MgSO
4.7H
2O
5Gram.
cSubstratum: contain flour 80 grams in every liter, yeast extract 10 grams and Vetsin 10 grams.
Embodiment 3: the pH value of producing gamma amino butyric acid
The method according to embodiment 2 contains flour 80 grams in every liter, the culture medium culturing of yeast extract 10 grams and Vetsin 10 grams is measured under the condition of different pH values the content of gamma amino butyric acid and Citrinin in the fermented liquid respectively.Result shown in the table 2 shows that monascus M022 and M1033 mutant strain under the pH of difference value condition, all can produce the gamma amino butyric acid of a large amount, and the content of Citrinin all very low (＜0.15ppm).
Table 2
The not test of "-" table
aThe content unit of gamma amino butyric acid is a mg/ml
*Citrinin content all is lower than detected value 0.15ppm
Claims (12)
1. a monascus (Monascus prupureus) mutant strain, wherein this mutant strain is monascus M1033, and it is preserved in American type culture collection on June 21st, 2002, and preserving number is PTA-4485.
2. method for preparing tunning, it is the monascus mutant strain of cultivating under suitable condition according to claim 1, wherein this tunning comprises gamma amino butyric acid and its citrinin content approximately less than 1.0ppm.
3. according to the method for claim 2, this tunning tool hypotensive activity wherein.
4. according to the method for claim 2, wherein this monascus mutant strain is to cultivate in solid or liquid nutrient medium.
5. according to the method for claim 4, wherein the pH value of this substratum is approximately between 3 to 9.
6. according to the method for claim 5, wherein the pH value of this substratum is approximately between 5 to 7.
7. according to the method for claim 2, wherein the citrinin content of this tunning is approximately less than 0.5ppm.
8. according to the method for claim 7, wherein the citrinin content of this tunning is approximately less than 0.15ppm.
9. according to the method for claim 3, wherein this tunning can be directly as the activeconstituents or the foodstuff additive of medicine.
10. according to the method for claim 3, it can comprise further that the purifying hypotensive activity becomes the step of branch, to get the hypotensive activity composition of purifying.
11. a pharmaceutical composition, it comprises the tunning that is prepared into by each method among the claim 2-10.
12. foodstuff additive, it comprises the tunning that is prepared into by each method among the claim 2-10.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021583706A CN100384981C (en) | 2002-12-30 | 2002-12-30 | Red rice mould mutant strain and its use in preparing fermented product with blood pressure reducing activity |
| HK04108090A HK1065332A1 (en) | 2002-12-30 | 2004-10-18 | Monascus prupureus mutants and their use in preparing fermentation products having hypotensive activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021583706A CN100384981C (en) | 2002-12-30 | 2002-12-30 | Red rice mould mutant strain and its use in preparing fermented product with blood pressure reducing activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1511942A CN1511942A (en) | 2004-07-14 |
| CN100384981C true CN100384981C (en) | 2008-04-30 |
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ID=34236989
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021583706A Expired - Lifetime CN100384981C (en) | 2002-12-30 | 2002-12-30 | Red rice mould mutant strain and its use in preparing fermented product with blood pressure reducing activity |
Country Status (2)
| Country | Link |
|---|---|
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| HK (1) | HK1065332A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100346162C (en) * | 2006-01-05 | 2007-10-31 | 北京联合大学应用文理学院保健食品功能检测中心 | Method for detecting citrinin content in red koji fermentation product |
| CN108641968B (en) * | 2018-05-24 | 2021-08-31 | 富乐顿生物工程科技（北京）有限公司 | Spatial monascus purpureus FuH-23-4 screening and application thereof in producing monascus pigment |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1174037A (en) * | 1996-09-26 | 1998-02-25 | 张茂良 | Monascus preparation for hyperlipemia and relevant cardiac and cerebral diseases |
| CN1373208A (en) * | 2001-02-28 | 2002-10-09 | 无锡轻工大学 | Variant strain of monoscus purpureus for generating lovastatin with high yield and its application |
-
2002
- 2002-12-30 CN CNB021583706A patent/CN100384981C/en not_active Expired - Lifetime
-
2004
- 2004-10-18 HK HK04108090A patent/HK1065332A1/en not_active IP Right Cessation
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1174037A (en) * | 1996-09-26 | 1998-02-25 | 张茂良 | Monascus preparation for hyperlipemia and relevant cardiac and cerebral diseases |
| CN1373208A (en) * | 2001-02-28 | 2002-10-09 | 无锡轻工大学 | Variant strain of monoscus purpureus for generating lovastatin with high yield and its application |
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|---|---|
| CN1511942A (en) | 2004-07-14 |
| HK1065332A1 (en) | 2005-02-18 |
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