CN104593267B - Monascus purpureus and its application in 1 DNJ is prepared - Google Patents
Monascus purpureus and its application in 1 DNJ is prepared Download PDFInfo
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Abstract
Application the invention discloses one plant of new strains monascus purpureus (Monascus purpureus) Zhejiang work red yeast rice 1 and its in 1 DNJ is prepared, monascus purpureus (Monascus purpureus) Zhejiang work red yeast rice 1, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date is on July 18th, 2014, and address is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number is：CGMCC No.9553；The fermented culture of monascus purpureus Zhejiang work red yeast rice 1 of the present invention, can obtain the culture containing 1 DNJ, 1 DNJ has α glucosidase inhibitory actives；The bacterial strain and its fermentation obtain the method containing 1 DNJ culture and had broad application prospects.
Description
(1) technical field
The present invention relates to one plant of new strains, more particularly to one plant can produce the monascus of 1-DNJ and answer
With the monascus purpureus (Monascus purpureus) that utilization can produce 1-DNJ is entered with grain raw materials such as rice
Row fermentation.
(2) background technology
Red yeast rice is tunning of the monascus on the grains such as rice, has the applicating history in over thousands of year in China.Red yeast rice
Application includes the fields such as brewageing, colouring and preservation for food, and Chinese medicine.According to relevant national standard (GB 2760, GB
4926), red yeast rice can be used as food additives；Issued according to the Ministry of Public Health《Mycophyta health food evaluation regulation》(defend method prison hair
(2001) No. 84), monascus (Monacus anka) and monascus purpureus (Monascus purpureus) belong to available for health care
The fungi strain of food；《Pharmacopoeia of People's Republic of China》Also red yeast rice is included by Chinese medicine.At present, it can be produced using monascus
Raw cholesteral biosynthesis inhibitor-do not draw Kelin (monacolin), has successfully developed the drop using red yeast rice as main component
Fat Chinese medicine and health products.Further new monascus product of the research and development with other effects, has positive to the development of red yeast rice industry
Facilitation.
Diabetes are the metabolic diseases being characterized with hyperglycaemia, according to《JAMA》(The Journal of
The American Medical Association) report：The whole world in 2013 about 3.82 hundred million diabetics, glycosuria
Disease has turned into that the whole world is the fourth-largest to cause the disease of death, and exploitation is cheap, effective prevention or improve the functional foods of diabetes
It is significant.The enteral various alpha-glucosidases of alpha-glucosidase restrainer energy Reverse transcriptase, slow down starch
The speed of glucose is decomposed into, so that slow down absorption of the enteron aisle to glucose, the hyperglycaemia of reduction after the meal.Natural phlorose
Glycosides enzyme inhibitor is present in animal, plant and microorganism, and alpha-glucosidase restrainer source strain, tool are screened from microorganism
There is wide industrial applications prospect.At present, it has been found that the 1-DNJ tool that some actinomyces and bacillus produce
There is preferable alpha-glucosaccharase enzyme inhibition.
The red yeast rice conventionally prepared, due to monascus produce and secrete a variety of extracellular hydrolases (including it is a variety of form sediment
Powder enzyme), so being commonly used for brewageing saccharifying agent (distiller's yeast), promote the decomposition of starch in fermentation raw material；Or as Chinese medicine, with it
Its medicinal material compatibility, promotes the digestion and absorption of food, such as《Compendium of Materia Medica》It is described that " red yeast rice cures mainly the promoting blood circulation that helps digestion, and invigorating the spleen is dry
Stomach ".The conventional red yeast rice of this explanation has the catalytic activity for promoting the carbohydrate breakdowns such as starch, typically without alpha-glucosaccharase
Enzyme inhibition activity.Although there is research to point out, red yeast rice has some improvement while blood fat is reduced to blood glucose, its
Active ingredient is unclear, and mechanism of action is also indefinite.Meanwhile, 1-DNJ can be produced currently without monascus,
And the report containing 1-DNJ is found in monascus product.
(3) content of the invention
It is an object of the present invention to provide one plant of new strains -- monascus purpureus (Monascus purpureus) Zhejiang work 1 and its system
The application of standby 1-DNJ.
The technical solution adopted by the present invention is：
The present invention relates to one plant of new strains -- monascus purpureus (Monascus purpureus) Zhejiang work red yeast rice 1, it is preserved in
China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date is on July 18th, 2014, and address is：North
The institute 3 of Jing Shi Chaoyang Districts North Star West Road 1, deposit number is：CGMCC No.9553.
The invention further relates to a kind of No. 1 application in 1-DNJ is prepared of monascus purpureus Zhejiang work red yeast rice,
Specific described application is made with No. 1 culture obtained through fluid nutrient medium or solid medium culture of monascus purpureus Zhejiang work red yeast rice
For strain, fermentation medium is seeded to, solid state fermentation is carried out under conditions of 25~40 DEG C of temperature, humidity 60%~90%, is sent out
After the completion of ferment, the tunning (being the red yeast rice containing 1-DNJ) containing 1-DNJ is obtained, fermentation is produced
Thing is isolated and purified, and obtains 1-DNJ；The fermentation medium is by eating grain, glucose, peptone and running water
The mass ratio of composition, the edible grain and glucose, peptone and running water is 1:0.01~0.1:0.015~0.15:0.2
~0.8 (preferably 1:0.03:0.01:0.3)；
The fluid nutrient medium quality final concentration is constituted：70~120 mesh rice meals or soluble starch 1%~4%, Portugal
Grape sugar or sucrose 0.5%~3%, peptone 0.3%~3%, NaNO30.1%~0.5%, KH2PO40.1%~0.5%, it is molten
Agent is running water, and pH value is natural；Preferred liquid culture medium quality final concentration is constituted：100 mesh rice meals 2%, glucose 2%,
Peptone 0.8%, NaNO30.2%, KH2PO40.15%, solvent is running water, and pH value is natural.
The solid medium is made up of rice, glucose, peptone and running water, the rice and glucose, albumen
The mass ratio of peptone and running water is 1:0.01:0.005:0.3.
Further, the edible grain is long-grained nonglutinous rice, polished rice, glutinous rice, red rice, black rice, millet, soya bean, barley or corn.
Further, the solid spawn and edible grain mass ratio are 0.005~0.05:1 (preferably 0.01:1), the liquid
Body strain volumetric usage is calculated as 0.1~0.3mL/g (preferably 0.1mL/g) with edible grain quality.
Further, the monascus purpureus Zhejiang work red yeast rice 1 is with 106~107The form of cfu/mL spore suspensions is seeded to liquid
The culture that culture medium or solid medium culture are obtained is as strain, and the spore suspension is by monascus purpureus Zhejiang work red yeast rice 1
Number it is seeded on EMA culture mediums, 32 DEG C are cultivated 7 days, is 10 by spore sterilized water adjustment concentration with spore under sterile washing6
~107cfu/mL；Every liter of malt extract powder agar medium (MEA)：Malt extract powder 20g, peptone 1.0g, glucose 20g,
Agar 15g, plus distilled water is to 1L.
Further, the preparation method of the strain is one of following：(1) by 106~107Cfu/mL monascus purpureus Zhejiang work is red
Bent No. 1 spore suspension is seeded to fluid nutrient medium with the inoculum concentration of volumetric concentration 10% (v/v), is cultivated 2 days at 32 DEG C, takes culture
Thing is used as liquid spawn；The fluid nutrient medium quality final concentration is constituted：100 mesh rice meals 2%, glucose 2%, peptone
0.8%, NaNO30.2%, KH2PO40.15%, solvent is running water, and pH value is natural；(2) by 106~107Cfu/mL monascus purpureus
Work red yeast rice No. 1 spore suspension in Zhejiang is seeded to solid medium, temperature be 30 DEG C, humidity be 80% under conditions of cultivate 10 days,
It regard culture as solid spawn；The spore suspension volumetric usage is calculated as 0.1mL/g with solid medium quality；The solid
Culture medium group is by rice, glucose, peptone and tap water group into, the rice and glucose, peptone and originally water quality
Than for 1:0.01:0.005:0.3.
Further, the tunning isolation and purification method is：By the tunning containing 1-DNJ at 55 DEG C
Vacuum drying, wears into 80 mesh powder, respectively with the ethanol water of volumetric concentration 70% of 5 times (v/w) 25 DEG C extract 3 times, every time
24h is extracted, extract solution merges, filtering, filter vacuum is concentrated into paste, obtains concentrate a；Concentrate a is subjected to silica gel column layer
Analysis, eluted successively with ethyl acetate, absolute ethyl alcohol, distilled water, collect distill water elution component, be concentrated in vacuo to it is dry,
Obtain concentrate b；By concentrate b again by neutral alumina column chromatography, first with n-hexane, then with ethyl acetate: petroleum ether (1:
1, v/v) elute, collect ethyl acetate: the eluent of petroleum ether, be concentrated in vacuo to dry, acquisition concentrate c；Concentrate c is utilized
Preparative TLC is chromatographed, with normal propyl alcohol: acetic acid: water (8:2:1, v/v) be solvent, using chloro- tolidine develop the color, reclaim with
Silica gel at the colour developing that standard items are consistent, is concentrated and dried after being dissolved with the ethanol water of volumetric concentration 50% and obtains the wild buttocks of 1- deoxidations
Mycin, the yield of this method is about 46%.The concentrate a, concentrate b and concentrate c are concentrate, for the ease of distinguishing
Concentrate that different step is obtained and name, letter itself is without implication.
Compared with prior art, the beneficial effects are mainly as follows：The present invention provides one plant of new strains -- and it is purplish red
Aspergillus Zhejiang work red yeast rice 1, fermented culture can obtain the culture containing 1-DNJ, 1-DNJ tool
There is alpha-glucosaccharase enzyme inhibition activity；The bacterial strain and its fermentation, which obtain the method containing 1-DNJ culture, to be had extensively
Wealthy application prospect.
(4) illustrate
Fig. 1 is the mass spectrogram that work red yeast rice No. 1 fermented product in monascus purpureus Zhejiang isolates and purifies obtained 1-DNJ.
Fig. 2 is based on the phylogenetic tree constructed by ITS.
Fig. 3 is the liquid chromatogram of 1-DNJ standard items and monascus purpureus Zhejiang No. 1 fermented product of work red yeast rice, a
For 1-DNJ standard items；B is No. 1 fermented product of monascus purpureus Zhejiang work red yeast rice.
(5) embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Experimental method in following embodiments, is conventional method unless otherwise specified.Examination used in following embodiments
Material is tested, unless otherwise specified, routine biochemistry reagent shop is all from and is commercially available.% in following embodiments, such as without special
Illustrate, be weight/mass percentage composition.
The separation and identification of the monascus purpureus Zhejiang work red yeast rice 1 of embodiment 1
First, the separation of Zhejiang work red yeast rice 1
Screening and culturing medium flat board is coated on after soil sample, plus sterilized water dilution are gathered near the grain processing plant in Hangzhou
On, 32 DEG C are cultivated 2 days, and the bacterium colony coating screening flat board that picking meets monascus morphological feature is further isolated and purified.Will wherein
The monascus strain that one plant growth is quick, acid resistance is strong is designated as Zhejiang work red yeast rice 1, and is identified using morphology and molecule.
The screening and culturing medium is constituted：10 ° of Bx brewer's worts, pH3.5, agar 1.5%.
2nd, the Morphological Identification of Zhejiang work red yeast rice 1
1st, culture medium
(1) every liter of Cha Shi yeast extract powders agar medium (CYA) composition：NaNO33.0g, K2HPO41.0g, KCl
0.5g, MgSO4·7Η2O 0.5g, FeSO4·7Η2O 0.0lg, yeast extract 5.0g, sucrose 20g, agar 15g, plus steam
Distilled water is to 1L.
(2) every liter of malt extract powder agar medium (MEA)：Malt extract powder 20g, peptone 1.0g, glucose 20g,
Agar 15g, plus distilled water is to 1L.
(3) every liter of complete medium (CM)：Sucrose 100g, yeast extract 3.0g, peptone 5.0g, NaNO32.0g,
K2HPO41.0g, MgSO4·7H2O 0.5g, KCl 0.5g, FeSO4·7H2O 0.01g, agar 20g, plus distilled water is to 1L.
3 kinds of culture mediums of above moist heat sterilization 20min at 121 DEG C.
2nd, the colonial morphology observation of Zhejiang work red yeast rice 1
By Zhejiang work red yeast rice, No. 1 is seeded on CYA culture mediums, and 30 DEG C are cultivated 25 days, and bacterium colony is flat, sparse, irregularly, surface
The usual flocciform of quality, there is a small amount of aerial hyphae once in a while, and just white is ripe and becomes pale red with bacterium colony for mycelium, it is aging after
It is orange red；
By Zhejiang work red yeast rice, No. 1 is seeded on MEA culture mediums, and 30 DEG C are cultivated 25 days, and bacterium colony is flat, sparse intensive to moderate,
Sometimes have a small amount of and short aerial hyphae, but white becomes orange with bacterium colony maturation at the beginning of generally flocciform, mycelium
Or red color tone, it is generally orange red；
By Zhejiang work red yeast rice, No. 1 is seeded on CM culture mediums, and 30 DEG C are cultivated 25 days, and colony growth is flat, and broken edge is in
Lava shape, has a small amount of aerial hyphae to be formed, and initial stage, white became orange with bacterium colony maturation, aging rear orange red or even reddish brown
Color.
3rd, the strain morphology feature of Zhejiang work red yeast rice 1：
Mycelium is sparse to abundant, mycelia erratically branch, and clear, colorless has oil droplet, and indivedual mycelia are very sturdy, generally
It is wide 3-5 μm, with many orange red excipuliform crystallizations on wall.Conidium list life or chaining, raw on mycelia top, and pyriform is extremely
It is spherical, 8~11 × 8~10 μm；Cleistothecium Dan Sheng is on the mycelia like handle, 45~46 μm of diameter, is coated with clear, colorless, thickness 2~4
μm, the mycoderm formed by mycelia like capsule is constituted；Ascus early stage disappears, and ascospore is wide oval 6~7 × 4.5~5 μm.
4th, the strain stability feature of Zhejiang work red yeast rice 1：
Respectively by Zhejiang work red yeast rice No. 1 be seeded on CM culture mediums and MEA culture mediums, under the conditions of 30 DEG C, through the continuous training of number generation
Support, its cultural characteristic and morphological feature have no significant change, the biological character of the bacterium is stable.
3rd, the ITS sequence analysis of Zhejiang work red yeast rice 1
1st, the amplification and sequencing of ITS sequence
Monascus Zhejiang No. 1 genomic DNA of work, the template reacted as PCR are extracted using CTAB methods；The amplification in ITS regions
Primer is ITS4：5 '-TCCTCCGCTTATTGATATGC-3 ', ITS5：5’-GGAAGTAAAAGTCGTAACAAGG-3’.PCR is anti-
The system is answered to be：500ng genomic DNAs, 50pmol primer Is TS4,50pmol primer I TS5,800 μM of dNTP mixtures, 3 μ L 10
× buffer, 1 μ L Pfu DNA Polymerase, plus ddH2O is to 30 μ L cumulative volumes.PCR response procedures are：First stage, 95
DEG C pre-degeneration 3min；Second stage, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension lmin, totally 30 circulations；3rd rank
Section, 72 DEG C of extension 7min.PCR primer, 1% Ago-Gel, the operation of 100v voltages are detected using agarose gel electrophoresis
20min, using gel imaging system analysis result.PCR primer is using DNA glue reclaims kit (raw work, Shanghai) purifying, purifying
ITS genetic fragments afterwards carry out DNA sequencing by Sangon Biotech (Shanghai) Co., Ltd., obtain 584bp nucleotide sequence
(see shown in SEQ ID NO.1).
2nd, the homologous comparison and the foundation of systematic evolution tree of ITS sequence
The ITS complete sequences bacterial strain higher with similitude in GenBank is compared, the higher sequence of homology is chosen
Compare and contribute.Using adjacent method (Neighbor-joining), with MEGA V5.2 programs, from Kimura-2-parameter
Gap model carries out Phylogenetic Analysis.Confidence level is repeated 1000 times using bootstrap analyses.Fig. 2 is based on constructed by ITS
Phylogenetic tree.
According to the Zhejiang work red yeast rice ITS analysis results of No. 1, with reference to above-mentioned Morphological Identification result, confirm that Zhejiang work red yeast rice 1 is
Monascus purpureus (Monascus purpureus), is named as monascus purpureus (Monascus purpureus) Zhejiang work red yeast rice 1.
No. 1 liquid spawn of monascus purpureus Zhejiang work red yeast rice of embodiment 2 prepares polished rice fermented product
(1) preparation of No. 1 liquid spawn of monascus purpureus Zhejiang work red yeast rice
By monascus purpureus Zhejiang work red yeast rice 1 on EMA culture mediums, 32 DEG C are cultivated 7 days, with spore under sterile washing, by spore
Son sterilized water adjustment concentration is 106~107Cfu/mL spore suspensions.
By 106~107Cfu/mL spore suspensions are seeded to fluid nutrient medium with 10% (v/v) inoculum concentration, in 32 DEG C of cultures
2 days, nutrient solution was taken as liquid spawn.
The fluid nutrient medium quality final concentration is constituted：100 mesh rice meals 1%, glucose 3%, peptone 1%,
NaNO30.3%, KH2PO40.1%, solvent is running water, and pH value is natural.
(2) preparation of No. 1 fermented product of monascus purpureus Zhejiang work red yeast rice
2g glucose, 5g peptones and 30mL running water are added into 100g polished rices, then under 0.15MPa high steams
Sterilize 30min, and fermentation medium is made.When fermentation medium temperature is cooled to 43 DEG C, liquid prepared by access 10mL steps (1)
Body strain, cultivates 12d under conditions of 25 DEG C of temperature, humidity 60%, cultivates fermenting mixture after terminating in 0.15MPa high pressures
Sterilize 30min under steam, and 55 DEG C of vacuum dryings obtain the red yeast rice containing 1-DNJ to constant weight after decompression.1- will be contained to take off
The red yeast rice of oxygen nojirimycin wears into 80 mesh powder in 55 DEG C of vacuum dryings, respectively with the ethanol water of volumetric concentration 70% of 5 times (v/w)
25 DEG C of solution is extracted 3 times, and 24h is extracted every time, and extract solution merges, filtering, is concentrated in vacuo to paste, obtains concentrate a；Will concentration
Thing a carries out silica gel (200-300 mesh) column chromatography, is eluted successively with ethyl acetate, absolute ethyl alcohol, distilled water, collects distillation
The component of water elution, is concentrated in vacuo to dry, acquisition concentrate b；Concentrate b is passed through into neutral alumina (200-300 mesh) post again
Chromatography, first with n-hexane, then with ethyl acetate: petroleum ether (1:1, v/v) elute, collect ethyl acetate: the eluent of petroleum ether,
It is concentrated in vacuo to dry, acquisition concentrate c；Concentrate c is chromatographed using preparative TLC, with normal propyl alcohol: acetic acid: water (8:2:1, v/
V) it is solvent, is developed the color using chloro- tolidine, reclaim silica gel at the colour developing being consistent with 1-DNJ standard items, uses
It is concentrated and dried after the dissolving of the ethanol water of volumetric concentration 50% and obtains alpha-glucosidase restrainer monomer, through mass spectrum (see figure
1) its molecular weight is analyzed for 163Da, is further carried out using high performance liquid chromatograph UV-detector and fluorescence detector qualitative
Analysis, sample is consistent with standard items retention time (see Fig. 3), and it is 1-DNJ to determine the inhibitor.
No. 1 solid spawn of monascus purpureus Zhejiang work red yeast rice of embodiment 3 prepares long-grained nonglutinous rice fermented product
(1) preparation of No. 1 bacterial strain solid spawn of monascus purpureus Zhejiang work red yeast rice
By monascus purpureus Zhejiang work red yeast rice 1 on EMA culture mediums, 32 DEG C are cultivated 7 days, with spore under sterile washing, by spore
Son sterilized water adjustment concentration is 106~107Cfu/mL spore suspensions.
It is 10 by concentration6~107Cfu/mL spore suspensions are seeded to solid medium with 10% (v/w) inoculum concentration, in temperature
Spend for 30 DEG C, humidity is to cultivate 10 days under conditions of 80%, when culture medium is all changed into red, micro- sem observation media surface
There is ascospore, culture mix is used as solid spawn.
Solid medium by rice, glucose, peptone and tap water group into, the rice and glucose, peptone and
Running water mass ratio is 1:0.01:0.005:0.3.
(2) preparation of No. 1 fermented product of monascus purpureus Zhejiang work red yeast rice
1g glucose is added into 100g long-grained nonglutinous rices, 2g peptones, 20ml running water sterilizes under 0.15MPa high steams
30min, is made fermentation medium, when culture medium temperature is cooled to about 43 DEG C, solid spawn 5g prepared by access step (1),
9d is cultivated under conditions of 35 DEG C of temperature, humidity 80%, culture goes out culture mix after terminating under 0.15MPa high steams
Bacterium 30min, 55 DEG C of vacuum dryings obtain the red yeast rice containing 1-DNJ to constant weight after decompression.Using high performance liquid chromatography
Method is analyzed, and the content of the 1-DNJ in long-grained nonglutinous rice fermentation red yeast rice is 1.79mg/g.(3) high-efficient liquid phase chromatogram technique analysis are red
1-DNJ in song
The red yeast rice containing 1-DNJ of 5.0g steps (2) preparation, plus 0.05mol/L HCl solution 15mL are weighed,
Ultrasonication 30min, 12000r/min centrifugation, collects supernatant, produces red yeast rice sample extracting solution.
1-DNJ standard solution and red yeast rice sample extracting solution perform the derivatization processing first：Take and treat derivatization
The μ L of solution 10 add 10 μ L sodium borate buffer liquids (0.2mol/L, pH8.5), 20 μ L FMOC-Cl acetonitriles in 1.5mL centrifuge tubes
Solution (5mmol/L), is mixed, 30 DEG C of reaction 20min；10 μ L glycine solutions (0.1mol/L) are added, 20min is reacted；Most
Afterwards plus the glacial acetic acid aqueous solutions of 950 μ L 0.1%, mixed liquor (0.22 μm) filtering of film, sample introduction HPLC analyses.
HPLC testing conditions are：Liquid chromatograph Waters2695, chromatographic column Agilent 4.6 × 250mm of SB-C18,
Mobile phase is acetonitrile: 0.1% glacial acetic acid (40:60, v/v), flow velocity 1.0mL/min, Detection wavelength 254nm, 25 DEG C of column temperature.
No. 1 liquid spawn of monascus purpureus Zhejiang work red yeast rice of embodiment 4 prepares fermented soybean product
(1) preparation of No. 1 liquid spawn of monascus purpureus Zhejiang work red yeast rice
It is 10 by concentration6~107Cfu/mL spore suspensions are seeded to fluid nutrient medium with 10% (v/v) inoculum concentration, 32
DEG C culture 2 days, take nutrient solution as liquid spawn.
The fluid nutrient medium quality final concentration is constituted：Soluble starch 4%, sucrose 0.5%, peptone 0.3%,
NaNO30.5%, KH2PO40.5%, solvent is running water, and pH value is natural.
(2) preparation of No. 1 fermented product of monascus purpureus Zhejiang work red yeast rice
10g glucose, 1.5g peptones and 80mL running water are added into 100g soybean, is then steamed in 0.15MPa high pressures
Sterilize 30min under vapour, and fermentation medium is made.When fermentation medium temperature is cooled to 43 DEG C, prepared by access 30mL steps (1)
Liquid spawn, cultivate 10d under conditions of 37 DEG C of temperature, humidity 90%, culture terminate after by fermenting mixture in 0.15MPa
Sterilize 30min under high steam, and 55 DEG C of vacuum dryings obtain the red yeast rice containing 1-DNJ to constant weight after decompression.Using
The content of 1-DNJ in high-efficient liquid phase chromatogram technique analysis, fermented soybean red yeast rice is 0.81mg/g.
No. 1 liquid spawn of monascus purpureus Zhejiang work red yeast rice of embodiment 5 prepares millet fermented product
(1) preparation of No. 1 liquid spawn of monascus purpureus Zhejiang work red yeast rice
It is 10 by concentration6~107Cfu/mL spore suspensions are seeded to fluid nutrient medium with 10% (v/v) inoculum concentration, 32
DEG C culture 2 days, take nutrient solution as liquid spawn.
The fluid nutrient medium quality final concentration is constituted：Soluble starch 2%, sucrose 2%, peptone 3%,
NaNO30.1%, KH2PO40.3%, solvent is running water, and pH value is natural.
(2) preparation of No. 1 fermented product of monascus purpureus Zhejiang work red yeast rice
8g glucose, 8g peptones and 40mL running water are added into 100g soybean, then under 0.15MPa high steams
Sterilize 30min, and fermentation medium is made.When fermentation medium temperature is cooled to 43 DEG C, liquid prepared by access 25mL steps (1)
Body strain, cultivates 9d under conditions of 40 DEG C of temperature, humidity 70%, cultivates fermenting mixture after terminating in 0.15MPa high pressures
Sterilize 30min under steam, and 55 DEG C of vacuum dryings obtain the red yeast rice containing 1-DNJ to constant weight after decompression.Using efficient
The content of 1-DNJ in liquid chromatography analysis, millet fermentation red yeast rice is 0.92mg/g.
No. 1 solid spawn of monascus purpureus Zhejiang work red yeast rice of embodiment 6 prepares glutinous rice fermented product
(1) preparation of No. 1 solid spawn of monascus purpureus Zhejiang work red yeast rice
It is 10 by concentration6~107Cfu/mL spore suspensions are seeded to solid medium with 10% (v/w) inoculum concentration, in temperature
Spend for 30 DEG C, humidity is to cultivate 10 days under conditions of 80%, when culture medium is all changed into red, micro- sem observation media surface
There is ascospore, culture mix is used as solid spawn.
Solid medium by rice, glucose, peptone and tap water group into, the rice and glucose, peptone and
Running water mass ratio is 1:0.01:0.005:0.3.
(2) preparation of No. 1 fermented product of monascus purpureus Zhejiang work red yeast rice
1g glucose, 15g peptones and 45mL running water are added into 100g glutinous rice, then in 0.15MPa high steams
Lower sterilizing 30min, is made fermentation medium.When fermentation medium temperature is cooled to 43 DEG C, solid prepared by access step (1)
Strain 0.5g, cultivates 15d under conditions of 25 DEG C of temperature, humidity 60%, and culture is high in 0.15MPa by fermenting mixture after terminating
Sterilize 30min under pressure steam, and 55 DEG C of vacuum dryings obtain the red yeast rice containing 1-DNJ to constant weight after decompression.Using height
Effect liquid phase chromatogram method is analyzed, and the content of the 1-DNJ in glutinous rice fermentation red yeast rice is 1.65mg/g.
No. 1 liquid spawn of monascus purpureus Zhejiang work red yeast rice of embodiment 7 prepares Black-rice Wine product
(1) preparation of No. 1 liquid spawn of monascus purpureus Zhejiang work red yeast rice
It is 10 by concentration6~107Cfu/mL spore suspensions are seeded to fluid nutrient medium with 10% (v/v) inoculum concentration, 32
DEG C culture 2 days, take nutrient solution as liquid spawn.
The fluid nutrient medium quality final concentration is constituted：Soluble starch 3%, glucose 1%, peptone 2%,
NaNO30.2%, KH2PO40.2%, solvent is running water, and pH value is natural.
(2) preparation of No. 1 fermented product of monascus purpureus Zhejiang work red yeast rice
5g glucose, 6g peptones and 50mL running water are added into 100g black rices, then under 0.15MPa high steams
Sterilize 30min, and fermentation medium is made.When fermentation medium temperature is cooled to 43 DEG C, liquid bacteria prepared by access step (1)
20mL is planted, 15d is cultivated under conditions of 28 DEG C of temperature, humidity 80%, is cultivated fermenting mixture after terminating in 0.15MPa high pressures
Sterilize 30min under steam, and 55 DEG C of vacuum dryings obtain the red yeast rice containing 1-DNJ to constant weight after decompression.Using efficient
The content of 1-DNJ in liquid chromatography analysis, Black-rice Wine red yeast rice is 0.73mg/g.
No. 1 solid spawn of monascus purpureus Zhejiang work red yeast rice of embodiment 8 prepares corn fermentation product
(1) preparation of No. 1 solid spawn of monascus purpureus Zhejiang work red yeast rice
It is 10 by concentration6~107Cfu/mL spore suspensions are seeded to solid medium with 10% (v/w) inoculum concentration, in temperature
Spend for 30 DEG C, humidity is to cultivate 10 days under conditions of 80%, when culture medium is all changed into red, micro- sem observation media surface
There is ascospore, culture mix is used as solid spawn.
Solid medium by rice, glucose, peptone and tap water group into, the rice and glucose, peptone and
Running water mass ratio is 1:0.01:0.005:0.3.
(2) preparation of No. 1 fermented product of monascus purpureus Zhejiang work red yeast rice
6g glucose, 10g peptones and 70mL running water are added into 100g corns, then in 0.15MPa high steams
Lower sterilizing 30min, is made fermentation medium.When fermentation medium temperature is cooled to 43 DEG C, solid prepared by access step (1)
Strain 3g, cultivates 15d under conditions of 32 DEG C of temperature, humidity 70%, cultivates fermenting mixture after terminating in 0.15MPa high pressures
Sterilize 30min under steam, and 55 DEG C of vacuum dryings obtain the red yeast rice containing 1-DNJ to constant weight after decompression.Using efficient
The content of 1-DNJ in liquid chromatography analysis, corn fermentation red yeast rice is 0.45mg/g.
Claims (8)
- Monascus purpureus 1. (Monascus purpureus) Zhejiang work red yeast rice 1, is preserved in Chinese microorganism strain preservation management committee Member's meeting common micro-organisms center, preservation date is on July 18th, 2014, and preservation address is：BeiChen West Road, Chaoyang District, BeiJing City 1 Institute 3, deposit number is：CGMCC No.9553.
- 2. work red yeast rice No. 1 application in 1-DNJ is prepared in monascus purpureus Zhejiang described in a kind of claim 1.
- 3. application as claimed in claim 2, it is characterised in that described application is trained for No. 1 with monascus purpureus Zhejiang work red yeast rice through liquid The culture of base or solid medium culture acquisition is supported as strain, fermentation medium is seeded to, in 25~40 DEG C of temperature, humidity Solid state fermentation is carried out under conditions of 60%~90%, after the completion of fermentation, the tunning containing 1-DNJ is obtained, will send out Ferment product is isolated and purified, and obtains 1-DNJ；The fermentation medium by edible grain, glucose, peptone and from Water is constituted, and the mass ratio of the edible grain and glucose, peptone and running water is 1:0.01~0.1:0.015~ 0.15:0.2~0.8；The fluid nutrient medium quality final concentration is constituted：70~120 mesh rice meals or soluble starch 1%~4%, glucose Or sucrose 0.5%~3%, peptone 0.3%~3%, NaNO30.1%~0.5%, KH2PO40.1%~0.5%, solvent is Running water, pH value is natural；The solid medium is made up of rice, glucose, peptone and running water, the rice and glucose, peptone and The mass ratio of running water is 1:0.01:0.005:0.3.
- 4. application as claimed in claim 3, it is characterised in that the edible grain be long-grained nonglutinous rice, polished rice, glutinous rice, red rice, black rice, Millet, soya bean, barley or corn.
- 5. application as claimed in claim 3, it is characterised in that the solid spawn and edible grain mass ratio be 0.005~ 0.05：1, the liquid spawn volume consumption is calculated as 0.1~0.3mL/g with edible grain quality.
- 6. application as claimed in claim 3, it is characterised in that the monascus purpureus Zhejiang work red yeast rice 1 is with 106~107cfu/mL The form of spore suspension is seeded to the culture of fluid nutrient medium or solid medium culture acquisition as strain；The spore hangs Liquid be by monascus purpureus Zhejiang work red yeast rice No. 1 be seeded on EMA culture mediums, 32 DEG C cultivate 7 days, with spore under sterile washing, by spore Son sterilized water adjustment concentration is 106~107cfu/mL。
- 7. application as claimed in claim 6, it is characterised in that the preparation method of the strain is one of following：(1) by concentration For 106~107Cfu/mL monascus purpureus Zhejiang No. 1 spore suspension of work red yeast rice is seeded to Liquid Culture with the inoculum concentration of volumetric concentration 10% Base, cultivates 2 days at 32 DEG C, takes culture as liquid spawn；Fluid nutrient medium quality final concentration is constituted：100 mesh rice meals 2%, glucose 2%, peptone 0.8%, NaNO30.2%, KH2PO40.15%, solvent is running water, and pH value is natural；(2) will Concentration is 106~107Cfu/mL monascus purpureus Zhejiang No. 1 spore suspension of work red yeast rice is seeded to solid medium, is 30 DEG C in temperature, Humidity is to cultivate to media surface and ascospore occur under conditions of 80%, regard culture as solid spawn；The spore Suspension volume consumption is calculated as 0.1mL/g with solid medium quality；The solid medium by rice, glucose, peptone and Tap water group is into the rice is 1 with glucose, peptone and running water mass ratio:0.01:0.005:0.3.
- 8. application as claimed in claim 3, it is characterised in that the tunning isolation and purification method is：1- deoxidations will be contained wild The tunning of buttocks mycin wears into 80 mesh powder in 55 DEG C of vacuum dryings, then with the ethanol water of volumetric concentration 70% at 25 DEG C Extraction, leaching liquor is filtered, and filter vacuum is concentrated into paste, obtains concentrate a；Concentrate a is subjected to silica gel column chromatography, successively Eluted with ethyl acetate, absolute ethyl alcohol, distilled water, collect the component of distillation water elution, be concentrated in vacuo to dry, concentrated Thing b；By concentrate b again by neutral alumina column chromatography, respectively with n-hexane, volume ratio is 1:1 ethyl acetate and oil Ether mixed liquor is eluted as eluant, eluent, collects the eluent containing target components, is concentrated in vacuo to dry, acquisition concentrate c；Will Concentrate c is chromatographed using preparative TLC, using volume ratio as 8:2:1 normal propyl alcohol, acetic acid and water mixed liquid is solvent, is collected Target components, are concentrated and dried after being dissolved with the ethanol water of volumetric concentration 50%, that is, obtain 1-DNJ.
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| Development of inhibitors against lipase and α-glucosidase from derivatives of monascus pigment;Jong Hoon Kim，等;《FEMS Microbiology Letters》;20071101;第276卷(第1期);第93页摘要，第1栏第1段，第98页参考文献部分第4段 * |
| 天然来源的α-葡萄糖苷酶抑制剂的研究进展;卜同，等;《海洋科学》;20101231;第34卷(第2期);第92-95页 * |
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