CN107190026A - A kind of method for improving Monascus secondary metabolite - Google Patents
A kind of method for improving Monascus secondary metabolite Download PDFInfo
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Abstract
The present invention provides a kind of method for improving Monascus secondary metabolite, this method be Monascus liquid state fermentation is initial or fermentation process in add 5 azacytidines.5 azacytidine is added in red yeast rice strains liquid fermentation culture 48h, and addition is 10‑6G/L~1g/L.The present invention can be such that the most of secondary metabolites of Monascus improve, easy, quick, and not have the generation of other harmful side products.
Description
Technical field
The invention belongs to microculture field, it is related to a kind of method for improving Monascus secondary metabolite.
Background technology
Monascus (Monascus purpureus Went.) Chinese nickname red yeast rice, red wine dregs, red rice, be present in trees,
Soil and deposit etc..It is the important microbial resources of China, the extensive natural products with bioactivity can be produced, had
Very high commercial value.The temperature range of monascus growth adaptation is very wide, general 15~42 DEG C of temperature, and optimum temperature is 25~30
DEG C, growth optimum pH is 3.5~5, is resistant to the ethanol at concentrations up to 10%.The well-grown on wort agar culture medium,
Be white at the beginning of bacterium colony, it is aging after become pale pink, purple or grey black, it is form red more.The existing more than one thousand years of the application of red yeast rice
History, its medical functions are early on the books in Chinese Ancient Books, available for making wine, vinegar processed, the colouring agent and flavor enhancement for cooking fermented bean curd,
Also Chinese medicine can be done.Now there are some researches show red yeast rice has reducing blood lipid, hypotensive, anti-oxidant, anticancer, antibacterial, antifatigue, prevention old
The multi-efficiencies such as dementia disease.
The secondary metabolite that Monascus is produced mainly has：Monascorubin, Monacolins classes compound, citrinin and its
His metabolite.Monascorubin is a kind of hybrid pigment, is mainly made up of haematochrome, uranidin and citraurin.On red yeast rice color
The biosynthesis of element, it is considered that possible process is：Polyketide synthases are catalyzed acetyl-CoA first and malonyl CoA generates oneself
Ketone chromophore, the medium chain fatty acid that hexanone chromophore produces with fatty acid synthesis pathway again is generated orange by transesterification
Element, citraurin passes through ammonification reaction and reduction formation haematochrome and uranidin respectively again.Current research shows, amino acid
It is the important factor in order in monascorubin building-up process, different amino acid classes and content can significantly affect monascorubin
Composition and yield.Monacolins class compounds have lipid-loweringing, active anticancer.It is annual right with increasing for hypertensive patient
MonacolinK demand also increases year by year.Therefore finding a kind of simple and efficient method improves its yield, just there is real meaning
Justice.
At present, the purpose for improving Monascus secondary metabolite is mainly reached by regulating and controlling Monascus fermentation process.Such as,
A kind of application number 201710071293.6, entitled invention for the method for promoting red yeast rice strains liquid fermentation to produce red yeast rice citraurin is special
Monascus seed culture fluid is inoculated with profit, this application in after liquid culture medium, is fermented 6-8 days at 30 DEG C, it is after fermentation starts
24h in added into fermentation culture surfactant (Span series, Tween series, TritonX-100, PEG it is serial or
Gleditsia sinensis), to promote Monascus synthetic active substance red yeast rice citraurin in growth course.Application number 201410117001.4, one
The biliquid phase fermentation method of coupling situ extracting fermentation red yeast rice citraurin is planted, passes through carbon nitrogen source component reasonable in design, control and trains
The content and Two Liquid Phases fermentation system of free amino acid in base are supported, the red yeast rice citraurin of synthesis is enriched to rapidly fat-soluble molten
In agent, so as to improve the production efficiency of red yeast rice citraurin.Application number 201610475354.0, one kind improves red in monascus purpureus
The fermentation process of element and uranidin yield, the invention extra addition histidine or tyrosine in without amino nitrogen source culture medium, can
Significantly improve the synthetic quantity of haematochrome or uranidin.
But above-mentioned prior art, method can only all improve the part secondary metabolite of Monascus, restricted application.
Not yet have while improving the relevant report of most of Monascus secondary metabolites at present.
The content of the invention
For above-mentioned the deficiencies in the prior art, the present invention, which provides one kind, makes the most of secondary metabolites of Monascus to carry
High simple, fast method.
To realize above-mentioned target, a kind of method for improving Monascus secondary metabolite of the present invention, methods described is red
The liquid state fermentation of aspergillus initially or in fermentation process adds 5-azacitidine.
The 5-azacitidine is added in red yeast rice strains liquid fermentation culture 48h, it is preferable that the 5-azacitidine is red
Added in aspergillus liquid state fermentation culture 24h.
The 5-azacitidine addition is 10-6G/L~1g/L, optimum addition is 10-2g/L。
The fermentation culture conditions are air bath, 33 DEG C, 200r/min cultures 18 days.
The Monascus is purplish red aspergillus M1 or purplish red aspergillus RP2.
Present inventor has been surprisingly found that during purplish red bent RP2 and M1 liquid state fermentation, adds 5-azacitidine
Afterwards, the secondary metabolite of two bacterial strains is improved, and (that is, monascorubin, citraurin, uranidin and MonacolinK yield is all
Lifted), and Electron microscope showed with the addition of the bacterial strain fold and filiform of 5-azacitidine and increases.Speculate, 5- azepine born of the same parents
Glycosides contributes to the generation of DNA methylation, and the DNA methylation for having a fungi can promote the raising of its metabolite；And fold and silk
Shape thing adds the surface area of cell, makes the secondary metabolite in cell body be easier to excrete, and reduces secondary generation in vivo
Thank to product accumulation, so as to increase secondary metabolite amount.
The present invention has the advantages that：
(1) yield of a variety of Monascus secondary metabolites can be greatly improved.
Of the invention maximum the characteristics of is exactly to be realized by adding a kind of material while improving monascorubin, citraurin, Huang
The purpose of pigment and MonacolinK yield.Present invention research shows to add the two bacterial strain Monascus M1 and red yeast rice of 5-azacitidine
Bacterium RP2 dry cell weight improves 7.7% and 5.1%, red yeast rice color than the bacterial strain without 5-azacitidine original culture medium respectively
In plain color valency haematochrome be respectively increased 19.6% and 15.6%, citraurin be respectively increased 16.3% and 20.6%, uranidin difference
Improve 16.4% and 15%；Monascus M1 MonacolinK output increaseds 58.6%.
(2) economical and efficient.
The inventive method in suitable incubation time only by adding appropriate 5-azacitidine, you can reaches while carrying
The purpose of high a variety of secondary metabolites, it is more economical, efficient relative to method of the prior art.
(3) applicability is wider.
It is typically to add different materials for certain secondary metabolite to improve yield to realize in the prior art
Purpose.Its is with strong points, but restricted application, if it is desired to obtain high yield secondary metabolite, it is necessary to individually send out
Ferment is produced.And the inventive method can just reach the purpose for improving a variety of metabolites simultaneously during one time fermentation.
(4) no coupling product.
The inventive method will not produce other harmful side products during the fermentation, will not increase the yield of citrinin.
Brief description of the drawings
Fig. 1 is the experiment of single factor result that purplish red aspergillus M1 adds the 5-azacitidine time.
Fig. 2 is the experiment of single factor result that purplish red aspergillus RP2 adds the 5-azacitidine time.
Fig. 3 is the experiment of single factor result that purplish red aspergillus RP2 adds 5-azacitidine concentration.
Fig. 4 is the experiment of single factor result that purplish red aspergillus M1 adds 5-azacitidine concentration.
Fig. 5 is purplish red aspergillus M1 experimental group and control group bacterium in 5-azacitidine optimum addition and addition time
The contrast of soma weight.
Fig. 6 is purplish red aspergillus RP2 experimental group and control group bacterium in 5-azacitidine optimum addition and addition time
The contrast of soma weight.
Fig. 7 is that purplish red aspergillus M1 experimental group and control group is red in 5-azacitidine optimum addition and addition time
Bent haematochrome (A), citraurin (B), the contrast of uranidin (C).
Fig. 8 is that purplish red aspergillus RP2 experimental group and control group is red in 5-azacitidine optimum addition and addition time
Bent haematochrome (A), citraurin (B), the contrast of uranidin (C).
Fig. 9 is the purplish red aspergillus M1 experimental group control group in 5-azacitidine optimum addition and addition time,
MonacolinK contrast.
Figure 10 is purplish red aspergillus M1 experimental group (a) and control in 5-azacitidine optimum addition and addition time
Group (b) efficient liquid phase figure.
Figure 11 is the Electronic Speculum after experimental group and control group culture 8 days in 5-azacitidine optimum addition and addition time
Figure.Wherein (a, c, e) is RP2 control strain figures, and (b, d, f) is the RP2 experimental strain figures of addition 5-azacitidine；(g、i、k)
For M1 control strain figures, (h, j, l) is the M1 experimental strain figures of addition 5-azacitidine.
Embodiment
With reference to specific test method and accompanying drawing technical scheme and its produced technique effect are done into
The elaboration of one step, the description below are merely to explain the present invention, but the present invention is not any limitation as in any way, based on this hair
Any conversion or replacement that bright training centre is made, belong to protection scope of the present invention.The method of the invention unless otherwise specified,
It is this area conventional method.Agents useful for same unless otherwise specified, can be obtained from commercial channels.
Embodiment 1
1st, bacterial strain, material
(Beijing Technology and Business University tests by purplish red aspergillus M1 (offer of China Microbiological resource collection) and purplish red aspergillus RP2
Room preservation) bacterial strain, the refrigerator preservation of -80 DEG C of laboratory.
Plating medium：PDA culture medium
RP2 bacterial strain seed liquid culture mediums：Long rice flour 40g/L, peptone 8g/L, bean cake powder 5g/L, KH2PO4 2g/L、
NaNO3 2g/L、MgSO4Under the conditions of 1g/L, 33 DEG C and 200r/min, 48h is shaken in air bath, zymotic fluid of transferring.
RP2 strain fermentation liquid culture mediums：Long rice flour 77g/L, glucose 75g/L, bean cake powder 2g/L, KH2PO4 0.5g/L、
NaNO31.8g/L、MgSO41g/L, corn steep liquor 3.5g/L, air bath shaking table, condition are set to 33 DEG C and 200r/min.
M1 bacterial strain seed liquid culture mediums：Glucose 30g/L, analysis for soybean powder 15g/L, MgSO4 1g/L KH 2PO4It is 2g/L, sweet
Oily 70g/L, peptone 10g/L, NaNO3Under the conditions of 2g/L, 30 DEG C and 200r/min, 48h is shaken in air bath, zymotic fluid of transferring.
M1 strain fermentation liquid culture mediums：Glycerine 90g/L, long rice flour 20g/L, peptone 10g/L, NaNO3 5g/L、
MgSO41g/L、ZnSO4 2g/L、KH 2PO 4Under the conditions of 2.5g/L, 30 DEG C and 150r/min, 48h is shaken in air bath, then adjusts and shake
Riffling part is 25 DEG C and 150r/min.
2nd, the time point of 5-azacitidine addition and addition
5-azacitidine mainly acts on the G2 phases of cell mitogen, the specific period of the G2 phases of current red yeast rice, not
Report is seen, so to determine optimal time.After red yeast rice M1 and red yeast rice RP2 ferments two days, logarithmic phase is initially entered, because
This, the G2 phases of red yeast rice must be in 48 hours of seed liquor sending and receiving zymotic fluid.This experiment was an addition time point with 4 hours,
Addition 5-azacitidine makes 5-azacitidine concentration reach 10 in 50ml zymotic fluid respectively-3G/L, to determine optimal addition
Opportunity.
After optimum time point is obtained, by adding the 5-azacitidine of various concentrations in 50ml zymotic fluids.Make fermentation
5-azacytidine concentration gradient is in liquid：1g/L、10-1g/L、10-2g/L、10-3g/L、10-4g/L、10-5g/L、10-6g/
L.To study 5-azacitidine optimum addition.
Step is as follows：
(1) above-mentioned Monascus Strains and culture medium are used.In 48 hours of Monascus seed liquor sending and receiving zymotic fluid, with 4 hours
For a time point, 5-azacitidine is added respectively in 50ml zymotic fluid, 5-azacitidine concentration in zymotic fluid is reached
10-3g/L.Monascus seed liquor condition of culture：Air bath, 33 DEG C, 200r/min 48 hours sending and receiving zymotic fluids of culture；Fermented and cultured bar
Part：Seed liquor is inoculated into fermentation medium by 10% (V/V) inoculum concentration, air bath, 33 DEG C, 200r/min cultures.Knot
Fruit is as depicted in figs. 1 and 2.
(2) in 24 hours after seed liquor sending and receiving zymotic fluid, the 5-azacitidine of various concentrations is added in 50ml zymotic fluids
In；The concentration gradient of 5-azacitidine is in zymotic fluid：1g/L、10-1g/L、10-2g/L、10-3g/L、10-4g/L、10-5g/L、
10-6g/L.As a result as shown in Figure 3 and Figure 4, when 5-azacitidine is in low concentration, with the increase of concentration, secondary metabolite
Yield increase, reach 10 in 5-azacitidine concentration-2During g/L, yield reaches maximum, continues to increase concentration, 5-azacitidine
Side effect start to occur, the yield of secondary metabolite gradually reduces.
3rd, addition 5-azacitidine experimental group and the control group contrast experiment without 5-azacitidine
Test group in 48h, adds 5-azacitidine, addition is 10 after liquid state fermentation culture starts-2g/L.Control group
Without 5-azacitidine, addition equivalent aqua sterilisa.Compare two groups of dry cell weights, pH value, Monascus Pigments color value,
MonacolinK yield, the 8th day electron microscopic observation mycelium morphology.
3.1st, red yeast rice dry cell weight compares
Will fermentation 2,5,8,10,12,14,16,18 days addition 5-azacitidine experimental group Monascus M1 and RP2 and without
5-azacitidine, control group the Monascus M1 and RP2 for adding equivalent aqua sterilisa, take 5ml zymotic fluids respectively, with 4 layers of filtered through gauze,
With the zymotic fluid on aseptic water washing gauze, untill the liquid flowed out in gauze becomes colorless, dried under the conditions of 40 DEG C to
Constant weight is simultaneously weighed.As a result as shown in Figure 5,6, the growth in beginning U-18496 to cell has a certain impact, and makes experimental group
Dry weight is significantly lower than control group.But from the point of view of general trend, 5-azacitidine has the influence for promoting cell growth.We are from Fig. 5
It can see with Fig. 6, the 8th day starts, add the experimental group of 5-azacitidine than the control group without addition 5-azacitidine,
Dry cell weight is significantly increased, and when fermented and cultured was by 12 days, reaches maximum.Afterwards with the extension of time, cell entrance declines
Move back the phase, the dry weight of bacterium starts to be gradually reduced.
3.2nd, Monascus pH value compares
Survey 2 respectively with pH meter, 5,8,10,12,14,16,18 days experimental groups and control group Monascus M1 and RP2 pH value,
As a result it is as shown in table 1.
The pH value of the test group of table 1 incubation time different with control group
5-azacitidine has faint influence to red yeast rice M1 pH as can be seen from Table 1, to red yeast rice RP2 almost without shadow
Ring.
3.3rd, Monascus Pigments color value compares
Will fermentation 2,5,8,10,12,14,16,18 days, experimental group and control group Monascus M1 and RP2 take 3ml to send out respectively
Zymotic fluid, add 70% ethanol 6ml, water-bath 1h, 4000r centrifugation 15min under the conditions of 60 DEG C, dilute corresponding multiple and (use
70% ethanol is diluted), Monascus color, orange, the color value of uranidin are surveyed under the conditions of 505nm, 465nm, 410nm respectively.With point
Light photometer determines its OD value under wavelength 505nm, and it is haematochrome color value that this value, which is multiplied by extension rate,；Under wavelength 465nm
Its OD value is determined, it is citraurin color value that this value, which is multiplied by extension rate,；Its OD value is determined under wavelength 410nm, this value is multiplied by dilute
It is uranidin color value to release multiple.Color value unit is represented with U/mL.
As a result as Figure 7-8,5-azacitidine is carried to red, orange, uranidin color value in red yeast rice M1 and red yeast rice RP2 pigments
Height has more significant effect.
3.4th, MonacolinK Yield comparisons
By fermentation 2,5,8,10,12,14,16,18 days experimental groups and control group Monascus, 5ml zymotic fluids are taken respectively, then add
Enter 75% methanol 15ml, the ultrasonication 30min under the conditions of 30 DEG C, 300W, lucifuge stands 6 hours, organic with 0.45 μm
Membrane filtration, filtrate is put into liquid phase bottle, surveys MonacolinK contents.
MonacolinK testing conditions：Shimadzu liquid phase, chromatographic column C18Post (150mm × 4.6mm), mobile phase methanol：
0.1% concentration phosphoric acid=75:25, flow velocity：1ml/min, detector：UV-detector, wavelength：237nm, temperature；30℃.
Liquid phase testing result adds the experimental group of 5-azacitidine than no addition 5-azacitidine as shown in Fig. 9,10
Control group, the raising of MonacolinK yield has remarkable result.
3.5th, the 8th day Electronic Speculum is cultivated to compare
(1) under the red yeast rice M1 and red yeast rice RP2 of culture 8 days, 4 DEG C of cryogenic conditions, 12000r/min centrifugation 5min collect thalline
Cell, is resuspended in 2.5% glutaraldehyde solution by cell and fixes 12 hours.
(2) with 0.1M PBS rinsings cell twice, abandoning supernatant.
(3) cell is carried out with the ethanol solution (30%, 50%, 70%, 80%, 90%, 100%) of various concentrations successively
Dehydration, every kind of concentration stands 12000r/min centrifugations 5min, abandoning supernatant under 10min, 4 DEG C of cryogenic conditions.
(4) respectively with isoamyl acetate and ethanol (v：V=1:1) solution and isoamyl acetate solution carry out ethanol to cell
Displacement, cell is resuspended in every kind of solvent and stands 10min, and 12000r/min centrifuges 5min, supernatant discarding under 4 DEG C of cryogenic conditions
Liquid.
(5) hmds is added, the mouth of pipe will be centrifuged beyond the Great Wall with absorbent cotton, 60 DEG C of oven dryings are placed in sample into powder
Last shape, electron microscopic observation.
As shown in figure 11, a-e is red yeast rice RP2 electromicroscopic photograph.Wherein, a (2000 ×), c (5000 ×), e (10000 ×)
It is that control group adds the sterilized water of equivalent instead of the mycelial photos of Monascus RP2 of 5-azacitidine.b(2000×)、d
(5000 ×), f (10000 ×) is the photo for the Monascus RP2 filaments that experimental group adds equivalent 5-azacitidine.As shown by data,
Red yeast rice RP2 control group spores are full, and surface is smooth, and mycelium filiform is less.Experimental group spore surface is coarse, there is more wrinkle
Line and projection, mycelium fold are more, and plumpness is bad.G-l is Monascus M1 electromicroscopic photograph.G (2000 ×), i (5000 ×),
K (10000 ×) is that control group adds the sterilized water of equivalent instead of the mycelial photos of 5-azacitidine Monascus M1, h (2000
×), j (5000 ×), l (10000 ×) is with the addition of the mycelial photos of 5-azacitidine experimental group Monascus M1.As a result table
Bright, the mycelium surface of Monascus M1 control group is smooth, not so much filiform and spore pellet is fuller,
There is unconspicuous wrinkle.The mycelial rough surface of Monascus M1 experimental groups, has many filiforms and spore pellet is not full, have
Wrinkle.
Experimental group thalline fold and filiform are more, add the surface area of cell, make the secondary metabolite in cell body
It is easier to excrete, internal secondary metabolite accumulation is reduced, so as to increase secondary metabolite amount.Explained simultaneously from principle
Confirm that present invention addition 5-azacitidine can improve the amount of secondary metabolite.
Claims (8)
1. a kind of method for improving Monascus secondary metabolite, it is characterized in that, methods described is the liquid state fermentation in Monascus
5-azacitidine is added in initial or fermentation process.
2. the method for Monascus secondary metabolite is improved as claimed in claim 1, it is characterized in that, the 5-azacitidine exists
Added in red yeast rice strains liquid fermentation culture 48h.
3. the method for Monascus secondary metabolite is improved as claimed in claim 2, it is characterized in that, the 5-azacitidine exists
Added in red yeast rice strains liquid fermentation culture 24h.
4. the method for the raising Monascus secondary metabolite as described in claim 1-3 is any, it is characterized in that, the 5- azepines
Cytidine addition is 10-6G/L~1g/L.
5. the method for Monascus secondary metabolite is improved as claimed in claim 4, it is characterized in that, the 5-azacitidine adds
Dosage is 10-2g/L。
6. the method for Monascus secondary metabolite is improved as claimed in claim 1, it is characterized in that, the fermentation culture conditions
For air bath, 33 DEG C, 200r/min cultures 18 days.
7. the method for the raising Monascus secondary metabolite as described in claim 1-6 is any, it is characterized in that, the Monascus
For purplish red aspergillus.
8. the method for Monascus secondary metabolite is improved as claimed in claim 7, it is characterized in that, methods described can be simultaneously
Improve monascorubin, citraurin, uranidin and MonacolinK yield.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913730A (en) * | 2018-07-10 | 2018-11-30 | 长江大学 | A kind of cultural method of Monascus high yield monascus purpureus |
| CN110331151A (en) * | 2019-04-11 | 2019-10-15 | 北京工商大学 | The construction method of purple Monascus mokH gene overexpression bacterial strain |
| CN110592156A (en) * | 2019-10-21 | 2019-12-20 | 北京工商大学 | Application of nicotinamide to improvement of monacolin K produced by monascus purpureus |
| CN110951801A (en) * | 2019-11-25 | 2020-04-03 | 北京工商大学 | Application of polyoxyethylene lauryl ether in production of monacolin K |
| CN115197981A (en) * | 2022-07-12 | 2022-10-18 | 北京工商大学 | Fermentation method for improving synthesis of yellow pigment and orange pigment in monascus purpureus |
| CN115261423A (en) * | 2022-07-12 | 2022-11-01 | 北京工商大学 | Fermentation method for changing anabolism mode of monascus pigment in monascus purpureus and improving synthetic amount |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105969673A (en) * | 2016-06-27 | 2016-09-28 | 北京工商大学 | Fermentation method for increasing yield of haematochrome and uranidin in monascus purpureus |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108913730A (en) * | 2018-07-10 | 2018-11-30 | 长江大学 | A kind of cultural method of Monascus high yield monascus purpureus |
| CN108913730B (en) * | 2018-07-10 | 2021-09-28 | 长江大学 | Method for culturing high-yield lovastatin by using monascus |
| CN110331151A (en) * | 2019-04-11 | 2019-10-15 | 北京工商大学 | The construction method of purple Monascus mokH gene overexpression bacterial strain |
| CN110592156A (en) * | 2019-10-21 | 2019-12-20 | 北京工商大学 | Application of nicotinamide to improvement of monacolin K produced by monascus purpureus |
| CN110951801A (en) * | 2019-11-25 | 2020-04-03 | 北京工商大学 | Application of polyoxyethylene lauryl ether in production of monacolin K |
| CN110951801B (en) * | 2019-11-25 | 2021-04-13 | 北京工商大学 | Application of polyoxyethylene lauryl ether in production of monacolin K |
| CN115197981A (en) * | 2022-07-12 | 2022-10-18 | 北京工商大学 | Fermentation method for improving synthesis of yellow pigment and orange pigment in monascus purpureus |
| CN115261423A (en) * | 2022-07-12 | 2022-11-01 | 北京工商大学 | Fermentation method for changing anabolism mode of monascus pigment in monascus purpureus and improving synthetic amount |
| CN115197981B (en) * | 2022-07-12 | 2023-09-22 | 北京工商大学 | Fermentation method for improving synthesis of yellow pigment and orange pigment in monascus purpureus |
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