CN110468158A - A kind of high yield type monascus strain culture medium and preparation method - Google Patents
A kind of high yield type monascus strain culture medium and preparation method Download PDFInfo
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Abstract
It is an object of the present invention to provide a kind of Monascus Strains cultural methods that monascorubin can be improved.The present invention is research bacterial strain with Monascus Mr △ lig4 bacterial strain, it will be by fermentation medium carbon source, fermentation medium pH, four influence factors of liquid state fermentation incubation time and liquid state fermentation cultivation temperature optimize, and then determine the best liquid state fermentation condition of the bacterial strain, the yield that this bacterial strain produces monascorubin is improved, is supported to provide strong data for the application of this bacterial strain in the future.
Description
Technical field
The present invention designs a kind of high yield type monascus strain culture medium and preparation method.
Background technique
Monascus: on classification of fungi, monascus belongs to Ascomycotina, Ascomycetes, Eurotiale, monascaceae.It is red
There are also many subspecies for aspergillus, can produce many secondary metabolites, theirs what is common is that all have polyketone structure.It has
Many branches have tabula between mycelia.And over time, thallus color will become the later period from the colourless of initial stage
Aubergine.After mycelia comes to the ripening period, thallus branch top can grow the conidium and orange-red cleistothecium of brown.I
For a long time, more than 1000 years existing so far, Monascus is taken as enzyme system to the historical origin that state utilizes about Metabolites ofMonascus
Agent, Successful utilization is in the food fermentation production of some Asian countries.At the same time, before monascorubin also has as one kind very much
Scape and the food additives for being full of potentiality, are used in the industrial production of the U.S. and some European Union member countries.Establish monascus ruber
The French scholar of category is that screening has separated two fungal strains from potato culture earliest, then, the researcher of countries in the world
It is all sequentially added in the research of Monascus.Although China is the big country of monascus product production and application, in basic research
Obviously fall behind.Many secondary metabolites that Monascus generates include monascorubin, Lovastatin, ergosterol, citrinin etc.
All there is special bioactivity.Wherein monascorubin has many healthcare functions, for example, reducing blood lipid, hypoglycemic, blood pressure lowering and
Inhibit tumour cell.Compared with chemical synthesis pigment, microbial fermentation production natural food colour by flexible fermentation condition,
Higher feed stock conversion and it is at low cost many advantages, such as, become a kind of development trend.Monascorubin: Monascus produces red
Bent pigment has long history and wide application prospect as a kind of natural food colour and preservative[7].Monascorubin
Belong to anthraquinone derivative, color is scarlet strong, is dissolved in second alcohol and water, highly-safe (to coexist with metal ion, reductant-oxidant
It can keep stability[8]), thermal stability is strong, has no toxic side effect, and strong to the coloring of protein.Often in pure mellow wine, sauce
For colouring when the production of the food such as oil, fermented bean curd, beans sauce, fish, meat, cake.It relies on stable yield, cheap, production speed
Spending the advantages that fast far surpasses other natural pigments.At the same time, the safety of synthetic dyestuff also receives unprecedented challenge.It cuts
To currently, we have found the monascorubins of nearly 100 kinds known structures.Through acute toxicity test, subacute toxicity
After test, chronic toxicity test and mutagenicity test, monascorubin all shows non-toxic also without aberration inducing effect[10].It is existing
Nowadays, monascorubin has relied on the plurality of advantages of oneself, becomes a kind of natural food colour favored by various countries, becomes us and gives birth to
Very common food color in work.But since the production capacity that Monascus produces monascorubin is lower, influence the hair of monascorubin
Exhibition and application.Nowadays, the matter of utmost importance that the yield of monascorubin is researchers at home and abroad how is improved, is currently known main
Have following two technological means: one is utilize technique for gene engineering screening, the higher red yeast rice bacteria strain of mutagenesis yield；It is another
Kind method is exactly the color value for improving Monascus by optimum culture condition and producing monascorubin fermentation liquid[16-17].Due to our mesh
The preceding research for Mutation Mechanism is not still apparent, it is difficult to by artificially controlling and workload is cumbersome, acquired mutant strain
Without stability, still there is again a possibility that this back mutation.Therefore, pass through monascorubin product obtained by genetic engineering
Technological means still has very big limitation and unknown difficulty at present.Monascus production is improved by optimum culture condition
Color value in monascorubin fermentation liquid is temporarily most commonly seen technological means.The technique fermentation method of monascorubin is divided into solid-state
Fermentation and liquid state fermentation.Compared with traditional solid state fermentation, liquid state fermentation is with short production cycle, consumes low, yield height, however main
The shortcomings that be produce monascorubin color value it is generally relatively low.Therefore, either domestic or external, all attempt by changing red yeast rice
Bacterium condition of culture and training method, to improve the color value of monascorubin as far as possible.
Summary of the invention
It is an object of the present invention to provide a kind of Monascus Strains cultural methods that monascorubin can be improved.The present invention is with Monascus Mr
△ lig4 bacterial strain is research bacterial strain, will be by fermentation medium carbon source, fermentation medium pH, liquid state fermentation incubation time and liquid
Four influence factors of state fermented and cultured temperature optimize, and then determine the best liquid state fermentation condition of the bacterial strain, improve this
Bacterial strain produces the yield of monascorubin, supports to provide strong data for the application of this bacterial strain in the future.One kind can be improved
The Monascus Strains cultural method of monascorubin: bacterial strain selects first: bacterial strain of the invention is Monascus Strains Mr △ lig4.Secondly it trains
Support base preparation: dextrose solid medium and dextrose broth.Dextrose solid medium (1 L): glucose 30-50
g；Beef extract 2-4 g；Peptone 6-9g；Yeast extract (winter 4-6g, summer 2-4g),；Sodium nitrate 1-3 g；Magnesium sulfate 0.5-1.5
g；Agar 10-30 g, tryptophan 5-15g, glycine 5-15g, serine 7-12g.(pH is adjusted to 4-6.0).Glucose solids training
It supports the preparation (1 L) of base: weighing glucose 30-50 g, beef extract 2-4g, peptone 7-9g, yeast extract (winter 4-6 g, summer
2-4 g), sodium nitrate 1-3 g, magnesium sulfate 0.5-1.5 g are added distilled water and are settled to 1 L in the beaker of 1 L, then use 0.05-
0.15 mol/L hydrochloric acid solution adjusts medium pH to 4-6.0.The culture medium finally is dispensed with four 250 mL reagent bottles, and
By 10-30 g agar, tryptophan 5-15g, glycine 7-15g, serine 7-15g are divided into four parts, pour into reagent bottle.Then
Reagent bottle is put into high-pressure steam sterilizing pan together with the thing that other needs sterilize, selects 121 DEG C, sterilize 10-30 min
Mode carries out high pressure steam sterilization.It is stored after cooling spare.Glucose inclined-plane solid medium: ultraviolet-sterilization is being had already passed through
The production that test tube slant culture medium is carried out on aseptic operating platform, is put into sterile behaviour for the dextrose culture-medium for bacterium of having gone out as early as possible
Make platform (if solidified, need heat make its thawing), waits when being cooled to temperature and being about 30 DEG C -40 DEG C, while hot to
Sterile empty test tube pours into the dextrose culture-medium of 2-3 mL, with guaranteeing nothing after the alcolhol burner flame calcination rubber stopper test tube mouth several seconds
Bacterium, after cover rubber stopper for its slant setting on a pipette, wait it completely cooling.It repeats above operation until knot
Beam.5-8 branch test tube is wrapped with brown paper to be fixed with rubber band again, is put into 4 DEG C of refrigerator cold-storages, is waited spare.Inclined-plane completes
Aseptic operating platform table top is cleared up afterwards.Dextrose broth: dextrose broth (1 L): glucose 30-50 g；Ox
Meat extract 2-4 g；Peptone 6-9g；Yeast extract (winter 4-6 g, summer 2-4g),；Sodium nitrate 1-3 g；Magnesium sulfate 0.5-1.5g,
Tryptophan 5-15g, glycine 5-15g, serine 7-12g.(pH is adjusted to 4.0-6.0).The preparation (1 of dextrose broth
L): weighing glucose 30-50 g, beef extract 2-4 g, peptone 7-9 g, yeast extract (winter 54-6g；Summer 2-4 g)；, nitre
Sour sodium 1-3g, tryptophan 5-15g, glycine 7-15g, serine 7-12g, magnesium sulfate 0.5-1.5g are added in the beaker of 1 L
Distilled water is settled to 1 L, then adjusts medium pH to 4.0-6.0 with 0.05-0.15 mol/L hydrochloric acid solution.Finally pour into 250
In mL conical flask, each conical flask pours into 150 mL solution.After covering rubber stopper, conical flask is wrapped into brown paper, uses rubber band
It is fixed, it finally puts into high-pressure steam sterilizing pan, selects 121 DEG C, sterilizing 10-30 min mode sterilizes.Main agents and
(1) 0.1 mol of preparation/L hydrochloric acid solution (1 L) of solution: the concentrated hydrochloric acid solution 8.3 that concentration is 36.5 % is measured with graduated cylinder
ML is poured into the beaker of clean dried after measurement, is diluted with distilled water, is then drained with glass bar by the solution after complete dilution
It pours into 1000 mL volumetric flasks, then is washed with distilled water beaker and glass bar two to three times, it should be noted that cleaning solution
It pours into volumetric flask, then adds distilled water to graduation mark very about 2 centimetres of top into volumetric flask, use rubber head dropper drop instead
Add, until concave meniscus lowest part and graduation mark keep horizontal, covers plug mixing, pour into reagent bottle, it is labelled spare.
(2) 70 %(v/v) ethanol solution (500 mL): measuring concentration with graduated cylinder is 95 % ethanol solution, 368.4 mL in clean beaker
It is interior, pure distilled water is added to 500 mL, after shaking up, transfers in reagent bottle, it is spare to post reagent label.Into one
Step ground, actication of culture: taking the bacterial strain to grow fine to be inoculated with, complete on the aseptic operating platform for have already passed through ultraviolet-sterilization
It is operated at following actication of culture: to avoid living contaminants, by the calcination on alcolhol burner flame of transfer needle whole body, until syringe needle burning is red
Afterwards, the inboard wall of test tube to be cooled that extendes back is waited (transfer needle to be placed on inboard wall of test tube, appropriate cooling is carried out and excessively high temperature is avoided to make
Monascus inactivation).With oese from one fritter red yeast rice of picking on Monascus inclined-plane, the glucose solids made are put it into
In test tube slant, so that it is scraped on inclined-plane and (is paid attention to needing the one side and contact of incline plane for having Monascus) with transfer needle promotion, protect
It demonstrate,proves after entire solid slope surface layer is all uniformly distributed Monascus and takes out red yeast rice block.Beyond the Great Wall before rubber stopper, calcination test tube mouth and
Rubber stopper.It repeats aforesaid operations and continues inoculation until terminating, remembering to be inoculated with a new test tube every time will calcination loop-carrier and cold
But.Inclined-plane solid medium after inoculation is placed in culture activation 7-10 days in 28 DEG C of mold incubator.It probably connects every time
Kind 15 guarantees sufficient every a batch of inoculation in 2 days.Period be also required to often observe bacterium growing way and whether microbiological contamination, once hair
Existing microbiological contamination, abandons in time.Further, seed culture: the test tube slant of culture 15 days or so is taken to be put into nothing from mold incubator
Bacterium station completes following operation on the aseptic operating platform for have already passed through ultraviolet-sterilization: with the liquid-transfering gun with sterilizing pipette tips
Inject 5 mL sterile waters in every test tube, then with by after alcolhol burner high temperature sintering and the thick loop-carrier that cools down is on inclined-plane
It scratches back and forth, this purpose is in order to which spore to be disengaged in sterile water from media surface.Again with the nothing for cutting pipette tips front end
Bacterium pipette tips draw spore suspension, and the sterile cone for having 8-10 bead is flowed by the funnel filtering with sterile lens wiping paper
In shape bottle.After the completion of to be filtered, stoppered with rubber stopper and wrap brown paper.In order to be collected into more spores as far as possible, need
It is 150 r/min that conical flask, which is put into revolving speed, shakes 20-30 min in the shaking table that temperature is 28 DEG C.Further, fermented and cultured:
Through by completing following operation on the aseptic operating platform of ultraviolet-sterilization: after shaking table, conical flask is rocked, by spore suspension point
In centrifuge tube after being packed into sterilizing, then put centrifuge 10 min of interior centrifugation of 8000 r/min into, after centrifugation, upper layer is clear
Liquid collects concentration spore liquid.Drawing 1 uL concentration spore liquid with liquid-transfering gun (will be concentrated spore liquid in the sterile water of 99 uL
100 times of dilution, and notice that dilution is uniform), the concentration spore liquid after dilution is counted on electron microscope using blood counting chamber
Spore.Spore count is calculated, so that it is determined that be added to the content in dextrose culture-medium.Concentration spore liquid equivalent is injected into
It is 150 r/min in revolving speed, temperature is that 28 DEG C of constant temperature incubation is shaken in the dextrose broth of the mL of 150 mL/250
Oscillation 12-16 days in bed.Finally, blood counting chamber count number spore: directly being counted under the microscope using blood counting chamber, be
A kind of common microbial enumeration method.The method has the advantages that directly and quick.Will by appropriate diluted bacteria suspension (or
Spore suspension) it is placed in the counting chamber between blood counting chamber glass slide and coverslip, it is counted under the microscope.Take out one
A clean blood counting chamber (blood counting chamber in our laboratories is 25*16 type), lid one opens 20 mm* above counting chamber
The coverslip of 20 mm is squeezed into the spore liquid that liquid-transfering gun draws about 10 μ L or so along the edge of coverslip, keeps spore liquid slow
It is slow to penetrate into counting chamber and be careful not to generate bubble, the extra spore liquid in coverslip edge is gently wiped completely, whole spores are waited
After son is deposited in blood count room, start microscopic counting.If 25 total spore counts of medium square are A, spore liquid is diluted to B,
So, total spore count in a block plaid (i.e. total spore count of 0.1 mm3) is A × B；Because of the cm of 1 mL=13=1000 mm3, 1
Sum=A × B × 1000 × 10=10000 A × B in mL suspension.The invention has the advantages that: pass through red yeast rice engineering strain
Industrial fermentation process conditions (culture medium prescription, initial p H, inoculum concentration, cultivation temperature, fermentation period etc.), will greatly improve
There is the recovery rate of potency monascorubin, 85% or more will be improved than originally.This experiment has determined best fermentation by experiment of single factor
Culture medium carbon source, the best liquid state fermentation incubation time of optimal initial fermentation medium pH and best liquid state fermentation cultivation temperature four
A factor.Optimised process is as follows: using glucose as carbon source, initial medium pH is 4.5,16 d of fermented incubation time, culture temperature
Degree is 28 DEG C.
Figure of description
Influence of Fig. 1 different carbon source to color value outside monascorubin spore.Shadow of Fig. 2 different carbon source to color value in monascorubin spore
It rings.Different initial influences of the pH to pigment color value outside spore of Fig. 3 fermentation liquid.The different initial pH of Fig. 4 fermentation liquid are to pigmentary color in spore
The influence of valence.Influence of Fig. 5 difference incubation time to pigment color value outside spore.Fig. 6 difference incubation time is to pigment color value in spore
Influence.Influence of Fig. 7 cultivation temperature to pigment color value outside spore.Influence of Fig. 8 cultivation temperature to pigment color value outside spore.
Specific embodiment
The present embodiment provides a kind of Monascus Strains cultural methods that monascorubin can be improved for embodiment 1: bacterial strain selects first
Select: bacterial strain of the invention is Monascus Strains Mr △ lig4.Secondly preparation of culture medium: inclined-plane solid medium and glucose solids are trained
Support base.Dextrose solid medium (1 L): 40 g of glucose；3 g of beef extract；8 g of peptone；Yeast extract (winter, summer) 5
G, 3 g；2 g of sodium nitrate；1 g of magnesium sulfate；Agar 20 g, tryptophan 10g, glycine 12g, serine 9g.(pH is adjusted to 5.0).
The preparation (1 L) of dextrose solid medium: 40 g of glucose, 3 g of beef extract, 8 g of peptone, yeast extract (winter 5 are weighed
G, g) 2 g of sodium nitrate, 1 g of magnesium sulfate were added distilled water and were settled to 1 L in the beaker of 1 L summer 3, then with 0.1 mol/
L hydrochloric acid solution adjusts medium pH to 5.0.The culture medium finally is dispensed with four 250 mL reagent bottles, and by 20 g agar, color
Propylhomoserin 10g, glycine 12g, serine 9g are divided into four parts, pour into reagent bottle.Then reagent bottle is needed to sterilize with other
Thing put into high-pressure steam sterilizing pan together, select 121 DEG C, sterilizing 20 min modes carry out high pressure steam sterilization.It is cooling
After store it is spare.Glucose inclined-plane solid medium: test tube slant is carried out on the aseptic operating platform for have already passed through ultraviolet-sterilization
The dextrose culture-medium for bacterium of having gone out is put into aseptic operating platform as early as possible and (if solidified, needs to add by the production of culture medium
Heat makes its thawing), it waits when being cooled to temperature and being about 30 DEG C -40 DEG C, pours into 2-3 mL's to sterile empty test tube while hot
Dextrose culture-medium, it is sterile with guaranteeing after the alcolhol burner flame calcination rubber stopper test tube mouth several seconds, after cover rubber stopper and tilted
It is placed on a pipette, waits it completely cooling.It repeats above operation until terminating.5-8 branch test tube is wrapped with brown paper
It is fixed again with rubber band, is put into 4 DEG C of refrigerator cold-storages, waited spare.Aseptic operating platform table top is cleared up after completing in inclined-plane.Portugal
Grape sugar liquors culture medium: dextrose broth (1 L): 40 g of glucose；3 g of beef extract；8 g of peptone；Yeast extract (the winter
Season, summer) 5 g, 3 g；2 g of sodium nitrate；Magnesium sulfate 1 g, tryptophan 10g, glycine 12g, serine 9g.(pH is adjusted to 5.0).
The preparation (1 L) of dextrose broth: 40 g of glucose, 3 g of beef extract, 8 g of peptone, yeast extract (winter are weighed；Summer
Season) 5 g；3 g, sodium nitrate 2 g, tryptophan 10g, glycine 12g, serine 9g, 1 g of magnesium sulfate are added in the beaker of 1 L
Distilled water is settled to 1 L, then adjusts medium pH to 5.0 with 0.1 mol/L hydrochloric acid solution.Finally pour into 250 mL conical flasks
In, each conical flask pours into 150 mL solution.After covering rubber stopper, conical flask is wrapped into brown paper, is fixed with rubber band, finally
It puts into high-pressure steam sterilizing pan, selects 121 DEG C, 20 min modes of sterilizing sterilize.The preparation of main agents and solution
(1) 0.1 mol/L hydrochloric acid solution (1 L): 8.3 mL of concentrated hydrochloric acid solution that concentration is 36.5 % is measured with graduated cylinder, is fallen after measurement
Enter in the beaker of clean dried, diluted with distilled water, is then drained with glass bar and the solution after complete dilution is poured into 1000 mL
In volumetric flask, then beaker and glass bar two are washed with distilled water to three times, it should be noted that cleaning solution will also pour into volumetric flask
In, then add distilled water to graduation mark very about 2 centimetres of top into volumetric flask, the dropwise addition of rubber head dropper is used instead, until concave meniscus
Lowest part and graduation mark keep horizontal, cover plug mixing, pour into reagent bottle, labelled spare.(2) 70 %(v/v)
Ethanol solution (500 mL): measuring concentration with graduated cylinder is 95 % ethanol solution, 368.4 mL in clean beaker, is added pure
Distilled water after shaking up, transfers in reagent bottle to 500 mL, it is spare to post reagent label.Further, strain is living
Change: taking the bacterial strain to grow fine to be inoculated with, following strain is completed on the aseptic operating platform for have already passed through ultraviolet-sterilization
Activation act: to avoid living contaminants, by the calcination on alcolhol burner flame of transfer needle whole body, until being waited cold after syringe needle burning is red
But inboard wall of test tube is protruded into afterwards (transfer needle to be placed on inboard wall of test tube, appropriate cooling is carried out and excessively high temperature is avoided to lose Monascus
It is living).With oese from one fritter red yeast rice of picking on Monascus inclined-plane, the glucose solids test tube slant made is put it into
It is interior, so that it is scraped on inclined-plane and (is paid attention to needing the one side and contact of incline plane for having Monascus) with transfer needle promotion, guarantees entire solid
Red yeast rice block is taken out after being all uniformly distributed Monascus on body inclined-plane surface layer.Beyond the Great Wall before rubber stopper, calcination test tube mouth and rubber stopper.Weight
Multiple aforesaid operations continue inoculation until terminating, and remembering to be inoculated with a new test tube every time will calcination loop-carrier and cooling.It will inoculation
Inclined-plane solid medium afterwards is placed in culture activation 7 days in 28 DEG C of mold incubator.Probably inoculation 15 every time, every 2
Its inoculation a batch guarantees sufficient.Period is also required to often observe the growing way of bacterium and whether microbiological contamination is lost in time once finding microbiological contamination
It abandons.Further, seed culture: the test tube slant of culture 15 days or so is taken to be put into aseptic operating platform from mold incubator,
Through by completing following operation on the aseptic operating platform of ultraviolet-sterilization: being infused in every test tube with the liquid-transfering gun with sterilizing pipette tips
Penetrate 5 mL sterile waters, then with by after alcolhol burner high temperature sintering and cooling thick loop-carrier scratches back and forth on inclined-plane, this mesh
Be in order to which spore to be disengaged in sterile water from media surface.Spore is drawn with the sterile pipette tips for cutting pipette tips front end again
Suspension flows into the sterile conical flask with 8 beades by the funnel filtering with sterile lens wiping paper.It is complete etc. to be filtered
Cheng Hou is stoppered with rubber stopper and is wrapped brown paper.In order to be collected into more spores as far as possible, needs conical flask being put into revolving speed and be
150 r/min, temperature are to shake 20 min in 28 DEG C of shaking table.Further, fermented and cultured: the sterile of ultraviolet-sterilization is had already passed through
Following operation is completed on station: after shaking table, rocking conical flask, and spore suspension is distributed into the centrifuge tube after sterilizing
It is interior, then put centrifuge 10 min of interior centrifugation of 8000 r/min into, after centrifugation, supernatant liquor, concentration spore liquid is received
Collection.Draw 1 uL concentration spore liquid with liquid-transfering gun (will be concentrated 100 times of spore liquid dilution, and pay attention to dilute in the sterile water of 99 uL
Release uniformly), the concentration spore liquid after dilution is counted into spore on electron microscope using blood counting chamber.Spore count is calculated, from
And determination will be added to the content in dextrose culture-medium.Concentration spore liquid equivalent is injected into the grape of the mL of 150 mL/250
In sugar liquors culture medium, it is 150 r/min in revolving speed, is vibrated 12 days in the constant-temperature shaking incubator that temperature is 28 DEG C.Finally, blood
Ball count plate count number spore: directly being counted under the microscope using blood counting chamber, is a kind of common microorganism count side
Method.The method has the advantages that directly and quick.Blood count will be placed in by appropriate diluted bacteria suspension (or spore suspension)
In counting chamber between onboard slide and coverslip, counted under the microscope.Take out clean blood counting chamber (I
Laboratory blood counting chamber be 25*16 type), lid one opens the coverslip of 20 mm*20 mm above counting chamber, uses liquid-transfering gun
The spore liquid for drawing about 10 μ L or so is squeezed into along the edge of coverslip, is slowly penetrated spore liquid to counting chamber and is paid attention to not
Bubble is generated, the extra spore liquid in coverslip edge is gently wiped completely, after waiting all spores to be deposited in blood count room,
Start microscopic counting.Comparative experiments design: test 1: on the basis of basal fermentation medium, by changing carbon source (malt
Sugar, glucose, soluble starch, sucrose), fermentation liquid initial pH value (4.0,4.5,5.0,5.5,6.0), fermented and cultured temperature
(8 d, 12 d, 16 d, 20 d), with last institute for degree (24 DEG C, 26 DEG C, 28 DEG C, 30 DEG C, 32 DEG C) and fermented incubation time
The monascorubin color value obtained is inspection target, carries out single factor experiment, every group of test to fermentation medium composition and condition of culture
It is repeated 3 times, test result takes its average value.
Influence of the different carbon source to monascorubin yield in fluid nutrient medium
Based on preliminary fermentation culture medium, respectively using glucose, soluble starch, sucrose, maltose as carbon source, other conditions
Keep identical.At 28 DEG C, 14 d of shaking table shaken cultivation is cultivated in the constant temperature vibration of 150 r/min.Measure monascorubin in fermentation liquid
Total color value and the total color value of mycelium pigment.
Test 2: influence of the different initial pH values to monascorubin yield in fermentation liquid
Based on preliminary fermentation culture medium, respectively with 4.0,4.5,5.0,5.5,6.0 for initial pH, other conditions are kept
It is identical.At 28 DEG C, 14 d of constant-temperature shaking incubator shaken cultivation of 150 r/min.Measure total color value of monascorubin in fermentation liquid
With the total color value of mycelium pigment.
Test influence of the 3 different fermentations times to monascorubin yield
Based on preliminary fermentation culture medium, respectively using 8 d, 12 d, 16 d, 20 d as fermentation time, at 28 DEG C, 150
The constant-temperature shaking incubator shaken cultivation of r/min, other conditions keep identical.Measure total color value of monascorubin and bacterium in fermentation liquid
The total color value of mycelial pigments.
Test influence of the 4 different fermentations temperature to monascorubin yield
Based on preliminary fermentation culture medium, respectively with 24 DEG C, 26 DEG C, 28 DEG C, 30 DEG C, 32 DEG C are fermentation temperature, in 150 r/
14 d of constant-temperature shaking incubator shaken cultivation of min, other conditions keep identical.Measure total color value of monascorubin in fermentation liquid
With the total color value of mycelium pigment and the total color value of mycelium pigment.
Data calculate: by concentration spore liquid after 100 times of dilution, carrying out blood counting chamber counting under an electron microscope.
Total spore count in a final block plaid is 125.According to calculation formula, it is known that sum=1.25 in spore liquid are concentrated in 1 mL
×107(a).In addition the concentration spore inoculating amount of every bottle of culture medium is 0.7 mL.
Data analysis: data such as table 1 and Fig. 1, Fig. 2 are shown, using glucose, soluble starch, sucrose, maltose as carbon source
When, the color value that sucrose and glucose produce monascorubin as two bottles of culture mediums of carbon source is higher, and soluble starch and maltose are made
Color value for two bottles of culture mediums production monascorubin of carbon source is lower.And if figure Fig. 2 is shown, soluble starch and maltose conduct
When carbon source, can not measure pigment in spore (fermentation liquid filter residue is very little, can not grind into powder, so pigment in spore can not be measured),
And when glucose is as carbon source, the color value highest of monascorubin, color value reaches 202.4 U/g in spore.It follows that sucrose and
Glucose is the high-quality carbon source of the culture medium of Monascus producing of Monascus rubber pigments by liquid fermentation.Wherein optimal carbon source is glucose.
Pigment color value inside and outside 1 spore of table
* light absorption value when extension rate is 2 is indicated
^ indicates light absorption value when extension rate is 4
# indicates that fermentation liquid filter residue can not measure pigment in spore very little
Test 5: influence data of the different initial pH values to monascorubin yield calculate in fermentation liquid: by concentration spore liquid dilution
After 100 times, blood counting chamber counting is carried out under an electron microscope.Total spore count in a final block plaid is 74.According to
2.2.7 the calculation formula in, it is known that sum=7.4 × 10 in spore liquid are concentrated in 1 mL6(a).In addition every bottle of culture medium is dense
Contracting spore inoculating amount is 1 mL.Data analysis: data such as Fig. 3,4 displays, with initial pH 4.0,4.5,5.0,5.5,6.0
When for independent variable, when pH is 4.5, color value highest in monascorubin spore, color value reaches 412 U/g in spore, and the outer pigment color value of spore is inclined
It is low, it is 1.252 U/mL, when pH is 5.5, the outer pigment color value highest of monascorubin spore is 2.045 U/mL, and color in spore
Plain color valence is also higher, is 336.25 U/g.PH is known at 4.5 and 5.5, monascorubin color value is generally higher.Due to mainly seeing
Pigment color value in spore, therefore it is the optimal pH of fermentation medium that initial pH, which is 4.5,.
Pigment color value inside and outside 2 spore of table
* extinction when extension rate is 5 is indicated
Test 6: influence data of the different fermentations time to monascorubin yield calculate: by concentration spore liquid in 100 times of dilution
Afterwards, blood counting chamber counting is carried out under an electron microscope.Total spore count in a final block plaid is 96.According to 2.2.7
In calculation formula, it is known that 1 mL be concentrated spore liquid in sum=9.6 × 106(a).In addition the concentration spore of every bottle of culture medium
Inoculum concentration is 0.8 mL.Data are analyzed as shown in Fig. 5,6 and table 3, and the speed of the chromogenic element of 8 d -20 d is opposite to be tended to slowly, can
Can be since pigment is most starting to breed rapidly in several days, the later period due to nutriment deficiency and monascorubin unstability,
Monascorubin yield is caused to decline.At the 16th day, monascorubin color value highest in measured spore reached 222.25 U/g.By
This it can be seen that, best fermented incubation time is 16 d.
Pigment color value inside and outside 3 spore of table
* light absorption value when extension rate is 2 is indicated
^ indicates light absorption value when extension rate is 4
Test 7: influence of the different fermentations temperature to monascorubin yield
Data calculate: by concentration spore liquid after 100 times of dilution, carrying out blood counting chamber counting under an electron microscope.Finally
Total spore count in one block plaid is 97.According to the calculation formula in 2.2.7, it is known that 1 mL be concentrated spore liquid in sum=
9.7×106(a).In addition the concentration spore inoculating amount of every bottle of culture medium is 0.8 mL.
Data analysis
As shown in Fig. 7,8 and table 4, for cultivation temperature at 28 DEG C, pigment color value reaches highest in spore, can reach 299 U/g.Culture
For temperature in 24 DEG C and 32 DEG C, pigment color value is lower in spore.It may be that it is normal to influence monascorubin since temperature is excessively high and too low
Breeding and production monascorubin.Therefore, best fermented and cultured temperature is 28 DEG C.
Pigment color value inside and outside 4 spore of table
* light absorption value when extension rate is 2 is indicated
^ indicates light absorption value when extension rate is 4
Summarize: the empirical factor for influencing Monascus fermenting and producing monascorubin has very much, such as carbon source, nitrogen source, medium pH, training
Support time, cultivation temperature, inoculation, shaking speed.Type and yield of the different fermentation culture conditions to Monascus production pigment
There is larger difference.Therefore, it is significant to monascorubin yield is improved to probe into optimal culture condition.This experiment passes through Dan Yin
Element experiment has determined Optimal compositions of fermentation medium carbon source, the best liquid state fermentation incubation time of optimal initial fermentation medium pH and most
Four factors of good liquid state fermentation cultivation temperature.Optimised process is as follows: using glucose as carbon source, initial medium pH is 4.5, fermentation
16 d of incubation time, cultivation temperature are 28 DEG C.The above is only the preferred embodiment of the present invention, it is noted that above-mentioned excellent
Embodiment is selected to be not construed as limitation of the present invention, protection scope of the present invention should be with claim limited range
It is quasi-.For those skilled in the art, without departing from the spirit and scope of the present invention, if can also make
Dry improvements and modifications, these modifications and embellishments should also be considered as the scope of protection of the present invention.
Claims (8)
1. a kind of high yield type monascus strain culture medium and preparation method, which is characterized in that this bacterial strain is Monascus Mr △ lig4
Bacterial strain can synthesize monascorubin, citraurin and uranidin.
2. high yield type monascus strain culture medium according to claim 1 and preparation method, which is characterized in that cultural method
For dextrose solid medium and dextrose broth.
3. high yield type monascus strain culture medium according to claim 2 and preparation method, which is characterized in that glucose is solid
Body culture medium (1 L): glucose 30-50 g；Beef extract 2-4 g；Peptone 6-9g；Yeast extract (winter 4-6g, summer 2-
4g),；Sodium nitrate 1-3 g；Magnesium sulfate 0.5-1.5 g；Agar 10-30 g, tryptophan 5-15g, glycine 5-15g, serine 7-
12g；PH is adjusted to 4-6.0).
4. high yield type monascus strain culture medium according to claim 2 and preparation method, which is characterized in that Glucose Liquid
Body culture medium (1 L): glucose 30-50 g；Beef extract 2-4 g；Peptone 6-9g；Yeast extract (winter 4-6 g, summer 2-
4g),；Sodium nitrate 1-3 g；Magnesium sulfate 0.5-1.5g, tryptophan 5-15g, glycine 5-15g, serine 7-12g；PH is adjusted to
4.0-6.0).
5. high yield type monascus strain culture medium according to claim 4 and preparation method, which is characterized in that Glucose Liquid
Body culture medium: dextrose broth (1 L): 40 g of glucose；3 g of beef extract；8 g of peptone；Yeast extract (winter, summer
Season) 5 g, 3 g；2 g of sodium nitrate；Magnesium sulfate 1 g, tryptophan 10g, glycine 12g, serine 9g；5.0) pH is adjusted to.
6. high yield type monascus strain culture medium according to claim 4 and preparation method, which is characterized in that Glucose Liquid
Body culture medium: dextrose broth (1 L): 40 g of glucose；3 g of beef extract；8 g of peptone；Yeast extract (winter, summer
Season) 5 g, 3 g；2 g of sodium nitrate；Magnesium sulfate 1 g, tryptophan 10g, glycine 12g, serine 9g；5.0) pH is adjusted to.
7. high yield type monascus strain culture medium according to claim 2 and preparation method, which is characterized in that culture medium is most
Good technique optimal parameter are as follows: using glucose as carbon source, initial medium pH is 4.5,16 d of fermented incubation time, and cultivation temperature is
28℃。
8. high yield type monascus strain culture medium according to claim 2 and preparation method, which is characterized in that trained in fermentation
It supports and adds tryptophan, glycine, serine in base, to improve monascorubin, citraurin and the yield of uranidin.
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|---|---|---|---|---|
| CN105018533A (en) * | 2015-07-28 | 2015-11-04 | 华南理工大学 | Method for obtaining extracellular water-soluble monascus yellow pigment through high-carbon source fermentation and application of method |
| CN105349438A (en) * | 2015-12-15 | 2016-02-24 | 武汉佳成生物制品有限公司 | Functional red yeast rice strain and production process thereof |
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| CN105018533A (en) * | 2015-07-28 | 2015-11-04 | 华南理工大学 | Method for obtaining extracellular water-soluble monascus yellow pigment through high-carbon source fermentation and application of method |
| CN105349438A (en) * | 2015-12-15 | 2016-02-24 | 武汉佳成生物制品有限公司 | Functional red yeast rice strain and production process thereof |
| CN106190873A (en) * | 2016-09-22 | 2016-12-07 | 光明乳业股份有限公司 | A kind of Mauve aspergillar bacterial strain and the method utilizing this strain fermentation production monascorubin |
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