CN108866119A - A kind of whitening cosmetics kojic acid producing process - Google Patents
A kind of whitening cosmetics kojic acid producing process Download PDFInfo
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Abstract
The invention discloses a kind of technical matters processes of kojic acid that whitening cosmetics are produced using aspergillus oryzae fermentation, bacterial strain passes through three inclined-plane culture, seed culture and fermented and cultured steps, by the fermentation liquid containing kojic acid be placed in water temperature condition be in 70 DEG C -80 DEG C of Rotary Evaporators rotary evaporation to there is a small amount of crystal to generate, and then it places it under the conditions of temperature is 2 DEG C and freezes, placed it in after crystallization to be obtained dry in freeze drying box.The process flow provides a kind of high yield, kojic acid fermentation manufacturing technique easy to operate in view of the defects existing in the prior art, thus overcome current kojic acid conversion ratio lower, the bad defect of quality.
Description
Technical field
The invention belongs to microbial fermentation new technical fields, in particular to a kind of to produce whitening cosmetics with aspergillus oryzae
The manufacturing technique method of kojic acid.Special Statement is needed, the application is that application No. is 201510649370.2 division Shens
Please.
Background technique
Kojic acid (5-hydroxy-2-hydroxamethy-1,4-pyrone), molecular formula C6H6O4, relative molecular mass 142.1, chemistry
Structural formula is shown in Fig. 1.Kojic acid is in prismatic, white or colourless acicular crystal, and 151 DEG C -154 DEG C of fusing point.Soluble easily in water, methanol, second
Alcohol, acetone are slightly soluble in ether, the cruel chloroform of acetic acid second and pyridine, are insoluble in benzene and other most of solvents.
Kojic acid (Kojicacid) also known as kojic acid bring up acid, are produced by a variety of fungi aerobic fermentations such as aspergillus, Penicillium
The raw Acidic Compounds with bactericidal effect.A series of subsequent researchs have shown that kojic acid is Skin Cell synthesis of melanin
(Melanin) the Enzyme specificity inhibitor of one tyrosinase related protein2 of key enzyme, is able to suppress the synthesis of melanin, it is determined that kojic acid tool
The unique effects for having nti-freckle, blocking pigmentation, making skin-whitening.
In fact, kojic acid is just present among the brewing of soy sauce, thick broad-bean sauce, drinks since ancient times, more in food fermentation industry
The presence of kojic acid can be detected in the fermented product of aspergillus fermentation.Prove that kojic acid is to people by prolonged research practice
It raises safe and harmless.The application of kojic acid accelerates the update of whitening cosmetics.At abroad, various brands contain kojic acid
The cosmetics of super quality are launched in succession.Kojic acid is just being widely used in food, daily use chemicals and medical industry simultaneously, and application range still exists
Continue to widen.
From the 1970s, many scholars study the fermenting and producing of kojic acid, such as the northern Tian Mufu of Japan, richness
Give up former filial piety, river hand Zhaoping, Brazilian M.T.Lin etc..Japan is main kojic acid producing country in the world and kojic acid consumption at present
State.The country begins one's study exploitation from the 1980s, has more units at present and achieves significant research achievement, and has
Other research unit carries out large-scale production into people's industrialization.
The patent of application number 01135102 provides a kind of kojic acid producing process, belongs to the preparation neck of oxygen-containing organic compound
Domain.Including starch saccharification, inoculation fermentation, purification kojic acid, it is characterised in that starch direct plunges into fermentor liquefaction and saccharification, liquid glucose
Fermented and cultured kojic acid is inoculated with after sterilized, after dissolving, decolourizing, decrease temperature crystalline mentions the multiple condensing crystallizing product of kojic acid clear liquid
The pure kojic acid crystal for obtaining finished product.Wherein starch direct plunges into fermentor, and additional amount is 5~15 μ/g starch alpha-amylase,
It is warming up to 70~100 DEG C of liquefaction and saccharification.The sterilisation temp of liquid glucose is 110~130 DEG C, and disinfecting time is 30~40 minutes, is connect
The temperature of kind fermentation is 32~36 DEG C.The concentration of the lysate of multiple condensing crystallizing product is 9~12Be ' when dissolution, decoloration, is taken off
Toner is the active carbon of 0.1~5% weight.Acidacidity > 6% is produced, the purity of obtained kojic acid is in 98% or more, Er Qiejing
Body is uniform.
The patent of application number 201120279865 discloses a kind of kojic acid fermentation equipment comprising fermentor, in fermentor
Equipped with blender and heated at constant temperature pipe, it is characterized in that：The Zymolysis Equipment further includes feed supplement tank and separator, the feed supplement tank
For containing fluid infusion of fermenting, feed supplement tank is connected with the material-feeding port on fermentor.The separator for dividing during the fermentation
From kojic acid, separator) it include cooler, bullet and collector, point of the inlet of cooler through liquid valve and fermentor
It is connected from liquid outlet, the liquid outlet of cooler is connected with the osculum of bullet upper end, and the big mouth of bullet lower end connects collector,
Collector side wall upper part is equipped with liquid outlet, and the liquid outlet of collector passes through back liquid valve and is connected with the separation liquid return hole of fermentor.This reality
Fermentation liquid concentration is controlled by fed-batch cultivation feed supplement with novel, and production concentration is controlled by continuous separation product, so as to
To improve yeasting, kojic acid fermentation yield is greatly improved, improves utilization rate of equipment and installations, reduces energy consumption.
The patent of application number 201310375463 discloses a kind of separation purifying technique of kojic acid fermentation liquid, using cation
Exchanger resin removes condensing crystallizing after impurity cationic, is recrystallized after kieselguhr adsorption decoloration, has obtained the song of high-purity
Acid crystal.The present invention overcomes traditional kojic acid extraction process purification difficult, the deficiencies of step is various, seriously polluted, higher cost,
It solves the deeper disadvantage of conventionally produced product colour using direct concentrated crystallization, it is brilliant to obtain white needles kojic acid
Body, light transmittance are 90%~96%, and purity reaches 99.0% or more, and entire technique extraction purification yield reaches 60% or more.
However there is presently no the researchs of complete set how to provide a kind of high yield, kojic acid fermenting and producing easy to operate
Technique, the process flow difficulty that the prior art provides is complicated, higher cost, and it is inconvenient that raw material are chosen, and causes kojic acid yield not
Situations such as complying with one's wishes.
Summary of the invention
Object of the present invention is to provide a kind of high yield, whitening spot-removing easy to operate in view of the defects existing in the prior art
Cosmetic kojic acid fermentation manufacturing technique, thus overcome current kojic acid conversion ratio lower, the bad defect of quality.
The present invention to achieve the above object, adopts the following technical scheme that：
It is a kind of of the invention to produce the technical matters of kojic acid using aspergillus oryzae fermentation and include the following steps：
Step S101, the fermentation starting strain used are aspergillus oryzae 2336 or aspergillus oryzae KA2308；
Step S102, takes inclined-plane culture；
Step S103, using seed culture；
Step S104, using fermented and cultured；
In step S105, the fermentation liquid containing kojic acid is obtained, the manufacture craft of first stage is completed；
In step S106, the fermentation liquid containing kojic acid is placed in the Rotary Evaporators that water temperature condition is 70 DEG C -80 DEG C and is revolved
Turn to be evaporated to a small amount of crystal generation,
In step s 107, it and then places it under the conditions of temperature is 2 DEG C and freezes, placed it in after crystallization to be obtained cold
Freeze drying in drying box, the crude kojic acid crystallization of aubergine is thus obtained using direct concentrated crystallization；
In step S108, using active carbon carry out decolorization, specifically use activated carbon dosage 3%, decolourize pH value for
3.5, heat preservation decoloration 45min is obtained at least under this process condition by parallel laboratory test three times under the conditions of bleaching temperature is 80 DEG C
77% percent of decolourization and at least 98% kojic acid yield；
In step S109, impurity is removed with strongly acidic styrene type cation exchange resin, specifically by crude kojic acid
Solution is drained into cation exchange column after being suitably concentrated with glass bar, when exchanging band close to ion exchange column bottom, is stopped
It is added to destainer, is then washed out in column with distilled water and remain kojic acid, be washed till eluent and FeCl3- HCl color developing agent, which does not develop the color, is
Only；
In step s 110, by through cation exchange resin removal partial impurities kojic acid solution by Rotary Evaporators into
The dehydrated alcohol of 3-4 times of kojic acid content is added into solution, fullys shake to be concentrated into 100g/L for the evaporation of row secondary rotating
40-50min is filtered after standing 10min with filter paper, to remove protein precipitation；
In step S111, filtrate liquid is placed in 2 DEG C of refrigerators and is crystallized, places it in freeze drier after crystal to be obtained
Middle secondary freeze drying, final to obtain kojic acid yield at least 79%, product purity is at least 98% kojic acid crystallization.
Wherein, solid slope culture medium used in inclined-plane culture step includes：Potato (peeling) 180-200g, grape
Sugared 18-20g, agar 18-20g, deoxysodium cholate 1.8-2g, calcium carbonate 2-5g, total amount 1000mL, pH value is naturally, then 130
Sterilize 30min under DEG C high temperature；
Wherein, the seed culture medium in step includes：Potato (peeling) 180-200g, glucose 18-20g deoxygenates gallbladder
Sour sodium 1.8-2g, glycerol 3-5g, cottonseed meal 2-5g, (NH4)2SO41-4g, total amount 1000mL, pH value is naturally, then 130
Sterilize 30min under DEG C high temperature；
Wherein, the fermentation medium in fermented and cultured step includes：Soluble starch 100g/L, peptone 0.5g/L, ferment
Female cream 0.5g/L, beef extract 0.5g/L, sodium nitrate 0.5g/L and ammonium sulfate 0.5g/L, soybean powder 1g/L, (NH4)2SO4 1-4g/
L, MgSO4·7H2O 0.5g/L, KH2PO41.0g/L, KCl 0.4g/L, FeSO40.01g/L, pH value 5.0-5.5, then exists
Sterilize 20min under 130 DEG C of high temperature, and 30 DEG C of cultivation temperature, shaking speed 200r/min, liquid amount 50mL/250mL, inoculum concentration
10%, continuously cultivate 6d-10d.
It was verified that there is raw material easily to purchase for process flow of the invention, cheap, culture medium is prepared conveniently, work of fermenting
Skill is easy, and kojic acid yield is higher compared with prior art, is suitble to the advantages such as industrialized production, while the kojic acid produced using this method
It is applied to better effect on whitening cosmetics.
Detailed description of the invention
Fig. 1 shows the technical matters of the invention that kojic acid is produced using aspergillus oryzae fermentation.
Specific embodiment
As shown in Figure 1, the technical matters of the invention for producing kojic acid using aspergillus oryzae fermentation mainly includes the following steps：
Step S101, the fermentation starting strain used are aspergillus oryzae 2336 or aspergillus oryzae KA2308, and wherein aspergillus oryzae is one
The bacterial strain that class produces complex enzyme can also produce amylase, carbohydrase, cellulase, phytase etc. in addition to producing protease.In amylase
Under the action of, straight chain, the amylopectin in raw material are degraded to dextrin and various low molecule carbohydrates, such as maltose, glucose；
Under the action of protease, stodgy macro-molecular protein is degraded to peptone, polypeptide and various amino acid, Er Qieke
So that the difficult mass degradation absorbed such as crude fibre, phytic acid in auxiliary material, improves nutritive value, health-care efficacy and digestibility, answers extensively
For fermentation industries such as food, feed, production kojic acid, wine brewing；
In step S102 to step S104, bacterial strain is by inclined-plane culture (S102), seed culture (S103) and fermentation training
(S104) three steps are supported,
The solid slope culture medium used in the inclined-plane culture step of step S102 includes：Potato (peeling) 180-
200g, glucose 18-20g, agar 18-20g, deoxysodium cholate 1.8-2g, calcium carbonate 2-5g, total amount 1000mL, pH value naturally,
Then sterilize 30min under 130 DEG C of high temperature；
Seed culture medium (g/L) in the seed culture step of step S103 includes：Potato (peeling) 180-200g,
Glucose 18-20g, deoxysodium cholate 1.8-2g, glycerol 3-5g, cottonseed meal 2-5g, (NH4)2SO41-4g, total amount 1000mL,
PH value is naturally, the 30min that then sterilizes under 130 DEG C of high temperature；
Fermentation medium in the fermented and cultured step of step S104 includes：Soluble starch 100g/L combines nitrogen source
2.5g/L (including peptone 0.5g/L, yeast extract 0.5g/L, beef extract 0.5g/L, sodium nitrate 0.5g/L and ammonium sulfate 0.5g/
L), soybean powder 1g/L, (NH4)2SO41-4g/L, MgSO4·7H2O 0.5g/L, KH2PO41.0g/L, KCl 0.4g/L,
FeSO40.01g/L, pH value 5.0-5.5, then sterilize under 130 DEG C of high temperature 20min, and 30 DEG C of cultivation temperature, shaking speed
200r/min, liquid amount 50mL/250mL, inoculum concentration 10% continuously cultivate 6d-10d；
The fermentation liquid containing kojic acid is obtained in step s105, completes the manufacture craft of first stage；
In step S106, the fermentation liquid containing kojic acid is placed in the Rotary Evaporators that water temperature condition is 70 DEG C -80 DEG C and is revolved
Turn to be evaporated to a small amount of crystal generation,
In step s 107, it and then places it under the conditions of temperature is 2 DEG C and freezes, placed it in after crystallization to be obtained cold
Freeze drying in drying box, the crude kojic acid crystallization of aubergine is thus obtained using direct concentrated crystallization；
In step S108, decolorization is carried out using active carbon.By activated carbon dosage, bleaching time, bleaching temperature,
Decolourize pH value investigation, specifically use activated carbon dosage 3%, decoloration pH value for 3.5, bleaching temperature be 80 DEG C under the conditions of heat preservation take off
Color 45min obtains at least 77% percent of decolourization under this process condition and at least 98% kojic acid is received by parallel laboratory test three times
Rate；
In step S109, impurity is removed with strongly acidic styrene type cation exchange resin, specifically by crude kojic acid
Solution is drained into cation exchange column after being suitably concentrated with glass bar, when exchanging band close to ion exchange column bottom, is stopped
It is added to destainer, is then washed out in column with distilled water and remain kojic acid, be washed till eluent and FeCl3- HCl color developing agent, which does not develop the color, is
Only；
In step s 110, by through cation exchange resin removal partial impurities kojic acid solution by Rotary Evaporators into
The dehydrated alcohol of 3-4 times of kojic acid content is added into solution, fullys shake to be concentrated into 100g/L for the evaporation of row secondary rotating
(purpose is the macromoleculars such as further removing protein) is filtered with filter paper after 40-50min, standing 10min, to remove protein etc.
Precipitating.
In step S111, filtrate liquid is placed in 2 DEG C of refrigerators and is crystallized, places it in freeze drier after crystal to be obtained
Middle secondary freeze drying, final to obtain kojic acid yield at least 79%, product purity is at least 98% kojic acid crystallization.
According to another aspect of the present invention, following experimentation is also disclosed,
(1) pass through the fermentation of experiment of single factor and response surface experiments design method to mutagenic obtained excellent kojic acid bacterial strain
Condition carries out the optimization of system, provides and lays the foundation for the industrialized production of kojic acid.
In order to inquire into the influence that promotor generates kojic acid under various concentration, in the basic fermentation medium of kojic acid respectively
The dehydrated alcohol that mass concentration is 0%, 2%, 4%, 6%, 8%, 10%, 12% is added, liquid amount is 50mL/250mL shaking flask,
The shaken cultivation 6-7d in shaking table under the conditions of 30 DEG C, 200r/min.
Under conditions of the dehydrated alcohol of addition certain mass concentration generates promotor as kojic acid in the fermentation medium, lead to
The method for gradually changing single-factor type in fermentation medium is crossed, research different culture medium ingredient generates the shadow of yield to kojic acid
It rings.
This experiment is by the carbon source kind in monistic change fermentation medium, with sucrose, glucose, maltose and can
The group of soluble starch is combined into combination carbon source, studies influence of the carbon source kind of different ratio amount to kojic acid yield, chooses optimal match
Proportion.Optimal fermentation medium components group becomes：Combination carbon source (carbon element content is on the basis of sucrose) 100g/L (including sucrose
70g/L, glucose 15g/L, maltose 10g/L and soluble starch 5g/L), yeast extract 2.0g/L, MgSO4·7H2O
0.5g/L, KH2PO41.0g/L, KCl 0.5g/L, FeSO4Sterilize 20min under the conditions of 5.0,130 DEG C of 0.01g/L, pH.
Combination nitrogen source is combined into the group of peptone, yeast extract, beef extract, sodium nitrate, ammonium sulfate simultaneously, studies different ratio
Optimal proportion amount is chosen in the influence that the nitrogen source type of amount generates kojic acid.Optimal fermentation medium components are：Soluble starch
100g/L, combination nitrogen source 2.5g/L (including peptone 0.5g/L, yeast extract 0.5g/L, beef extract 0.5g/L, sodium nitrate 0.5g/L
With ammonium sulfate 0.5g/L), soybean powder 1g/L, (NH4)2SO41-4g/L, MgSO4·7H2O 0.5g/L, KH2PO41.0g/L
KCl0.4g/L, FeSO40.01g/L, pH value 5.0-5.5,130 DEG C of sterilizing 20min.30 DEG C of cultivation temperature, shaking speed 200r/
Min, liquid amount 50mL/250mL, inoculum concentration 10% detect kojic acid yield in fermentation liquid after cultivating 6d.Practice have shown that use with
The culture medium and condition of upper component can be improved bacterial strain performance and stablize, and fast growing is suitable for industrialized production.
(2) removal etc. of the decoloration process and foreign ion of the extraction to kojic acid in fermentation liquid and fermentation liquid is ground
Study carefully, to improve kojic acid recovery rate and product purity.
By the fermentation liquid containing kojic acid be placed in water temperature condition be in 70 DEG C of Rotary Evaporators rotary evaporation to there is a small amount of crystalline substance
Body freezes under the conditions of placing it in 2 DEG C after generating, and dry in freeze drying box, acquisition aubergine is placed it in after crystallization to be obtained
Crude kojic acid crystallization.
The decolorizing effect of active carbon is also influenced by different objective factors to a certain extent.It is taken in the present invention a certain amount of
Kojic acid crude crystal is soluble in water, is made the crude kojic acid solution of 100g/L, system research activated carbon dosage, to de-inking solution pH
The influence to active carbon decolorizing effect such as value, heat preservation bleaching temperature, bleaching time.
It is purer in order to be made since easily with metal ions such as iron, magnesium coloured complex reaction occurs for kojic acid
Kojic acid crystal needs to remove some impurity cationics with cation exchange resin.New resin is due to containing oligomer, swelling
The substances such as agent, pigment, alcohol-soluble, will lead to its impurity when contacting with water, bronsted lowry acids and bases bronsted lowry etc. can be transferred in water, dirty using initial stage
Contaminate effluent quality.Therefore, new resin will be pre-processed before use, be converted to specified ion species.The present invention first uses
95% ethyl alcohol carries out immersion treatment for 24 hours to cation exchange resin, is rinsed later with clear water to resin, is washed till wash-off water
It is limpid without muddy, free from admixture until.Then with the HCI and NaOH of 4-5% successively alternate immersion 2-4 hours in exchange column,
It is eluted until washing out water close to neutrality, is so repeated 2-3 times, the volume of each soda acid dosage with a large amount of distilled water between soda acid
It is 2 times of cation exchange resin resin volume.Last time processing is carried out with the HCI solution of 4-5%, and dosage is that cation is handed over
4 times for changing resin.Acid solution is drained, is eluted with clear water to neutrality, is dried at room temperature, for use.
When its exchange capacity is close to or up to saturation to resin in use, resin just needs to carry out regeneration treatment,
Method is, by the ion elution on the resin exchanged to, the cation exchange groups of resin to be made to be restored to exchange with solution appropriate elution
Preceding state is ready for the ion exchange in next period.Make cation exchange resin with the hydrochloric acid of 3%-6% in the present invention
Regeneration.
The present invention gives the following example to test the optimal selection of above method technique.
The measurement of 1 reduced sugar of embodiment
Reducing sugar test uses DNS method.DNS method i.e. 3,5- dinitrosalicylic Acid Colorimetry, the principle that the method measures are
3,5- dinitrosalicylic acids under the conditions of neutral or meta-alkalescence with amino-compound-that brownish red is reduced into after reduced sugar heat together
3- amino -5-NITROSALICYLIC ACID.In a certain range, the amount of reduced sugar and the color of reaction solution are proportional.
(1) preparation of standard solution and color developing agent
Glucose standards solution (lg/L)：Accurately weighing specification is analytically pure DEXTROSE ANHYDROUS (in hot air drier 100
Drying to constant weight at DEG C) 1.00g, it is dissolved in 1000mL distilled water.
3,5- dinitrosalicylic acid solutions：6.5g 3 is weighed, 5- dinitrosalicylic acid is dissolved in a small amount of distilled water, after dissolution
It is transferred in 1000mL volumetric flask, 2mol/L sodium hydroxide solution 325mL is added, adds glycerine 45g, shakes up, after cooling
It is settled to 1000mL, is then contained in brown reagent bottle, is placed in refrigerator after 5d stand-by.
(2) production of glucose standard curve
10 20mL test tubes are taken, reagent is added, each pipe solution is sufficiently mixed uniformly to be placed on to heat in boiling water bath and is boiled
5min is cooled to room temperature immediately with the cold water of flowing after taking-up, then appropriate distilled water constant volume is added into every pipe and shakes up, with pipe
1 is blank control, and the absorbance of each pipe is surveyed under 540nm wavelength.Using glucose mg number as abscissa, with 540 value of absorbance A BS
Absorbance-glucose concentration curve is drawn for ordinate.
(3) measurement of reduced sugar
By fermentation liquid loaded on 20min is centrifuged under the conditions of 6000r/min in centrifuge tube, supernatant is taken to be diluted 4 times, remaining
Operating method and step are identical as production glucose standard curve.Three repetitions are surveyed every time, take its average value.
The measurement of 2 kojic acid of embodiment
The preparation of color developing agent：22.5mL concentrated hydrochloric acid is taken, FeCl is added310g is settled to 1000mL with distilled water.Kojic acid mark
The preparation of quasi- liquid：Kojic acid standard sample 0.1g is accurately weighed in 100mL volumetric flask, distilled water is settled to after completely dissolution
100mL is to get 1mg/mL kojic acid titer.
The titer of 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL totally 5 concentration gradients is taken to be sub-packed in 100mL appearance respectively
In measuring bottle, then it is separately added into 2.0mLFeCl3- HCl color developing agent is settled to 100mL, and shaking up repeatedly reacts it sufficiently.With distillation
Water replaces kojic acid titer, and distilled water after same amount color developing agent is added to be settled to 100mL as blank, with UV-1700 UV, visible light
Spectrophotometer measures the ABS500 value of the kojic acid titer of concentration gradient at 500nm.Every group parallel determination 3 times, with quality
Concentration X (mg/mL) is abscissa, and Y (ABS500 value) is that ordinate draws standard curve.
Fermentation liquid is centrifuged (6000r/min, 20min), and supernatant is diluted 10 times, takes 1mL in 100mL volumetric flask, adds
Enter 2mLFeCl3- HCl color developing agent is settled to 100mL after sufficiently shaking up.On UV-1700 ultraviolet-uisible spectrophotometer in
Its absorption value is surveyed at ABS500.Make same treatment with fermentation liquid, makees blank control not access the fermentation liquid of strain.According to kojic acid
Standard curve equation of linear regression calculates kojic acid yield.
Influence of the 3 dehydrated alcohol concentration of embodiment to kojic acid fermentation concentration
The dehydrated alcohol that different quality concentration is added in kojic acid fermentation medium, the results show that in kojic acid fermented and cultured
Ethyl alcohol is added in base has good effect to promotion fermentation and acid, and the ethyl alcohol of different quality concentration generates facilitation not to kojic acid
Together：Ethyl alcohol mass concentration has different degrees of facilitation within the scope of 2%-10%, to acid is produced, wherein when ethyl alcohol quality is dense
When degree is 6%, kojic acid yield is up to 51.24g/L, and yield improves 15%, also reaches highest to the utilization rate of glucose；When ethyl alcohol is dense
When degree reaches 10%, the addition that continues of ethyl alcohol produces inhibiting effect to kojic acid generation, and when concentration of alcohol reaches 12%, kojic acid is obtained
Rate is even lower than original yield, while residual sugar content also reaches highest in fermentation liquid, and the utilization rate of reduced sugar is lower.
It can be seen that the utilization rate of kojic acid yield and glucose in just from kojic acid yield and residual sugar content general trend
The relationship of ratio：Kojic acid yield is higher, and residual sugar amount is lower, i.e., higher to the utilization rate of glucose；Kojic acid yield is lower, to glucose
Utilization rate is also lower.
The investigation of 4 carbon source of embodiment
Respectively using sucrose, glucose, maltose and soluble starch as carbon source, shadow of the research different carbon source to kojic acid yield
It rings.Fermentation medium components group becomes：Carbon source (carbon element content is on the basis of sucrose) 100g/L, yeast extract 2.0g/L is combined,
MgSO4·7H2O 0.5g/L, KH2PO41.0g/L, KCl 0.5g/L, FeSO4It goes out under the conditions of 5.0,130 DEG C of 0.01g/L, pH
Bacterium 20min.
Fermentation liquid is centrifuged (6000r/min, 20min), and supernatant is diluted 10 times, takes 1mL in 100mL volumetric flask, adds
Enter 2mLFeCl3-HCl color developing agent, is settled to 100mL after sufficiently shaking up.On UV-170O ultraviolet-uisible spectrophotometer in
Its absorption value is surveyed at ABS500.Make same treatment with fermentation liquid, makees blank control not access the fermentation liquid of strain.According to kojic acid
Standard curve equation of linear regression calculates kojic acid yield.
Different carbon source has a great impact to the generation of kojic acid.When using soluble starch as carbon source, kojic acid yield is maximum,
Kojic acid yield when higher than glucose as carbon source, and said from economics point, the market price of soluble starch is more than grape
Sugar is low, therefore starch is selected to ferment the optimum carbon source for generating kojic acid as this variant with double benefit.
The investigation of 5 nitrogen source of embodiment
Respectively using albumen arteries and veins, yeast extract, beef extract, sodium nitrate, ammonium sulfate as nitrogen source, research different nitrogen sources generate kojic acid
Influence.Fermentation medium components are：Soluble starch 100g/L combines nitrogen source 2.5g/L, MgSO4·7H2O 0.5g/L,
KH2PO41.0g/L, KCl 0.5g/L, FeSO45.0,130 DEG C of sterilizing 20min of 0.01g/L, pH.
Fermentation liquid is centrifuged (6000r/min, 20min), and supernatant is diluted 10 times, takes 1mL in 100mL volumetric flask, adds
Enter 2mLFeCl3- HCl color developing agent is settled to 100mL after sufficiently shaking up.On UV-170O ultraviolet-uisible spectrophotometer in
Its absorption value is surveyed at ABS500.Make same treatment with fermentation liquid, makees blank control not access the fermentation liquid of strain.According to kojic acid
Standard curve equation of linear regression calculates kojic acid yield.
The selection result shows that in kojic acid fermentation medium, the kojic acid yield generated under the conditions of inorganic nitrogen-sourced is significantly lower than
Organic nitrogen source, and bacterial strain mycelial growth rate is also slowly more than under the conditions of organic nitrogen source during fermented and cultured.Having
In machine nitrogen source, the kojic acid yield using peptone as nitrogen source is maximum, therefore finally selected albumen arteries and veins is given birth to as the fermentation of this variant
Produce the optimum nitrogen source of kojic acid.
6 active carbon decolorization of embodiment
The decolorizing effect of active carbon is also influenced by different objective factors to a certain extent.It is taken in this experiment a certain amount of
Kojic acid crude crystal is soluble in water, is made the crude kojic acid solution of 100g/L, system research activated carbon dosage, to de-inking solution pH
The influence to active carbon decolorizing effect such as value, heat preservation bleaching temperature, bleaching time.
Using the crude kojic acid solution containing 100g/L as research object, take 100mL solution in 5 triangular flasks respectively, separately
The active carbon for being separately added into kojic acid content 1%, 3%, 5%, 7%, 9% outside carries out heat preservation decoloration to crude kojic acid solution, keeps the temperature
Bleaching temperature is 60 DEG C, and time 30min, pH are natural.
It takes the crude kojic acid solution of 100mL100g/L in 5 triangular flasks respectively, investigates the different pH value of solution to activity
The influence of carbon decoloring effect；Take the crude kojic acid solution that 100mL concentration is 100g/L in 5 triangular flasks respectively, examination is different
Influence of the bleaching temperature to active carbon decoloring；Take the crude kojic acid solution that 100mL concentration is 100g/L in 5 triangular flasks respectively
In, examine or check influence of the different heat preservation bleaching temperatures to active carbon decoloring.
Although the detailed description and description of the specific embodiments of the present invention are given above, it should be noted that
We can carry out various equivalent changes and modification to above embodiment according to the concept of the present invention, and generated function is made
It, should all be within protection scope of the present invention when with the spirit still covered without departing from specification and attached drawing.The above, only
Presently preferred embodiments of the present invention is not intended to limit the invention, according to the technical essence of the invention to above embodiments institute
Any trickle amendment, equivalent replacement and the improvement made, all should be included in the scope of protection of the technical solution of the present invention.
Claims (1)
1. a kind of technical matters process for being produced kojic acid using aspergillus oryzae fermentation, is mainly included the following steps：
Step S101, the fermentation starting strain used are aspergillus oryzae 2336 or aspergillus oryzae KA2308；
Step S102, takes inclined-plane culture；
Step S103, using seed culture；
Step S104, using fermented and cultured；
In step S105, the fermentation liquid containing kojic acid is obtained, the manufacture craft of first stage is completed；
In step S106, the fermentation liquid containing kojic acid is placed in rotate in the Rotary Evaporators that water temperature condition is 70 DEG C -80 DEG C and is steamed
A small amount of crystal has been sent to generate,
In step s 107, it and then places it under the conditions of temperature is 2 DEG C and freezes, it is dry that freezing is placed it in after crystallization to be obtained
It is dry in dry case, the crude kojic acid crystallization of aubergine is thus obtained using direct concentrated crystallization；
In step S108, decolorization is carried out using active carbon, specifically use activated carbon dosage 3%, pH value of decolourizing for 3.5,
Heat preservation decoloration 45min obtains at least 77% by parallel laboratory test three times under this process condition under the conditions of bleaching temperature is 80 DEG C
Percent of decolourization and at least 98% kojic acid yield；
In step S109, impurity is removed with strongly acidic styrene type cation exchange resin, specifically by crude kojic acid solution
It is drained into cation exchange column after appropriate concentration with glass bar, when exchanging band close to ion exchange column bottom, stops being added
To destainer, is then washed out in column with distilled water and remain kojic acid, be washed till eluent and FeCl3Until-HCl color developing agent does not develop the color；
In step s 110, the kojic acid solution through cation exchange resin removal partial impurities is carried out two by Rotary Evaporators
The dehydrated alcohol of 3-4 times of kojic acid content is added into solution, 40- fullys shake to be concentrated into 100g/L for secondary rotary evaporation
50min is filtered after standing 10min with filter paper, to remove protein precipitation；
In step S111, filtrate is placed in 2 DEG C of refrigerators and is crystallized, placed it in after crystal to be obtained secondary in freeze drier
Freeze-drying, final to obtain kojic acid yield at least 79%, product purity is at least 98% kojic acid crystallization；
Wherein, solid slope culture medium used in inclined-plane culture step includes：Peeled potatoes 180-200g, glucose 18-
20g, agar 18-20g, deoxysodium cholate 1.8-2g, calcium carbonate 2-5g, total amount 1000mL, pH value is naturally, then in 130 DEG C of height
The lower 30min that sterilizes of temperature；
Wherein, the fermentation medium in fermented and cultured step includes：Soluble starch 100g/L, peptone 0.5g/L, yeast extract
0.5g/L, beef extract 0.5g/L, sodium nitrate 0.5g/L and ammonium sulfate 0.5g/L, soybean powder 1g/L, (NH4)2SO41-4g/L,
MgSO4·7H2O 0.5g/L, KH2PO41.0g/L, KCl 0.4g/L, FeSO40.01g/L, pH value 5.0-5.5, then 130
Sterilize 20min under DEG C high temperature, and 30 DEG C of cultivation temperature, shaking speed 200r/min, liquid amount 50mL/250mL, inoculum concentration 10%,
Continuous culture 6d-10d.
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| CN112481139B (en) * | 2020-12-22 | 2022-08-05 | 华东理工大学 | Culture medium for producing emodin by using marine fungus aspergillus flavus HN4-13 and preparation method thereof |
| CN113735824B (en) * | 2021-09-07 | 2023-06-20 | 中国科学院成都生物研究所 | PROTAC for targeted degradation of tyrosinase and application thereof |
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