CN100347312C - Method for producing aurantin by liquid-state fermentation of monacolin - Google Patents
Method for producing aurantin by liquid-state fermentation of monacolin Download PDFInfo
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Abstract
红曲菌液态发酵生产橙色素的方法，属于生物工程和色素制备技术领域。本发明以一株高产色素红曲霉菌9903为出发菌株，生产以橙色素为主的红曲色素，并提高橙色素含量及色调(OD465nm/OD510nm)，确定了最佳发酵培养基配方，在15l的自动发酵罐中，以玉米淀粉和无机氮源硫酸铵为碳、氮源的发酵液色价达到121.7U/ml，色调值达到1.13；发酵动力学的初步研究表明，橙色素的生产与菌体生长有一定的偶联关系；提供了几种橙色素的精制方法：由发酵液直接进行喷雾干燥得到粉末状的橙色素产品，是显橙色调的多种色素的混合物，其橙色素色价达1030.75U/g，色调值达1.99；经有机溶剂萃取后进行喷雾干燥，色价达2000U/g以上，色调值达2.45；或经过板层析或柱层析精制后得到纯的橙色素针状结晶。
The invention discloses a method for producing orange pigment by liquid state fermentation of Monascus bacterium, belonging to the technical field of bioengineering and pigment preparation. The present invention uses a strain of high-yield pigment Monascus 9903 as the starting strain to produce monascus pigment mainly composed of orange pigment, and increase the orange pigment content and color tone (OD 465nm /OD 510nm ), and determine the optimum fermentation medium formula. In a 15l automatic fermentation tank, the color value of the fermented liquid with cornstarch and inorganic nitrogen source ammonium sulfate as carbon and nitrogen sources reached 121.7U/ml, and the hue value reached 1.13; preliminary research on fermentation kinetics showed that the production of orange pigment There is a certain coupling relationship with the growth of bacteria; several refining methods of orange pigment are provided: the powdery orange pigment product is obtained by direct spray drying of the fermentation broth, which is a mixture of various pigments with an orange hue. The color value reaches 1030.75U/g, and the hue value reaches 1.99; after being extracted by organic solvent, it is spray-dried, and the color value reaches more than 2000U/g, and the hue value reaches 2.45; or it can be purified by plate chromatography or column chromatography to obtain pure orange Plain needle crystal.
Description
Technical field
A kind of method of producing aurantin by liquid-state fermentation of monacolin belongs to biotechnology and pigment preparing technical field.
Background technology
Monascorubin determines that at present six kinds of compositions of structure are: citraurin (erythema red colouring agent for food, also used as a Chinese medicine element, red colouring agent for food, also used as a Chinese medicine rubine element), yellow pigment (red colouring agent for food, also used as a Chinese medicine element, yellow red colouring agent for food, also used as a Chinese medicine element), purpurin (erythema red colouring agent for food, also used as a Chinese medicine amine and red colouring agent for food, also used as a Chinese medicine rubine amine).Its research is comparatively ripe, and mostly its application on food is to utilize haematochrome in the monascorubin as tinting material, is a kind of hybrid pigment.
In the hybrid pigment of monascorubin, citraurin is a kind of the most garish pigment.If be used for food or other needs pigmented product, can increase a kind of alternative bright-coloured tone for it.And Gong Huimei and L.Martinkova etc. mention citraurin and have certain biocidal property, can suppress gram-positive microorganism and intestinal bacteria, it may be that monascorubin and protein substance combine that its principle is considered to, to such an extent as to make protein denaturation, bacteria growing inhibiting plays the anticorrosion effect of Denging.Therefore it is a kind of main antibacterial substance in the red colouring agent for food, also used as a Chinese medicine, and its biocidal property and absorbancy are positive phase change.In addition, Carels and Shepherp think that haematochrome, yellow pigment are all got through chemical oxidation by citraurin, and promptly citraurin is an important intermediate during monascorubin synthesizes.
Citraurin is an inter-level in the monascorubin biosynthetic pathway, and character is more active, and easily oxidized or reduction forms yellow pigment or haematochrome.Monascorubin as a kind of orange hue, only limit to sample production at present at home and abroad, the scale operation of all being unrealized, in the application in food and other field, do not obtain sufficient development research yet, but because of its color comparatively unique, eye-catching, having a extensive future in the tinting material sector application, is the monascorubin kind that is worth exploitation.
Summary of the invention
The present invention seeks to method in order to seek a kind of High-efficient Production red colouring agent for food, also used as a Chinese medicine citraurin and can to produce in enormous quantities.Main technique route: utilize Monascus anka Nakazawa et sato, change culture medium prescription and fermentation condition by deep ventilation fermentation and produce monascorubin based on orange hue (erythema red colouring agent for food, also used as a Chinese medicine element or red colouring agent for food, also used as a Chinese medicine rubine element).
Technical solution of the present invention: the liquid submerged fermentation and the spray drying process that adopt the red colouring agent for food, also used as a Chinese medicine citraurin.
(1) bacterial classification: be the bacterial classification that sets out with monascus ruber (Monascus spp.) 9903, at " food and biotechnology " the 21st volume the 1st phase P43-47, in January, 2002 is open, is provided by biotechnology institute of Southern Yangtze University for this bacterial classification.
(2) substratum
Slant medium: potato substratum.
Basis fermention medium: KH
2PO
4, 1～5g/L; K
2HPO
4, 1～5g/L; MgSO
47H
2O, 0.2～0.8g/L; CaCl
2, 0.05～0.2g/L; FeSO
47H
2O, 0.005～0.02g/L; ZnSO
47H
2O, 0.005～0.02g/L; MnSO
4H
2O, 0.015～0.06g/L, NaNO
3, 1～4g/L, pH3.0～6.0.
(3) conditions of flask fermentation:
Substratum: add W-Gum 40～80g/L in the basic fermention medium as carbon source, ammonium sulfate 2～6g/L is as nitrogenous source, initial pH3.0～6.0, liquid amount 100mL/500mL triangular flask, substratum is inoculated inoculum size spore suspension 4mL through after the gelatinization, 30 ℃ of reciprocating type shaking tables, stroke 10cm, shaking culture, fermentation time 3.5～5.5 days.
(4) 15L ferment tank:
Ventilation 20～40L/min, 28～32 ℃ of culture temperature, rotating speed 200～400r/min, inoculum size 5%, cultivated 90～120 hours, substratum is with the used substratum of shake flask fermentation, and carries out gelatinization before use, and effective liquid amount of fermentor tank is 10L, end liquid measure is 7L, stream adds the substratum of gelatinization more during the fermentation, and fermenting began to flow add operation in 18 hours, and flow acceleration is 0.03L/h.
Best fermentation condition is: shake flask fermentation and the used substratum of 15L ferment tank are to add W-Gum 40g/L in the basic fermention medium as carbon source, and ammonium sulfate 2g/L is as nitrogenous source, initial pH6.0; The shake flask fermentation time is 4.0 days, and the 15L ferment tank time is 108 hours.
(5) spraying drying:
Fermented liquid carries out high-pressure homogeneous, and pressure is 3kgf/cm
2, or colloidal mill with bacterial cell disruption after, add and to help the siccative dextrin, the amount that adds dextrin is: dextrin: fermented liquid counts 0.02～0.06 with Kg/L, 170～200 ℃ of inlet temperature, 70～100 ℃ of temperature outs, sprinkler pressure 1～4kgf/cm
2, flow velocity 1.5～3.0L/h, spraying drying obtains Powdered citraurin product (I).
Or (6) make with extra care step (4) gained fermented liquid:
Fermented liquid through ultrasonic disruption or high-pressure homogeneous after with cytoclasis, discharge the pigment in the born of the same parents, 5000r/min, 20min high speed centrifugation get mycelium, extract with methyl alcohol or ethanol, and extracting does not have obviously orange 3 times to solution, filter the back merging filtrate, add and help the siccative dextrin, the amount that adds dextrin is: dextrin: fermented liquid counts 0.02～0.06 with Kg/L, carries out spraying drying, the same step of spraying drying condition (5), Dry Sack are heightened the Powdered citraurin product (II) of some.
Or the merging filtrate after the extraction is proceeded the plate layer chromatography separation, select for use G-60 type silica gel as the plate layer chromatography medium: vacuum concentration to orange plain color valency is 3000～4500U/mL, concentrated solution is used for plate layer chromatography for the first time, developping agent is a toluene: ethyl acetate: formic acid=7: 3: 1 scrapes apparent orange bands of a spectrum R
f==0.7941, again with methyl alcohol or ethanol lixiviate 3 times, 5000r/min, 20min high speed centrifugation, the supernatant liquor rotation is concentrated into orange plain color valency 3000～4500U/mL, concentrated solution is used for plate layer chromatography for the second time, and developping agent is a toluene: ethyl acetate: formic acid=8: 2: 1 scrapes apparent orange bands of a spectrum R
f=0.7458, methyl alcohol or ethanol lixiviate 3 times, 5000r/min, 20min high speed centrifugation, supernatant concentration gets citraurin needle crystal (III).
Or the merging filtrate after the extraction is proceeded column chromatography for separation, select for use Qingdao to produce silica gel as the column chromatography medium: the pillar model is 28mm * 500mm, maximum applied sample amount is the citraurin concentrated solution 4～8mL of look valency at 3000～4500U/mL, moving phase is toluene: ethyl acetate: formic acid=7: 3: 1, flow rate control is between 0.5～1.5mL/min, collect and show orange effluent liquid, the effluent liquid rotation is concentrated into orange plain color valency 3000～4500U/mL, concentrated solution is used for plate layer chromatography, developping agent is a toluene: ethyl acetate: formic acid=8: 2: 1, scrape and show orange bands of a spectrum Rf=0.7458, methyl alcohol or ethanol lixiviate 3 times, 5000r/min, 20min high speed centrifugation, supernatant concentration get citraurin needle crystal (III).
Use the solvent lixiviate in the citraurin process for refining, the citraurin product that food service industry is used all carries out lixiviate with ethanol, and the citraurin product that chemical industry and other industry are used carries out lixiviate with methyl alcohol.
Beneficial effect of the present invention: the present invention adopts a plant height to produce the Monascus anka Nakazawa et sato 9903 of pigment, for producing monascorubin based on citraurin, and improve citraurin content and carried out the optimization of fermentation condition optimization and liquid-state fermentation technology condition, reach in the hope of large-scale industrial production and on the food color Application and Development, do element task for later citraurin.In addition, to the drying process with atomizing of citraurin and refiningly also study, obtained purer citraurin, wished to be its developing purposes more widely, as be applied in beverage, the makeup products such as (as lipsticks).
The shake flat experiment of red colouring agent for food, also used as a Chinese medicine high yield citraurin is determined the bacterial classification of high yield citraurin and is the culture medium prescription of only nitrogen source with inorganic nitrogen-sourced; The liquid submerged fermentation of red colouring agent for food, also used as a Chinese medicine citraurin is determined the operating procedure parameter of suitability for industrialized production red colouring agent for food, also used as a Chinese medicine citraurin; The preparation parameter index of the spray-dired red colouring agent for food, also used as a Chinese medicine citraurin of single stage method product: three jars of average stable reaching more than the 100U/mL of look valency (15L jar) of red colouring agent for food, also used as a Chinese medicine citraurin are produced in liquid state fermentation, and the spray-dried back of fermented liquid products obtained therefrom look valency is 1000U/g, tone OD
465nm/OD510Nm is greater than 1.
Fermentation cylinder for fermentation liquid look valency at 15L reaches 121.7U/mL, and tone value reaches 1.13; Make with extra care the citraurin product that approach obtains not homochromy valency and tone through difference, citraurin product (I) the look valency that direct spraying drying obtains reaches 1030.75U/g, and tone value reaches 1.99; Up to more than the 2000U/g, tone is up to 2.45 through extraction then spray-dired citraurin product (II) look valency; Product (III) has obtained pure citraurin needle crystal.
Description of drawings
Fig. 1 citraurin product (III) full wavelength scanner figure.
The liquid chromatogram of Fig. 2 citraurin product (III).
The mass spectrum of Fig. 3 citraurin product (III).
Embodiment
Carbon source is to producing the influence of citraurin
Selecting glucose, sucrose, W-Gum, sticky rice flour, maltose and dextrin is that carbon source experimentizes.Carbon source concentration 50g/L, other proportionings of substratum are seen basic fermention medium.With W-Gum during as the carbon source of 9903 strain fermentations, orange plain color valency and biomass all are higher than far away when adopting other carbon sources, illustrate that W-Gum not only contributes the growth of thalline probably, the more important thing is the synthetic of this secondary metabolite of citraurin played a positive role, this influence of W-Gum may be that the composition of W-Gum is complicated, and contained inorganic or organic composition is also more, more complete for the monascus nutritive ingredient, thereby more help the generation of the secondary metabolite of citraurin.Determine that thus W-Gum is the best citraurin carbon source of producing.Then because of containing organic nitrogen source, the citraurin that causes a part to be generated combines with amino acid and generates haematochrome in the sticky rice flour.
The W-Gum of different concns has chosen 20,40,60 respectively to the influence that monascus 9903 produces citraurins in the substratum, five concentration levels of 80g/L, the different proportionings of attempting W-Gum and glucose in addition.By height and the tone of considering orange plain color valency, finally selecting concentration for use is the carbon source of the W-Gum of 40～80g/L as substratum.
Nitrogenous source is to producing the influence of citraurin
Experimental result shows that different nitrogen sources is different because of the nitrogenous source kind to the influence of citraurin output, and color and luster also has very big-difference.In 8 kinds of nitrogenous sources of experiment, four kinds of organic nitrogen sources (peptone, monosodium glutamate, corn steep liquor, yeast extract paste) produce orange plain color valency and nearly all are lower than product citraurin when using four kinds inorganic nitrogen-sourced (ammonium sulfate, ammonium nitrate, ammonium chloride, SODIUMNITRATE), and it is red that color all shows, tone (OD
465nm/ OD
510nm) all less than 1.And use when inorganic nitrogen-sourced, the look valency of ammonium nitrate and SODIUMNITRATE is lower again with respect to ammonium sulfate and ammonium chloride, and its tone is less than 1.When using ammonium sulfate not only the look valency be higher than other nitrogenous source far away, and tone is also higher relatively, reaches 1.68, therefore selects it as producing the citraurin nitrogenous source.Determined further again simultaneously that the ammonium sulfate concentrations when the product citraurin is the highest is 2～6g/L.
When using organic nitrogen source, orange plain color valency is low, and major cause is to contain other more amino acid in the organic nitrogen source, and this can further synthesize haematochrome with the citraurin that is generated, and citraurin is reduced.
Ammonium sulfate is made nitrogenous source, and look costly reason mainly is that sulfate ion was present in the fermented liquid after ammonium ion was utilized, and can make fermented liquid remain on acidity.This acidity can make Monascus anka Nakazawa et sato be in better physiological status.
The initial pH of substratum is to producing the influence of citraurin
In the fermenting process of Monascus anka Nakazawa et sato, most of times of fermented liquid are to be in the acid range, therefore under weakly acidic substratum starting condition, relatively are easy to the growth of thalline, and it is also higher relatively to produce orange plain color valency, and along with the pH value raises, orange plain color valency descends gradually.Tone is then on a declining curve always with the rising of pH value.Determine that according to experimental result the initial pH of substratum is 3.0～6.0.
Different sample times are to producing the influence of citraurin
On the basis of above test, measure the growth curve that Monascus anka Nakazawa et sato 9903 produces citraurin, we as can be seen before 3 days citraurin output increase in time and increase fast, about 3～5.5 days, be in steady production phase, rapid decline was just arranged after 5.5 days, this shows that citraurin should belong to a secondary metabolism intermediate product of Monascus anka Nakazawa et sato 9903, therefore we will be taken at the centre between 3～5.5 days sample time, get 3.5～4.5 days.
Four factors, the three horizontal quadratures experiment of medium component
Selecting carbon source, nitrogenous source, initial pH, fermentation time is the investigation factor, and all the other compositions are with basic fermention medium.Investigating index is that 465nm institute colour examining valency is orange plain color valency.The arrangement of factor and level sees Table 1, experimental program and the results are shown in Table 2.
Table 1 level of factor table
| Factor | Level | ||
| 1 | 2 | 3 | |
| A: carbon source concentration/gL -1B: nitrogen concentration/gL -1C: initial pH | 40 2 3.0 | 60 4 4.5 | 80 6 6.0 |
| D: fermentation time/d | 3.5 | 4.0 | 4.5 |
Table 2 Orthogonal experiment results
| 3 three orange plain color valency sum/UmL of III=level -1 | 196.21 | 179.54 | 219.13 | 177.50 | Better horizontal A 1B 1C 3D 2 |
| Extreme difference R | 16.54 | 39.38 | 54.69 | 17.64 | Better levels A 1B 1C 3D 2 |
The extreme difference R that compares four kinds of factors, the extreme difference maximum of C, next is B, D, the extreme difference minimum of A, illustrate that the initial pH of substratum produces the citraurin ability to Monascus anka Nakazawa et sato 9903 and has the greatest impact, next is to add nitrogen concentration and fermentation time, and the influence of adding in the variation range of carbon source concentration in experiment is not obvious.Experimental result, selecting best level of factor is A
1B
1C
3D
2, i.e. W-Gum 40g/L, ammonium sulfate 2g/L, initial pH value is 6.0, fermentation time is 4.0 days, with this as optimal medium prescription of the present invention.
The optimization of fermentation parameter
Adopt the optimal medium prescription of embodiment 1 orthogonal experiment gained, carried out the optimization of 15L fermentor tank deep layer cultivation operational condition.Red colouring agent for food, also used as a Chinese medicine liquid state fermentation is aerobic fermentation, so dissolved oxygen level is important parameters, and it directly influences the especially output of secondary metabolite citraurin of tunning.By changing the dissolved oxygen condition that ventilation and mixing speed form high and low two kinds of different levelss, investigate monascus produces citraurin under these two kinds different dissolved oxygen conditions situation, found that under the low dissolved oxygen situation, the climax time of producing citraurin obviously lags behind under the high dissolved oxygen situation, and the ultimate capacity of citraurin is also obviously low. and visible dissolved oxygen is bigger to the influence that red koji fermentation produces citraurin.The optimum operation processing condition of finally determining monascus 9903 liquid state fermentations product citraurin on the basis of experiment are: ventilation 20～40L/min, and 28～32 ℃ of culture temperature, rotating speed 200～400r/min, inoculum size 5% was cultivated 90～120 hours.
Liquid batch fermentation
The process of growth of monascus in 15L liquid state fermentation jar roughly can be divided into 3 stages: and the cessation of growth cessation phase (0～18h), all do not increase substantially at this stage biomass, orange plain color valency, citrinin content; Second stage (18～108h), biomass increases sharply, and citraurin output also is accompanied by thalli growth and improves rapidly simultaneously, Citrinin then begin growth pole slowly (18～90h), then increase (90～108h) again rapidly; The last stage (108～120h), how the dry weight of thalline does not almost increase, and the decrease to some degree of citraurin output, tone is some reduction thereupon also.
As seen, in the generation and the thalli growth basic synchronization of citraurin in early stage, the then too big variation of nothing of citrinin content illustrates that the production of citraurin and the growth of thalline have certain coupling.But in the later stage, citrinin content but increased rapidly when citraurin content reduced, reason may be that the relevant enzyme gene of Citrinin synthetic in early stage begins to start, or through the Citrinin precursor of certain hour after the synthetic and accumulation, the formation unit of Citrinin molecule just begins to be assembled into the Citrinin molecule, citraurin growth then is and thalli growth link coupled mutually that during to the thalline fast growing period, citraurin is also synthetic in a large number.The reduction of the minimizing of later stage citraurin and tone value in addition, reason may be that the amino acid in citraurin and the substratum reacts, and have generated water miscible haematochrome.
Liquid flow adds fermentation, and stream adds the influence of the W-Gum of gelatinization to citraurin output
Fed-batch technique can be eliminated substrate and product inhibition.Therefore we have attempted the method that stream adds the substratum of gelatinization, in the hope of improving citraurin output.On the basis of above experiment, adopting effective liquid amount is the fermentor tank of 10L, end liquid measure is 7L, begin to flow add operation during fermentation 18h, flow acceleration is 0.03L/h, under this operational condition, final citraurin rate ratio batch fermentation has improved 18%, as seen, utilize stream can overcome too high and the inhibition that causes of initial nutrient concentration, thereby improve citraurin growing amount and raw material availability effectively the growth of thalli growth and citraurin with fermented liquid.Three jars of average stablizing of the orange plain color valency of final fermented liquid reach 121.7U/mL, and tone value reaches 1.13.
The extraction of embodiment 3. citraurins and refining
The direct spray-drying process production of citraurin
Operating procedure parameter condition when determining the citraurin fermented liquid and directly carry out spraying drying through experimental study: fermented liquid carries out high-pressure homogeneous, and pressure is 3kgf/cm
2, or colloidal mill with bacterial cell disruption after, add and to help the siccative dextrin, the amount that adds dextrin is: dextrin: fermented liquid counts 0.02～0.06 with Kg/L, 170～200 ℃ of inlet temperature, 70～100 ℃ of temperature outs, sprinkler pressure 1～4kgf/cm
2, flow velocity 80～120mL/h.Finally obtain citraurin product (I) under this operational condition, its look valency is 1030.75U/g, and tone is 1.99, and the pigment yield reaches 66.67%.
The condensing crystal of citraurin and spraying drying.
The effect that different extraction agents extract the red colouring agent for food, also used as a Chinese medicine citraurin compares
Measure the citraurin fermented liquid of certain volume respectively, carry out ultrasonic disruption or other bacterial cell disruption method, behind clarifixator, colloidal mill etc., 5000r/min, 20min high speed centrifugation get mycelium, extract with 15mL normal hexane, hexanaphthene, ethyl acetate, methylene dichloride, chloroform, toluene, acetone, methyl alcohol, ethanol or formic acid respectively.Carry out frozen centrifugation behind the 2h, pipette and be used for ultraviolet spectrophotometer in 300～600nm interscan after supernatant liquor suitably dilutes, survey its look valency at 410nm, 465nm and 510nm place, found that when using acetate best to the extraction efficiency of citraurin, the look valency is up to 3240.625U/g, but when in the pigment sample, adding extraction agent, find that exothermic phenomenon is arranged, illustrate that acetate might reaction take place with pigment, be difficult to therefore guarantee that the look valency that records is exactly the content of the citraurin of nature.When using normal hexane or hexanaphthene to extract, based on yellow pigment and citraurin, haematochrome does not almost have in the extraction phase, if therefore from considering the isolating angle of pigment, hexanaphthene or normal hexane are more satisfactory selections, but the two is relatively low to the extraction yield of citraurin.When using ethyl acetate, chloroform or methylene dichloride, the extraction yield of citraurin is higher relatively, but the extraction yield of yellow pigment is also very high simultaneously, especially ethyl acetate.And when using methyl alcohol and ethanol, not only the extraction yield of citraurin is very high, and the extraction yield of yellow pigment is extremely low, very favourable to separating like this, therefore we select for use methyl alcohol or ethanol as the optimum extractant that extracts citraurin, this will see the use field of final citraurin product, if be used in food service industry, then select nontoxic ethanol as extraction agent, then select for use methyl alcohol as extraction agent if be applied to chemical industry and other industry, because with respect to ethanol, methyl alcohol is all very high to the percentage extraction and the final look valency that extracts of citraurin.After finally carrying out spraying drying with the citraurin product (II) after the organic solvent extraction, orange plain color valency reaches more than the 2000U/g, and tone value reaches 2.45.
The selection of plate layer chromatography used silica gel and developping agent for the first time
It is better that experiment find to use silica gel to carry out the effect of plate layer chromatography, and the silica gel of different size is compared, the silica gel works very well in homemade Qingdao, so we have selected the plate layer chromatography medium of the G-60 type silica gel of Qingdao marine chemical industry group as this patent research.What at first consider for the selection of developping agent is that most of pigment and citraurin in the red colouring agent for food, also used as a Chinese medicine extraction liquid are separated, on the basis of experiment, we have selected with toluene: ethyl acetate: formic acid=7: 3: 1 is developping agent, monascorubin is carried out an initial gross separation, finally obtain the orange bands of a spectrum of Rf=0.7941.
The selection of the used developping agent of plate layer chromatography for the second time
By the first time plate layer chromatography the result as can be seen monascorubin be kind of a hybrid pigment, only ordinary light according under just can see 18 kinds, and under ultraviolet ray, can see the fluorescent substance of can't see under a lot of ordinary light photographs.Citraurin can separate fully with haematochrome, but adjacent with yellow pigment very near, overlaps, and needs further to separate.Therefore we have carried out plate layer chromatography for the second time, and for employed different developping agents, adopt hexanaphthene: chloroform: pigment can not separate during Virahol (6: 4: 3), on the contrary pigment is had certain dissemination.Use toluene: ethyl acetate: formic acid (8: 2: 1) is that separating effect is best, isolates 8 colour bands altogether, the final R that gets
f=0.7458 citraurin bands of a spectrum have finally obtained pure citraurin product (III) by rectification flow, and the mass spectrum that is used for next step is identified usefulness.
Determining of column chromatography for separation citraurin method
Column chromatography silica gel is selected the column chromatography silica gel of the technical grade of Qingdao product for use, the pillar model is 28mm * 500mm, maximum applied sample amount is the citraurin concentrated solution 4～8mL of look valency at 3000～4500U/mL, moving phase is toluene: ethyl acetate: formic acid=7: 3: 1, flow rate control is between 0.5～1.5mL/min, collect and show orange effluent liquid, the effluent liquid rotation is concentrated into orange plain color valency 3000～4500U/mL, concentrated solution is used for plate layer chromatography, developping agent is a toluene: ethyl acetate: formic acid=8: 2: 1, scrape and show orange bands of a spectrum Rf=0.7458, methyl alcohol or ethanol lixiviate 3 times, 5000r/min, 20min high speed centrifugation, supernatant concentration get citraurin needle crystal (III).This method is compared with the plate layer chromatography method, and treatment capacity is big, and method is simple, is easy to suitability for industrialized production.
The mass spectrum of citraurin product (III) is identified
With citraurin crystal dissolve with methanol, suitably be used for ultraviolet spectrophotometer in 300～600nm interscan after the dilution, scan as shown in Figure 1, the maximum absorption wavelength that gets citraurin is 465nm.
We have carried out qualitative detection with the method for mass spectrum inspection to prepared citraurin.Fig. 2 is the citraurin liquid chromatogram, know the peak that target substance goes out in the time of may be for 16.86min by figure, its positive ion mode mass spectrum as shown in Figure 3, can the clear M+Na of finding out (molecular weight is 377.6) and the quasi-molecular ion peak of M+H (molecular weight is 355.6) by Fig. 3, determine that its molecular weight is 354D, identical with the molecular weight of the erythema red colouring agent for food, also used as a Chinese medicine element of reporting on the document, its peak is an one-component.Be it can also be seen that by Fig. 2 that in addition main component is erythema red colouring agent for food, also used as a Chinese medicine element really in the citraurin product of preparation, from peak area normalization method meter, erythema red colouring agent for food, also used as a Chinese medicine element has accounted for 85.93% of sample total amount.Therefore can think purelyr, can be used for researchs such as its character.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI473612B (en) * | 2010-12-23 | 2015-02-21 | Food Industry Res & Dev Inst | Novel monascuspurpurones, preparation process thereof, and uses of the monascuspurpurones |
| CN106755171A (en) * | 2017-02-09 | 2017-05-31 | 江南大学 | A kind of method for promoting red yeast rice strains liquid fermentation to produce red yeast rice citraurin |
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| CN104946718B (en) * | 2014-03-26 | 2018-03-20 | 江南大学 | A kind of biliquid phase fermentation method for being coupled in-situ fermentation extraction red yeast rice citraurin |
| CN104878053B (en) * | 2015-05-26 | 2018-04-13 | 南昌大学 | A kind of preparation method of Metabolites ofMonascus |
| CN105734091A (en) * | 2016-04-19 | 2016-07-06 | 天津科技大学 | Method for producing high-purity monascine and ankafaflavin through monascus liquid-state fermentation |
| CN106432261A (en) * | 2016-09-23 | 2017-02-22 | 湛江嘉诚科技服务有限公司 | Technology for drying monascus yellow pigment through spray dryer |
| CN106636249B (en) * | 2016-12-30 | 2020-03-24 | 江南大学 | Method for promoting monascus liquid state fermentation to produce monascus yellow pigment |
| CN107723320B (en) * | 2017-09-08 | 2020-09-18 | 广东科隆生物科技有限公司 | Method for directly preparing crystal monascus orange pigment |
| CN110437644B (en) * | 2019-08-19 | 2020-08-07 | 长江大学 | Separation and purification method of monascus orange pigment |
| CN112143759B (en) * | 2020-09-16 | 2022-06-17 | 长江大学 | Method for improving yield of orange pigment in monascus mycelium and application |
| CN114507698A (en) * | 2020-11-17 | 2022-05-17 | 天津科技大学 | A kind of liquid fermentation method for improving the yield of monascus orange pigment |
| CN112457685B (en) * | 2020-12-01 | 2022-05-03 | 广东肇庆星湖生物科技股份有限公司 | Extraction and purification method of monascus orange pigment |
| CN113201230B (en) * | 2020-12-23 | 2023-02-10 | 天津科技大学 | A kind of red yeast rice pigment product and preparation method thereof |
-
2005
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Non-Patent Citations (2)
| Title |
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| 红曲中橙色素的研究 宫慧梅等.食品研究与开发，第3期 2002 * |
| 食品与生物技术 顾玉梅等.食品与生物技术，第21卷第1期 2002 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI473612B (en) * | 2010-12-23 | 2015-02-21 | Food Industry Res & Dev Inst | Novel monascuspurpurones, preparation process thereof, and uses of the monascuspurpurones |
| CN106755171A (en) * | 2017-02-09 | 2017-05-31 | 江南大学 | A kind of method for promoting red yeast rice strains liquid fermentation to produce red yeast rice citraurin |
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| CN1814783A (en) | 2006-08-09 |
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