CN105734091A - Method for producing high-purity monascine and ankafaflavin through monascus liquid-state fermentation - Google Patents
Method for producing high-purity monascine and ankafaflavin through monascus liquid-state fermentation Download PDFInfo
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- CN105734091A CN105734091A CN201610244335.7A CN201610244335A CN105734091A CN 105734091 A CN105734091 A CN 105734091A CN 201610244335 A CN201610244335 A CN 201610244335A CN 105734091 A CN105734091 A CN 105734091A
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Abstract
The invention relates to a method for producing high-purity monascine and ankafaflavin through monascus liquid-state fermentation. The method comprises the steps of seed culture, fermentation, centrifugation, drying, smashing, ultrasonic-assisted extraction, crude extract concentration, predissociation, concentration, refining and drying. Yellow pigment is specifically extracted from monascus fermentation products through normal hexane with low polarity, interference of red pigment and orange pigment on yellow pigment separation is reduced as much as possible, and the purity of monascine and ankafaflavin produced through fermentation is improved. By the adoption of the method, efficient and cost-effective preparation of monascine and ankafaflavin can be achieved, and the purity, calculated with the area normalization method, of produced monascine and ankafaflavin can be 99% or more.
Description
Technical field
The invention belongs to technical field of health care food, especially a kind of method being produced high-purity Monascin and ankaflavin by monascus liquid fermentation method.
Background technology
Monas cuspurpureus Went is the exported product of China's tradition, characteristic, and the application of monascorubin be there has also been the history of thousand by China.Research in recent years finds, except as food color, monascorubin also has the biological activitys such as antiinflammatory, antioxidation, antibacterial, cholesterol reducing, mutation and antitumor, it is also possible in preparing solaode and biological printing and dyeing etc., have boundless application prospect.Flavochrome therein--Monascin and ankaflavin are proved to especially to be had the effects such as anticancer, antiinflammatory, suppression tumor cell and receives significant attention.
In monascorubin, generally comprise three kinds of pigments red, orange, yellow simultaneously, its similar structure, close polarity bring difficulty to the separation and purification of pigment, the particularly existence of citraurin, the separation and purification of meeting severe jamming flavochrome, therefore, the market price of Monascin and ankaflavin is high, and rare its sterling, limit their application and popularization.Therefore, prepare highly purified Monascin and ankaflavin is possible not only to the demand that meets food industry to flavochrome natural, nontoxic, it is also possible to the bioactivity research for both pigments contributes.
In recent years, monascorubin being easily separated and in the research of purification, many employing ethanol are Extraction solvent, and the extraction of pigment is not had specificity by ethanol, adds the difficulty of purification；Cui Li etc.[1]Utilize high performance liquid chromatography that monascorubin has been separated.Zheng Yunquan etc.[2]Adopt high-speed countercurrent chromatography separation purification monascorubin.These researchs, on purification process, all adopt the method that single-stage is refining, and the separating effect of citraurin and flavochrome is inconspicuous.
By contrasting, the inventive method comparatively exclusively extracts flavochrome by adopting the solvent that normal hexane isopolarity is less from red koji fermentation thing, recycling silica gel chromatography is to crude extract pre-separation, avoid the interference that flavochrome is separated by citraurin, then with high speed adverse current chromatogram, flavochrome pre-separation thing is refined, reduce separating difficulty, and the method is more simple on technology path, efficiently.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art part, a kind of method being produced high-purity Monascin and ankaflavin by monascus liquid fermentation method is provided, the method comparatively exclusively extracts flavochrome by adopting the normal hexane that polarity is less from red koji fermentation thing, crude extract is carried out pre-separation by recycling silica gel chromatography, avoid the interference that flavochrome is separated by citraurin, then with high speed adverse current chromatogram, flavochrome pre-separation thing is refined, reduce separating difficulty, it is low that Monascin prepared by employing the method and ankaflavin have cost, purity is high, the features such as safety non-toxic.
This invention address that the technical scheme that the problems referred to above adopt is as follows:
A kind of method being produced high-purity Monascin and ankaflavin by monascus liquid fermentation method, step is as follows:
(1) seed culture: be inoculated into by monascus sp bacteria strain in the seed culture medium after sterilizing, cultivates 1-4d in 20-35 DEG C of shaking table, obtains seed liquor；
Fermentation: by step (1) in cultured seed liquor be inoculated in fermentation medium with the ratio of 3%--8% (v/v), 30 DEG C of shake-flask culture 6d, obtain fermentation liquid；
(3) being centrifuged: be centrifuged by fermentation liquid, centrifugal condition is 3500r/min, centrifugal 20min, obtains fermented product precipitation；
(4) dry: dry at fermented product is deposited in 40-60 DEG C to constant weight, obtain the fermented product dried；
(5) pulverize: dry fermented product mortar is ground, cross 100 mesh sieves, obtain the fermented product powder dried；
(6) ultrasonic assistant extracts: dry fermented product powder being added normal hexane, carries out ultrasonic assistant extraction, solid-liquid ratio is 1:20-120g:mL, extracts 1-4 time, and ultrasonic power is 50-100W, extracts 15-45min, obtains normal hexane crude extract；
(7) crude extract concentration: by the vacuum concentration at 40 DEG C of normal hexane crude extract, obtain concentrated solution；
(8) pre-separation: concentrated solution silica gel chromatographic column is carried out pre-separation, obtain yellow eluent, the post separation condition of described pre-separation is particularly as follows: the silica column of 100 order fineness is as fixing phase, and mobile phase is normal hexane and ethyl acetate, described normal hexane: the volume ratio of ethyl acetate is 2-8:1；
(9) concentration: yellow eluent vacuum at 40 DEG C is concentrated into Powdered, obtains pigment powder；
(10) refine: preparation high speed adverse current chromatogram phase up and down, stratification, described up and down by normal hexane: methanol: the volume ratio 10:2.5-9:7.5-1 of water is formulated, lower phased soln by the upper phase of pigment powder 5mL and 5mL, with high speed adverse current chromatogram, Monascin and ankaflavin are refined, detection wavelength is 405nm, collects 0-200min eluent, obtains Monascin and ankaflavin；
(11) dry: by Monascin and ankaflavin eluent, vacuum at 40 DEG C is concentrated into Powdered, obtains high-purity Monascin and ankaflavin.
And, described step (1) middle seed culture medium is: glucose 6g, peptone 2g, NaNO31g, MgSO4·7H2O0.5g, KH2PO41g, tap water 100mL.
And, described step (2) middle fermentation medium is: rice meal 5g, NaNO30.3g, MgSO4·7H2O0.1g, KH2PO40.15g, tap water 100mL.
And, described step (6) in ultrasonic assistant extract optimum extraction condition be: ultrasonic assistant extract solid-liquid ratio be 1:90 (g:mL), extract 3 times, ultrasonic power be 100W, extract 15-45min.
And, the defining method of the optimum extraction condition that described ultrasonic assistant extracts is as follows:
Using the absorbance under 405nm wavelength as evaluation index, test, by single factor experiment and response surface, the optimum condition obtaining normal hexane ultrasound assisted extraction flavochrome；
(1) experiment of single factor
1. the extraction time impact on normal hexane extraction flavochrome
Accurately weigh 0.050g Monas cuspurpureus Went mycelia powder, put in 10mL centrifuge tube, add 5mL normal hexane；15min, 30min, 45min, 60min is extracted respectively at 100W ultrasonic assistant when；3500r/min, centrifugal 20min；Take supernatant, be settled to 5mL, after diluting 30 times, measure the absorbance under 405nm respectively, repeat 3 times, seek its meansigma methods；
2. the extraction time impact on normal hexane extraction flavochrome
Accurately weigh 0.050g Monas cuspurpureus Went mycelia powder, be respectively put in 4 10mL centrifuge tubes, each addition 5mL normal hexane；Extracting 30min, 3500r/min when 100W ultrasonic assistant, centrifugal 20min takes clear liquid, as once extracting；Respectively the Monas cuspurpureus Went mycelia powder in above-mentioned 4 centrifuge tubes is extracted 1 time, 2 times, 3 times and 4 times, merge the extracting solution of corresponding test tube, dilute suitable multiple, make absorption values between 0.2～0.8, measure the absorbance of now 405nm；Repeat 3 times, seek its meansigma methods；
3. the liquid ratio impact on normal hexane extraction flavochrome
Accurately weigh 0.050g Monas cuspurpureus Went mycelia powder, be respectively put in 6 10mL centrifuge tubes, be separately added into the normal hexane of 20,40,60,80,100,120 times of volumes；30min is extracted in 100W ultrasonic assistant when；Hereafter the centrifugal 20min of 3500r/min；Taking supernatant, be settled to 10mL, dilute 30 times, measure the absorbance under 405nm wavelength respectively, each time conditions repeats 3 times；
4. the power impact on normal hexane extraction flavochrome is extracted
Accurately weigh 0.050g Monas cuspurpureus Went mycelia powder, be respectively put in 3 10mL centrifuge tubes, each addition 5mL normal hexane；Respectively at 90W, 70W, 50W ultrasonic assistant when, 30min is extracted after covering centrifuge tube lid；3500r/min, centrifugal 20min；Take supernatant, be settled to 5mL, after diluting 30 times, measure the absorbance under 405nm respectively, repeat 3 times；
(2) Box-Behnken experiment
Box-Benhnken design method can design by experiment, finally gives regression equation and is analyzed to seek the combination of optimum, thus solving the statistical method of Multivariable；With extraction time, solid-liquid ratio and three factors of extraction time for independent variable, DesignExpert8.0.6 is utilized to design Three factors-levels response surface experiments, being obtained by normal hexane is that extractant extracts the optimal conditions of flavochrome in Monascus anka Nakazawa et sato filament, namely ultrasonic assistant extract solid-liquid ratio be 1:90 (g:mL), extract 3 times, ultrasonic power is 100W, extract 15-45min.
And, described step (8) in pre-separation post separate optimum condition be: the silica column of 100 order fineness is as fixing phase, and mobile phase is normal hexane and ethyl acetate, described normal hexane: the volume ratio of ethyl acetate is 4:1.
And, the defining method of the optimum condition that the post of described pre-separation separates is as follows:
(1) in thin-layer developing cylinder, prepare the developing solvent of n-hexane-ethyl acetate 2:1,4:1,6:1,8:1 respectively；Take n-hexane extract capillary tube point sample on lamellae, after the ethanol volatilization of pigment to be dissolved, lamellae is placed in expansion cylinder, when the liquid level of developing solvent will arrive chromatoplate top, chromatoplate is taken out, is placed in shady and cool ventilation place and makes ethanol volatilize, by natural light and ultraviolet analysis instrument for three purposed, separation case according to pigment, calculates the Rf value of corresponding pigment；When mobile phase ratio is n-hexane-ethyl acetate 2:1, its polarity is bigger than normal, and citraurin and flavochrome are almost eluted simultaneously, and hangover is serious；When mobile phase ratio is n-hexane-ethyl acetate 6:1, although citraurin and flavochrome can separate, but its development rate is too slow, and in post separates, development rate can expend more mobile phase slowly, owing to utilizing silica gel chromatography simply pigment to be carried out pre-separation, it is not necessary to separate accurately very much, therefore final selective flow is n-hexane-ethyl acetate 4:1 mutually；
(2) silica gel chromatography pre-separation
With normal hexane extraction 1g monascus mycopowder, then by normal hexane concentrate drying；Utilize the silica gel adsorption resin of 100 order fineness, monascus normal hexane crude extract is carried out pre-separation；Collect and detect the component of different time sections eluent, simultaneously using acetonitrile as blank；1, the corresponding 100-200mL respectively of the eluent in 2, No. 3 brown bottles, 200-350mL, the eluent of 350mL-500mL, the suitableeest testing conditions of red pigment, citraurin, flavochrome three class pigment it is respectively provided with during its Liquid Detection, detection wavelength respectively 540nm, 480nm, 390nm, reference wavelength is 350nm, 330nm, 270nm respectively；Contrast by eluent and blank solution, it was demonstrated that the normal hexane crude extract of Monascus mycelium is after the silica gel chromatographic column of 100 order fineness separates, and the yellow color component collected is mainly Monascin and ankaflavin.
And, described step (10) in up and down by normal hexane: methanol: the volume ratio 10:7:3 of water is formulated.
And, described step specifically comprising the following steps that (10)
(1) flavochrome mensuration of apportionment ratio k value in normal hexane-methanol-water separation system
Selection system: normal hexane-methanol-water；
Configuration criteria: ensure that the final volume of phase up and down is close, because of than normal hexane, water and methanol polarity are close, therefore keep normal hexane constancy of volume, adjust the proportioning of first alcohol and water, finally make the cumulative volume of first alcohol and water add and close with the volume of normal hexane, so can ensure that upper and lower phase volume is close in separation system process for preparation, thus avoiding the waste of organic solvent, simultaneously again can the polarity of across-the-board regulation separation system；
10mL centrifuge tube is proportionally prepared phase up and down, is sufficiently mixed, after being layered, take 4mL upper and lower phase respectively, be respectively charged in two other clean 10mL centrifuge tube；A small amount of pigment crude extract solid is added lower phase, ultrasonic Treatment, after being completely dissolved, takes 1mL, citraurin content yellow with HPLC detection, it is designated as C respectively11、C12；The remaining lower phase containing pigment adds the upper phase of 3L；Taking phase under 1mL after being sufficiently mixed again, detect with HPLC, content is designated as C21, C22；
After balancing each other up and down, pigment is at the content/pigment in upper phase of the partition coefficient=pigment of phase up and down content in lower phase；Namely pigment is at biphase middle apportionment ratio k=(C1n-C2n)/C1n× 100% (n=1 or n=2)；
Measure according to the method described above Monascin, rubropunctatin, ankaflavin and monascorubin difference up and down mutually in distribution ratio, when utilizing high speed adverse current chromatogram that mixture is easily separated, in theory, k value can reach the separating effect of optimum when 0.95～1.5, obtains up and down by normal hexane: methanol: the volume ratio 10:7:3 of water is formulated for optimum；
With on 5mL mutually and after being dissolved under 5mL, being easily separated with HSCCC；
(2) the separation of high speed adverse current chromatogram
1. the preparation of high speed adverse current chromatogram phase up and down
Three kinds of reagent of designated volume ratio are added in large beaker, seals with sealed membrane after being sufficiently stirred for Glass rod, stand so that it is layering；Pouring out phase after system balance, the part being layered mutually up and down utilizes separatory funnel will separate mutually up and down after standing；Choosing phase for fixing phase, lower phase is mobile phase, and ultrasonic degassed 30min, carefully moves, in order to avoid being mixed into bubble respectively；
2. the preparation of high speed adverse current chromatogram
Open computer, circulator bath system, adverse current chromatogram main frame and detector successively；Ensureing that power supply, holding wire consolidate, pipeline is closed；Design temperature is 25 DEG C, and detection wavelength is 405nm；
Open pump, pump into fixing phase with the speed of 10mL/min, when fixing from pipeline go out post mouth flow out after termination of pumping；Switching pump head, is inserted in mobile phase, regulates adverse current chromatogram engine speed, selects FWD pattern, and rotating speed is slowly adjusted to 900r/min, after engine speed is stable, pumps into lower phase with the speed of 2mL/min；
3. the separation of sample and collection
After HSCCC is ready, the sample pigment powder 5mL that will separate dissolving with the mixed liquor of phase under 5mL mutually, ultrasonic assistant dissolves, to without obvious granule；If there being obvious granule, then by mixture with 0.45 μm of organic membrane filtration, load in clean 20mL syringe；
Mixed liquor in mouth and out mouth are inserted respectively sampling injector and empty syringe, termination of pumping；Six-way valve handle is quickly pushed load shelves；Promoting empty syringe, to sampling injector, liquid level rises, and does not have bubble, is drained by the gas of sample circle afterbody, pulls sky syringe, and the sample in sampling injector is sucked quantitative loop；
Six-way valve handle being allocated to inject gear, turn on pump, opens HSCCC computer software and automatic collector simultaneously, detection wavelength is 405nm, collects 0-200min eluent, obtains Monascin and ankaflavin.
Advantages of the present invention and having the beneficial effect that
1. this method is by adopting the less normal hexane of polarity comparatively exclusively to extract flavochrome from red koji fermentation thing, decreases the interference that flavochrome is separated by red, citraurin as far as possible, improves the purity of Monascin that the production of Fermentation Condition of Monascus spp method obtains and ankaflavin.
2. this method utilizes silica gel chromatography to crude extract pre-separation, orange polarity being sufficiently close to, flavochrome are separately, it is to avoid the interference that flavochrome is separated by citraurin.
3. this method uses high speed adverse current chromatogram that flavochrome pre-separation thing is refined, reduce separating difficulty, simultaneously, adopt the method prepare Monascin and ankaflavin cost is low, purity is high, safety non-toxic, can realize Monascin and ankaflavin efficient, inexpensively prepare, the Monascin prepared and ankaflavin, calculate through area normalization method, and purity is up to more than 99%.
Accompanying drawing explanation
Fig. 1 is headpin eluent liquid chromatographic detection result figure in the present invention；
Fig. 2 is No. 2 bottle eluent liquid chromatographic detection result figure in the present invention；
Fig. 3 is No. 3 bottle eluent liquid chromatographic detection result figure in the present invention；
Fig. 4 is empty eluent liquid chromatographic detection result figure of the present invention；
Wherein, in Figs. 1-4, upper figure is sample testing result figure of (detection wavelength 540nm, reference wavelength 350nm) under the testing conditions of monascus red pigment；Middle figure is sample testing result figure of (detection wavelength 480nm, reference wavelength 330nm) under the testing conditions of Monas cuspurpureus Went citraurin；Figure below is the testing result figure of (detection wavelength 390nm, reference wavelength 270nm) under the testing conditions of monascus yellow pigment；
Fig. 5 is the spectrogram of four kinds of pigments in the present invention；Wherein, Fig. 5-1 is the spectrogram of Monascin (Monascin, yellow)；Fig. 5-2 is the spectrogram of rubropunctatin (Rubropunetatin, orange)；Fig. 5-3 is the spectrogram of ankaflavin (Ankaflavin, yellow)；Fig. 5-4 is the spectrogram of monascorubin (Monascorubrin, orange)；
Fig. 6 is the separating resulting figure of HSCCC after silicagel column pre-separation in the present invention；
Fig. 7 is No. 26 collecting pipe liquid chromatographic detection result figure in the present invention；
Fig. 8 is No. 32 collecting pipe liquid chromatographic detection result figure in the present invention；
Fig. 9 is No. 51 collecting pipe liquid chromatographic detection result figure in the present invention；
Figure 10 is No. 63 collecting pipe liquid chromatographic detection result figure in the present invention；
Figure 11 is that in the present invention, Monascin marks product and purification product liquid chromatogram comparison diagram；
Figure 12 is the mass spectrum of purification Ankaflavin in the present invention；
Figure 13 be in the present invention extraction time extraction effect of ankaflavin affected figure；
Figure 14 is that in the present invention, the extraction effect of ankaflavin is affected figure by extraction time；
Figure 15 be in the present invention solvent make consumption on the extraction effect of ankaflavin affect figure；
Figure 16 is that in the present invention, monascus yellow pigment extraction effect is affected figure by ultrasonic power.
Detailed description of the invention
Below by specific embodiment, the invention will be further described, and following example are illustrative, is not determinate, it is impossible to limit protection scope of the present invention with this.
The method used in the present invention, if no special instructions, is the conventional method of this area；The reagent used in the present invention, if no special instructions, is the conventional reagent of this area.
The monascus used in the present invention is M9 or monascus M3 or other monascus, described monascus is M9 article (Stimulatoryeffectsofbluelightonthegrowth, monascinandankaflavinproductioninMonascus, BiotechnolLett (2015) 37:1043 1048DOI10.1007/s10529-014-1763-3, ChangluWang, DiChen, MianhuaChen, YurongWang, ZhenjingLi, engjuanLi；Or, Optimizationofsubmergedfermentationmedium, forcitrinin-freemonascinproductionbyMonascus, PreparativeBiochemistryandBiotechnology, DiChen, YuanXue, MianhuaChen, ZhenjingLi&ChangluWang；nullOr，EffectsofbluelightonpigmentbiosynthesisofMonascus，JournalofMicrobiology(2016)Vol.54,No.4,pp.305–310，DOI10.1007/s12275-016-6011-1，DiChen,ChunmaoXue,MianhuaChen,ShufenWu,ZhenjingLi,Disclosed in andChangluWang)，Described monascus M3 is patent publication us (CN201110065942.4，There is the monascus M3 bacterial strain of efficient cholesterol degradation ability) in disclosed.
Embodiment 1
A kind of method being produced high-purity Monascin and ankaflavin by monascus M3 liquid fermentation method, step is as follows:
1. seed culture: in the seed culture medium after monascus M3 inoculation to sterilizing, 3d will be cultivated in 30 DEG C of shaking tables, obtain seed liquor；
Described seed culture medium is: glucose 6g, peptone 2g, NaNO31g, MgSO4·7H2O0.5g, KH2PO41g, tap water 100mL；
2. fermentation: by step (1) in cultured seed liquor be inoculated in fermentation medium with the ratio of 5% (v/v), 30 DEG C of shake-flask culture 6d, obtain fermentation liquid；
Described fermentation medium is: rice meal 5g, NaNO30.3g, MgSO4·7H2O0.1g, KH2PO40.15g, tap water 100mL；
3. being centrifuged: be centrifuged by fermentation liquid, centrifugal condition is 3500r/min, centrifugal 20min, obtains fermented product precipitation；
4. dry: fermented product is deposited at 60 DEG C and dries to constant weight, obtain the fermented product dried；
5. pulverize: dry fermented product mortar is ground, cross 100 mesh sieves, obtain the fermented product powder dried；
6. ultrasonic assistant extracts: dry fermented product powder is added normal hexane, and ultrasonic assistant extracts, and solid-liquid ratio is 1:20-120g:mL, extracts 1-4 time, and ultrasonic power is 50-100W, extracts 15-45min, obtains normal hexane crude extract；
7. crude extract concentration: by the vacuum concentration at 40 DEG C of normal hexane crude extract, obtain concentrated solution；
8. pre-separation: concentrated solution silica gel chromatographic column is carried out pre-separation, obtain yellow eluent, the post separation condition of described pre-separation is particularly as follows: the silica column of 100 order fineness is as fixing phase, and mobile phase is normal hexane and ethyl acetate, described normal hexane: the volume ratio of ethyl acetate is 2-8:1；
9. concentration: yellow eluent vacuum at 40 DEG C is concentrated into Powdered, obtains pigment powder；
10. refine: preparation high speed adverse current chromatogram phase up and down, stratification, described up and down by normal hexane: methanol: the volume ratio 10:2.5-9:7.5-1 of water is formulated (such as, above-mentioned mutually concrete up and down it is formulated as follows: normal hexane, methanol and water are poured in the large beaker of, ultimately form the two phase liquid being made up of three kinds of solvents, namely it is poured on after together by three kinds of solvents to be finally divided into upper and lower two-layer, superincumbent is just called phase, phase under being called below.Up and down mutually in all existing normal hexane also have methanol also to have water, characteristic according to each component, the normal hexane contained in upper phase is relatively more, the first alcohol and water contained in lower phase is relatively more), by the lower phased soln of the upper phase of pigment powder 5mL and 5mL, with high speed adverse current chromatogram, Monascin and ankaflavin being refined, detection wavelength is 405nm, collect 0-200min eluent, obtain Monascin and ankaflavin；
(11) dry: by Monascin and ankaflavin eluent, vacuum at 40 DEG C is concentrated into Powdered, obtains high-purity Monascin and ankaflavin.
Embodiment 2
A kind of method being produced high-purity Monascin and ankaflavin by monascus M9 liquid fermentation method, step is as follows:
1. seed culture: in the seed culture medium after monascus M9 inoculation to sterilizing, 4d will be cultivated in 20 DEG C of shaking tables, obtain seed liquor；
Described seed culture medium is: glucose 6g, peptone 2g, NaNO31g, MgSO4·7H2O0.5g, KH2PO41g, tap water 100mL；
2. fermentation: by step (1) in cultured seed liquor be inoculated in fermentation medium with the ratio of 3% (v/v), 30 DEG C of shake-flask culture 6d, obtain fermentation liquid；
Described fermentation medium is: rice meal 5g, NaNO30.3g, MgSO4·7H2O0.1g, KH2PO40.15g, tap water 100mL；
3. being centrifuged: be centrifuged by fermentation liquid, centrifugal condition is 3500r/min, centrifugal 20min, obtains fermented product precipitation；
4. dry: fermented product is deposited at 40 DEG C and dries to constant weight, obtain the fermented product dried；
5. pulverize: dry fermented product mortar is ground, cross 100 mesh sieves, obtain the fermented product powder dried；
6. ultrasonic assistant extracts: dry fermented product powder is added normal hexane, and ultrasonic assistant extracts, and solid-liquid ratio is 1:20-120g:mL, extracts 1-4 time, and ultrasonic power is 50-100W, extracts 15-45min, obtains normal hexane crude extract；
7. crude extract concentration: by the vacuum concentration at 40 DEG C of normal hexane crude extract, obtain concentrated solution；
8. pre-separation: concentrated solution silica gel chromatographic column is carried out pre-separation, obtain yellow eluent, the post separation condition of described pre-separation is particularly as follows: the silica column of 100 order fineness is as fixing phase, and mobile phase is normal hexane and ethyl acetate, described normal hexane: the volume ratio of ethyl acetate is 2-8:1；
9. concentration: yellow eluent vacuum at 40 DEG C is concentrated into Powdered, obtains pigment powder；
10. refine: preparation high speed adverse current chromatogram phase up and down, stratification, described up and down by normal hexane: methanol: the volume ratio 10:2.5-9:7.5-1 of water is formulated (such as, above-mentioned mutually concrete up and down it is formulated as follows: normal hexane, methanol and water are poured in the large beaker of, ultimately form the two phase liquid being made up of three kinds of solvents, namely it is poured on after together by three kinds of solvents to be finally divided into upper and lower two-layer, superincumbent is just called phase, phase under being called below.Up and down mutually in all existing normal hexane also have methanol also to have water, characteristic according to each component, the normal hexane contained in upper phase is relatively more, the first alcohol and water contained in lower phase is relatively more), by the lower phased soln of the upper phase of pigment powder 5mL and 5mL, with high speed adverse current chromatogram, Monascin and ankaflavin being refined, detection wavelength is 405nm, collect 0-200min eluent, obtain Monascin and ankaflavin；
(11) dry: by Monascin and ankaflavin eluent, vacuum at 40 DEG C is concentrated into Powdered, obtains high-purity Monascin and ankaflavin.
Embodiment 3
A kind of method being produced high-purity Monascin and ankaflavin by monascus liquid fermentation method, step is as follows:
1. the preparation of fermented product powder
(1) seed culture: be inoculated into by monascus sp bacteria strain in the following seed culture medium after sterilizing, described culture medium is: glucose 6g, peptone 2g, NaNO31g、MgSO4·7H2O0.5g、KH2PO41g, tap water 100mL, shake-flask culture 3d at 30 DEG C.
(2) fermentation: being inoculated in following fermentation medium by cultured seed liquor in step (1) with the ratio of 5% (v/v), described culture medium is: rice meal 5g, NaNO30.3g, MgSO4·7H2O0.1g, KH2PO40.15g, tap water 100mL, 30 DEG C of shake-flask culture 6d, obtain fermentation liquid.
(3) centrifugal: being centrifuged by cultured fermentation liquid in step (2), centrifugal condition is 3500r/min, centrifugal 20min, obtain fermented product precipitation.
(4) dry: the fermented product of gained to be deposited at 60 DEG C and dries to constant weight, obtain the fermented product dried.
(5) pulverize: dry fermented product mortar is ground, cross 100 mesh sieves, obtain the fermented product powder dried.
2. determine optimum extraction condition
Using the absorbance under 405nm wavelength as evaluation index, test, by single factor experiment and response surface, the optimum condition obtaining normal hexane ultrasound assisted extraction flavochrome.It is 1:90 (g:mL) that ultrasonic assistant extracts solid-liquid ratio, extracts 3 times, and ultrasonic power is 100W, extracts 15-45min.
(1) experiment of single factor
1. the extraction time impact on normal hexane extraction flavochrome
Accurately weigh 0.050g Monas cuspurpureus Went mycelia powder, put in 10mL centrifuge tube, add 5mL normal hexane.15min, 30min, 45min, 60min is extracted respectively at 100W ultrasonic assistant when.3500r/min, centrifugal 20min.Take supernatant, be settled to 5mL, after diluting 30 times, measure the absorbance under 405nm respectively, repeat 3 times, seek its meansigma methods.Result is shown in Figure 13, along with the increase of extraction time, flavochrome extracted amount substantially in first raising the trend reduced afterwards, extraction time about 30min place can reach maximum.Therefore, at about 30min, the result of normal hexane extraction flavochrome can be optimized.
2. the extraction time impact on normal hexane extraction flavochrome
Accurately weigh 0.050g Monas cuspurpureus Went mycelia powder, be respectively put in 4 10mL centrifuge tubes, each addition 5mL normal hexane.Extracting 30min, 3500r/min when 100W ultrasonic assistant, centrifugal 20min takes clear liquid, as once extracting.Respectively the Monas cuspurpureus Went mycelia powder in above-mentioned 4 centrifuge tubes is extracted 1 time, 2 times, 3 times and 4 times, merge the extracting solution of corresponding test tube, dilute suitable multiple, make absorption values between 0.2～0.8, measure the absorbance of now 405nm.Repeat 3 times, seek its meansigma methods.Result is shown in Figure 14, wherein 1,2,3 respectively normal hexane extraction monascus mycelia 1 time, 2 times, 3 times time, absorbance during solution dilution 30 times, 4 is solution after extracting 4 times absorbance when being converted to dilution 30 times.As can be seen here, liquid ratio 100:1,100W ultrasound assisted extraction 30min extraction conditions under, be decreased obviously from the absorbance of extracting solution when starting for the 4th time, illustrate that now flavochrome extracts close to completely.Therefore select 2,3,4 variablees as normal hexane extraction flavochrome to be optimized extracting result.
3. the liquid ratio impact on normal hexane extraction flavochrome
Accurately weigh 0.050g Monas cuspurpureus Went mycelia powder, be respectively put in 6 10mL centrifuge tubes, be separately added into the normal hexane of 20,40,60,80,100,120 times of volumes.30min is extracted in 100W ultrasonic assistant when.Hereafter the centrifugal 20min of 3500r/min.Taking supernatant, be settled to 10mL, dilute 30 times, measure the absorbance under 405nm wavelength respectively, each time conditions is in triplicate.Result is shown in that in Figure 15, figure, measured value is result when being converted to dilution 30 times.As can be seen here, along with Extraction solvent makes the increase of consumption, the extracted amount of pigment also increases, it is considered to solvent make consumption and the cost of later stage concentration and evaporation, select about 100 times normal hexane amounts that extraction effect can be made to reach a good level.
4. the power impact on normal hexane extraction flavochrome is extracted
Accurately weigh 0.050g Monas cuspurpureus Went mycelia powder, be respectively put in 3 10mL centrifuge tubes, each addition 5mL normal hexane.Respectively at 90W, 70W, 50W ultrasonic assistant when, 30min is extracted after covering centrifuge tube lid.3500r/min, centrifugal 20min.Take supernatant, be settled to 5mL, after diluting 30 times, measure the absorbance under 405nm respectively, repeat 3 times.As shown in figure 16, within the scope of 50-100W, power is little on the impact of normal hexane extraction monascus yellow pigment, therefore is left out the power impact on extracting flavochrome in separation and Extraction process afterwards for result.
(2) Box-Behnken experiment
Box-Benhnken design method can design by experiment, finally gives regression equation and is analyzed to seek the combination of optimum, thus solving the statistical method of Multivariable.With extraction time, solid-liquid ratio and three factors of extraction time for independent variable, DesignExpert8.0.6 is utilized to design Three factors-levels response surface experiments, being desirably to obtain one utilizes normal hexane to extract the optimal conditions of flavochrome in Monascus anka Nakazawa et sato filament for extractant, namely ultrasonic assistant extract solid-liquid ratio be 1:90 (g:mL), extract 3 times, ultrasonic power is 100W, extract 15-45min.
Concrete Box-Behnken experimental design and related test results are in Table 1 and table 2.
Table 1Box-Behnken experimental design and experimental result table
Table 2Box-Behnken tests regression model analysis of variance table
By experimental data being carried out the multinomial regression fit of secondary, it is thus achieved that the multiple regression equation of dependent variable is by monascorubin response value under 405nm: Y=0.77+0.12A+0.15B+0.070C-0.084B2.In liquid ratio, extraction time and three variablees of extraction time, the minimum liquid ratio of prioritizing selection, minimum extraction time and minimum extraction time.Obtain normal hexane extraction flavochrome complete time extraction conditions be: liquid ratio 89.8, extraction time 2.89, extraction time 15min, it is contemplated that absorbance is 0.62543.After rounding, it is determined that optimum extraction conditions is liquid ratio 90:1, extracts three times, and extraction time is 15min.(adopting response surface experiments to be only used to determine the extraction conditions of the best, to be not only see the reciprocal action between several factor).
Dry fermented product powder being added normal hexane, carries out ultrasonic assistant extraction, solid-liquid ratio is 1:90 (g:mL), extract 3 times, ultrasonic power is 100W, extract 15-45min, obtains normal hexane crude extract；
By the vacuum concentration at 40 DEG C of normal hexane crude extract, obtain concentrated solution.
3. the silica gel chromatography pre-separation to pigment
(1) thin layer chromatography determines separation condition
Thin-layer developing cylinder is prepared the developing solvent of n-hexane-ethyl acetate 2:1,4:1,6:1,8:1 respectively.Take n-hexane extract capillary tube point sample on lamellae, after the ethanol volatilization of pigment to be dissolved, lamellae is placed in expansion cylinder, when the liquid level of developing solvent will arrive chromatoplate top, chromatoplate is taken out, is placed in shady and cool ventilation place and makes ethanol volatilize, by natural light and ultraviolet analysis instrument for three purposed, separation case according to pigment, calculates the Rf value (Rf value) of corresponding pigment.Its result is as shown in table 3, from table 3 it is observed that when mobile phase ratio is n-hexane-ethyl acetate (2:1), its polarity is bigger than normal, and citraurin and flavochrome are almost eluted simultaneously, and hangover is serious；When mobile phase ratio is n-hexane-ethyl acetate (6:1), although citraurin and flavochrome can separate, but its development rate is too slow, and in post separates, development rate can expend more mobile phase slowly, owing to utilizing silica gel chromatography simply pigment to be carried out pre-separation, it is not necessary to separate accurately very much, therefore final selective flow is n-hexane-ethyl acetate (4:1) mutually.
(2) silica gel chromatography pre-separation
With normal hexane extraction 1g monascus mycopowder, then by normal hexane concentrate drying.Utilize the silica gel adsorption resin of 100 order fineness, monascus normal hexane crude extract is carried out pre-separation.Collect and detect the component of different time sections eluent, simultaneously using acetonitrile as blank.1, the eluent of corresponding 100-200mL, 200-350mL, the 350mL-500mL respectively of the eluent in 2, No. 3 brown bottles, its Liquid Detection result is as shown in Figure 1, Figure 2, Figure 3 and Figure 4.Fig. 1-Fig. 4 is respectively provided with red pigment, citraurin, flavochrome, the suitableeest testing conditions (detection wavelength respectively 540nm, 480nm, 390nm, reference wavelength is 350nm, 330nm, 270nm respectively) of three class pigments.Contrast by eluent and blank solution, it was demonstrated that the normal hexane crude extract of Monascus mycelium is after silica gel chromatographic column separates, and the yellow color component collected is mainly Monascin (Monascin) and ankaflavin (Ankaflavin).Yl moiety is concentrated and is spin-dried for, obtain yellow powdery solid.
Concentrated solution silica gel chromatographic column is carried out pre-separation, obtain yellow eluent, the post separation condition of described pre-separation is particularly as follows: the silica column of 100 order fineness is as fixing phase, and mobile phase is normal hexane and ethyl acetate, described normal hexane: the volume ratio of ethyl acetate is 4:1；
Yellow eluent vacuum at 40 DEG C is concentrated into Powdered, obtains pigment powder.
4. high speed adverse current chromatogram refining pigment
(1) flavochrome mensuration of apportionment ratio k value in normal hexane-methanol-water separation system
Selection system: normal hexane-methanol-water.
Configuration criteria: ensure that the final volume of phase up and down is close, because of than normal hexane, water and methanol polarity are close, thus keep normal hexane constancy of volume, adjust first alcohol and water proportioning, finally make the cumulative volume of first alcohol and water add and close with the volume of normal hexane.So can ensure that upper and lower phase volume is close in separation system process for preparation, thus avoiding the waste of organic solvent, simultaneously again can the polarity of across-the-board regulation separation system.
10mL centrifuge tube is proportionally prepared phase up and down, is sufficiently mixed, after being layered, take 4mL upper and lower phase respectively, be respectively charged in two other clean 10mL centrifuge tube.A small amount of pigment crude extract solid is added lower phase, ultrasonic Treatment, after being completely dissolved, takes 1mL, citraurin content yellow with HPLC detection, it is designated as C respectively11、C12.The remaining lower phase containing pigment adds the upper phase of 3L.Taking phase under 1mL after being sufficiently mixed again, detect with HPLC, content is designated as C21, C22, the two kinds of citraurins wherein studied and the spectrogram of two kinds of flavochrome are as shown in Figure 5.
After balancing each other up and down, pigment is at the content/pigment in upper phase of the partition coefficient=pigment of phase up and down content in lower phase.Namely pigment is at biphase middle apportionment ratio k=(C1n-C2n)/C1n× 100% (n=1 or n=2).
Measure according to the method described above four kinds of pigments difference up and down mutually in distribution ratio, result is as shown in table 4.When utilizing high speed adverse current chromatogram that mixture is easily separated, in theory, k value is at about 1 separating effect that can reach optimum.Four kinds of pigments k value under these two kinds of systems is as shown in table 5.With on 5mL mutually and after being dissolved under 5mL, being easily separated with HSCCC.
The different developing solvent expansion effect table to citraurin and flavochrome of table 3
4 four kinds of pigments of table are at the distribution ratio table of the upper and lower phase system of difference
Note: wherein "-" represents that corresponding going up in phase or lower phase can not detect because relative detector response is too low.
5 four kinds of pigments of table are at the distribution ratio table of the suitableeest upper and lower phase system
(2) separation of high speed adverse current chromatogram
1. the preparation of high speed adverse current chromatogram phase up and down
Three kinds of reagent of designated volume ratio are added in large beaker, seals with sealed membrane after being sufficiently stirred for Glass rod, stand so that it is layering.Pouring out phase after system balance, the part being layered mutually up and down utilizes separatory funnel will separate mutually up and down after standing.Choosing phase for fixing phase, lower phase is mobile phase, and ultrasonic degassed 30min, carefully moves, in order to avoid being mixed into bubble respectively.
2. the preparation of high speed adverse current chromatogram
Open computer, circulator bath system, adverse current chromatogram main frame and detector successively.Ensureing that power supply, holding wire consolidate, pipeline is closed.Design temperature is 25 DEG C, and detection wavelength is 405nm.
Open pump, pump into fixing phase with the speed of 10mL/min, when fixing from pipeline go out post mouth flow out after termination of pumping.Switching pump head, is inserted in mobile phase, regulates adverse current chromatogram engine speed, selects FWD pattern, and rotating speed is slowly adjusted to 900r/min, after engine speed is stable, pumps into lower phase with the speed of 2mL/min.
3. the separation of sample and collection
After HSCCC is ready, the sample pigment powder 5mL that will separate carries out with the mixed liquor of phase under 5mL dissolving mutually (select mutually each 5mL up and down so to consider: to be dissolved in lower phase time when sample is bad, general equivalent up and down mutually go dissolve, and sample size general when being HSCCC at ordinary times can be 10mL), ultrasonic assistant dissolves, to without obvious granule.If there being obvious granule, then by mixture with 0.45 μm of organic membrane filtration, load in clean 20mL syringe.
Mixed liquor in mouth and out mouth are inserted respectively sampling injector and empty syringe, termination of pumping.Six-way valve handle is quickly pushed load shelves.Promoting empty syringe, to sampling injector, liquid level rises, and does not have bubble, is drained by the gas of sample circle afterbody, pulls sky syringe, and the sample in sampling injector is sucked quantitative loop.
Six-way valve handle is allocated to inject gear, turn on pump, opens HSCCC computer software and automatic collector simultaneously, collect effluent.
As shown in Figure 6, appearance time is about 52-126min to high speed adverse current chromatogram separating resulting, and corresponding collecting pipe is 26-63 pipe.The collecting pipe that wherein 52-64min absworption peak is corresponding is 26-32 pipe, and the collecting pipe that 102-126min absworption peak is corresponding is 51-63 pipe.It will be appreciated from fig. 6 that the normal hexane pigment crude extract after HSCCC chromatographic isolation can be divided into two sections of flavochrome well.
Take No. 26 collecting pipes and No. 32 collecting pipes, No. 51 collecting pipes and No. 63 collecting pipes respectively, detect flavochrome therein with HPLC.Testing result is such as shown in Fig. 7, Fig. 8, Fig. 9 and Figure 10.
By Fig. 7 to Figure 10 it can be seen that under three class pigment testing conditions red in monascus, orange, yellow, all only have single absworption peak, after deduction reference signal, peak area ratio is all higher than 99%, it is possible to illustrate, pigment now is single flavochrome.
5. dry: by Monascin and ankaflavin eluent, vacuum at 40 DEG C is concentrated into Powdered, obtains high-purity Monascin and ankaflavin.
The related test results of the present invention:
Being contrasted by the liquid chromatogram of Monascin (Monascin) pigment after obtaining by liquid fermentation monascus and be purified with commercially available Monascin (Monascin) standard substance, result is as shown in figure 11.Standard substance appearance time is 9.747min, and the appearance time of purification product is 9.796min, contrasts both spectrograms, it is possible to illustrate that this material is Monascin (Monascin)., standard curve obtaining in 0.23g pigment normal hexane crude extract, the quality of purification Monascin (Monascin) is 1.88mg meanwhile.
Owing to not having the standard substance of ankaflavin (Ankaflavin), therefore adopting the means of LC-MS to carry out the ankaflavin (Ankaflavin) of purification Identification, Figure 12 is the mass spectrum of purification ankaflavin (Ankaflavin).
Shown by Figure 12 positive ion mode mass spectrum, [M+H]+Molecular weight (m/z) is the quasi-molecular ion peak of 387.42, and negative ion mode mass spectrum shows [M-H]-, molecular weight (m/z) is the quasi-molecular ion peak of 385.31, it is determined that this component molecular amount is 386D, meanwhile, in conjunction with spectrogram, it is determined that this material is the ankaflavin (Ankaflavin) in monascorubin.
With traditional vacuum concentrating instrument, gained ankaflavin (Ankaflavin) is concentrated into liquid volatilize, weighs centrifuge tube and ankaflavin (Ankaflavin) gross weight m with ten thousand/balance1, divide 4 times with 10mL hplc grade methanol and pigment cleaned, weigh the quality m of dry centrifuge tube2, with the quality m before washing1Deduct the quality m after washing2, it is the quality of ankaflavin (Ankaflavin).Finally from 0.23g flavochrome normal hexane crude extract, prepare ankaflavin (Ankaflavin) 4.8mg.
Leading reference
[1] Cui Li, Hu Xiaodan, Zhang Dequan .HPLC method measures the Monascin in monascorubin and Monascus anka flavin [J] simultaneously. Food Science, and 2009,30 (08): 163-166.
[2] Zheng Yunquan, Li Yongning, Wang Awan, etc. high-speed countercurrent chromatography separation purification monascus pigment component [J]. Food Science, 2010,31 (20): 192-195.
Claims (9)
1. the method being produced high-purity Monascin and ankaflavin by monascus liquid fermentation method, it is characterised in that: step is as follows:
(1) seed culture: be inoculated into by monascus sp bacteria strain in the seed culture medium after sterilizing, cultivates 1-4d in 20-35 DEG C of shaking table, obtains seed liquor；
Fermentation: by step (1) in cultured seed liquor be inoculated in fermentation medium with the ratio of 3%--8% (v/v), 30 DEG C of shake-flask culture 6d, obtain fermentation liquid；
(3) being centrifuged: be centrifuged by fermentation liquid, centrifugal condition is 3500r/min, centrifugal 20min, obtains fermented product precipitation；
(4) dry: dry at fermented product is deposited in 40-60 DEG C to constant weight, obtain the fermented product dried；
(5) pulverize: dry fermented product mortar is ground, cross 100 mesh sieves, obtain the fermented product powder dried；
(6) ultrasonic assistant extracts: dry fermented product powder being added normal hexane, carries out ultrasonic assistant extraction, solid-liquid ratio is 1:20-120g:mL, extracts 1-4 time, and ultrasonic power is 50-100W, extracts 15-45min, obtains normal hexane crude extract；
(7) crude extract concentration: by the vacuum concentration at 40 DEG C of normal hexane crude extract, obtain concentrated solution；
(8) pre-separation: concentrated solution silica gel chromatographic column is carried out pre-separation, obtain yellow eluent, the post separation condition of described pre-separation is particularly as follows: the silica column of 100 order fineness is as fixing phase, and mobile phase is normal hexane and ethyl acetate, described normal hexane: the volume ratio of ethyl acetate is 2-8:1；
(9) concentration: yellow eluent vacuum at 40 DEG C is concentrated into Powdered, obtains pigment powder；
(10) refine: preparation high speed adverse current chromatogram phase up and down, stratification, described up and down by normal hexane: methanol: the volume ratio 10:2.5-9:7.5-1 of water is formulated, lower phased soln by the upper phase of pigment powder 5mL and 5mL, with high speed adverse current chromatogram, Monascin and ankaflavin are refined, detection wavelength is 405nm, collects 0-200min eluent, obtains Monascin and ankaflavin；
(11) dry: by Monascin and ankaflavin eluent, vacuum at 40 DEG C is concentrated into Powdered, obtains high-purity Monascin and ankaflavin.
2. the method being produced high-purity Monascin and ankaflavin by monascus liquid fermentation method according to claim 1, it is characterised in that: described step (1) middle seed culture medium is: glucose 6g, peptone 2g, NaNO31g, MgSO4·7H2O0.5g, KH2PO41g, tap water 100mL.
3. the method being produced high-purity Monascin and ankaflavin by monascus liquid fermentation method according to claim 1, it is characterised in that: described step (2) middle fermentation medium is: rice meal 5g, NaNO30.3g, MgSO4·7H2O0.1g, KH2PO40.15g, tap water 100mL.
4. the method being produced high-purity Monascin and ankaflavin by monascus liquid fermentation method according to claim 1, it is characterised in that: described step (6) in the optimum extraction condition that extracts of ultrasonic assistant be: ultrasonic assistant extract solid-liquid ratio be 1:90 (g:mL), extract 3 times, ultrasonic power is 100W, extract 15-45min.
5. the method being produced high-purity Monascin and ankaflavin by monascus liquid fermentation method according to claim 4, it is characterised in that: the defining method of the optimum extraction condition that described ultrasonic assistant extracts is as follows:
Using the absorbance under 405nm wavelength as evaluation index, test, by single factor experiment and response surface, the optimum condition obtaining normal hexane ultrasound assisted extraction flavochrome；
(1) experiment of single factor
1. the extraction time impact on normal hexane extraction flavochrome
Accurately weigh 0.050g Monas cuspurpureus Went mycelia powder, put in 10mL centrifuge tube, add 5mL normal hexane；15min, 30min, 45min, 60min is extracted respectively at 100W ultrasonic assistant when；3500r/min, centrifugal 20min；Take supernatant, be settled to 5mL, after diluting 30 times, measure the absorbance under 405nm respectively, repeat 3 times, seek its meansigma methods；
2. the extraction time impact on normal hexane extraction flavochrome
Accurately weigh 0.050g Monas cuspurpureus Went mycelia powder, be respectively put in 4 10mL centrifuge tubes, each addition 5mL normal hexane；Extracting 30min, 3500r/min when 100W ultrasonic assistant, centrifugal 20min takes clear liquid, as once extracting；Respectively the Monas cuspurpureus Went mycelia powder in above-mentioned 4 centrifuge tubes is extracted 1 time, 2 times, 3 times and 4 times, merge the extracting solution of corresponding test tube, dilute suitable multiple, make absorption values between 0.2～0.8, measure the absorbance of now 405nm；Repeat 3 times, seek its meansigma methods；
3. the liquid ratio impact on normal hexane extraction flavochrome
Accurately weigh 0.050g Monas cuspurpureus Went mycelia powder, be respectively put in 6 10mL centrifuge tubes, be separately added into the normal hexane of 20,40,60,80,100,120 times of volumes；30min is extracted in 100W ultrasonic assistant when；Hereafter the centrifugal 20min of 3500r/min；Taking supernatant, be settled to 10mL, dilute 30 times, measure the absorbance under 405nm wavelength respectively, each time conditions repeats 3 times；
4. the power impact on normal hexane extraction flavochrome is extracted
Accurately weigh 0.050g Monas cuspurpureus Went mycelia powder, be respectively put in 3 10mL centrifuge tubes, each addition 5mL normal hexane；Respectively at 90W, 70W, 50W ultrasonic assistant when, 30min is extracted after covering centrifuge tube lid；3500r/min, centrifugal 20min；Take supernatant, be settled to 5mL, after diluting 30 times, measure the absorbance under 405nm respectively, repeat 3 times；
(2) Box-Behnken experiment
Box-Benhnken design method can design by experiment, finally gives regression equation and is analyzed to seek the combination of optimum, thus solving the statistical method of Multivariable；With extraction time, solid-liquid ratio and three factors of extraction time for independent variable, DesignExpert8.0.6 is utilized to design Three factors-levels response surface experiments, being obtained by normal hexane is that extractant extracts the optimal conditions of flavochrome in Monascus anka Nakazawa et sato filament, namely ultrasonic assistant extract solid-liquid ratio be 1:90 (g:mL), extract 3 times, ultrasonic power is 100W, extract 15-45min.
6. the method being produced high-purity Monascin and ankaflavin by monascus liquid fermentation method according to claim 1, it is characterized in that: described step (8) in pre-separation post separate optimum condition be: the silica column of 100 order fineness is as fixing phase, mobile phase is normal hexane and ethyl acetate, described normal hexane: the volume ratio of ethyl acetate is 4:1.
7. the method being produced high-purity Monascin and ankaflavin by monascus liquid fermentation method according to claim 6, it is characterised in that: the defining method of the optimum condition that the post of described pre-separation separates is as follows:
(1) in thin-layer developing cylinder, prepare the developing solvent of n-hexane-ethyl acetate 2:1,4:1,6:1,8:1 respectively；Take n-hexane extract capillary tube point sample on lamellae, after the ethanol volatilization of pigment to be dissolved, lamellae is placed in expansion cylinder, when the liquid level of developing solvent will arrive chromatoplate top, chromatoplate is taken out, is placed in shady and cool ventilation place and makes ethanol volatilize, by natural light and ultraviolet analysis instrument for three purposed, separation case according to pigment, calculates the Rf value of corresponding pigment；When mobile phase ratio is n-hexane-ethyl acetate 2:1, its polarity is bigger than normal, and citraurin and flavochrome are almost eluted simultaneously, and hangover is serious；When mobile phase ratio is n-hexane-ethyl acetate 6:1, although citraurin and flavochrome can separate, but its development rate is too slow, and in post separates, development rate can expend more mobile phase slowly, owing to utilizing silica gel chromatography simply pigment to be carried out pre-separation, it is not necessary to separate accurately very much, therefore final selective flow is n-hexane-ethyl acetate 4:1 mutually；
(2) silica gel chromatography pre-separation
With normal hexane extraction 1g monascus mycopowder, then by normal hexane concentrate drying；Utilize the silica gel adsorption resin of 100 order fineness, monascus normal hexane crude extract is carried out pre-separation；Collect and detect the component of different time sections eluent, simultaneously using acetonitrile as blank；1, the corresponding 100-200mL respectively of the eluent in 2, No. 3 brown bottles, 200-350mL, the eluent of 350mL-500mL, the suitableeest testing conditions of red pigment, citraurin, flavochrome three class pigment it is respectively provided with during its Liquid Detection, detection wavelength respectively 540nm, 480nm, 390nm, reference wavelength is 350nm, 330nm, 270nm respectively；Contrast by eluent and blank solution, it was demonstrated that the normal hexane crude extract of Monascus mycelium is after the silica gel chromatographic column of 100 order fineness separates, and the yellow color component collected is mainly Monascin and ankaflavin.
8. the method being produced high-purity Monascin and ankaflavin by monascus liquid fermentation method according to claim 1, it is characterised in that: described step (10) in up and down by normal hexane: methanol: the volume ratio 10:7:3 of water is formulated.
9. the method being produced high-purity Monascin and ankaflavin by monascus liquid fermentation method according to claim 1, it is characterised in that: described step specifically comprising the following steps that (10)
(1) flavochrome mensuration of apportionment ratio k value in normal hexane-methanol-water separation system
Selection system: normal hexane-methanol-water；
Configuration criteria: ensure that the final volume of phase up and down is close, because of than normal hexane, water and methanol polarity are close, therefore keep normal hexane constancy of volume, adjust the proportioning of first alcohol and water, finally make the cumulative volume of first alcohol and water add and close with the volume of normal hexane, so can ensure that upper and lower phase volume is close in separation system process for preparation, thus avoiding the waste of organic solvent, simultaneously again can the polarity of across-the-board regulation separation system；
10mL centrifuge tube is proportionally prepared phase up and down, is sufficiently mixed, after being layered, take 4mL upper and lower phase respectively, be respectively charged in two other clean 10mL centrifuge tube；A small amount of pigment crude extract solid is added lower phase, ultrasonic Treatment, after being completely dissolved, takes 1mL, citraurin content yellow with HPLC detection, it is designated as C respectively11、C12；The remaining lower phase containing pigment adds the upper phase of 3L；Taking phase under 1mL after being sufficiently mixed again, detect with HPLC, content is designated as C21, C22；
After balancing each other up and down, pigment is at the content/pigment in upper phase of the partition coefficient=pigment of phase up and down content in lower phase；Namely pigment is at biphase middle apportionment ratio k=(C1n-C2n)/C1n× 100% (n=1 or n=2)；
Measure according to the method described above Monascin, rubropunctatin, ankaflavin and monascorubin difference up and down mutually in distribution ratio, when utilizing high speed adverse current chromatogram that mixture is easily separated, in theory, k value can reach the separating effect of optimum when 0.95～1.5, obtains up and down by normal hexane: methanol: the volume ratio 10:7:3 of water is formulated for optimum；
With on 5mL mutually and after being dissolved under 5mL, being easily separated with HSCCC；
(2) the separation of high speed adverse current chromatogram
1. the preparation of high speed adverse current chromatogram phase up and down
Three kinds of reagent of designated volume ratio are added in large beaker, seals with sealed membrane after being sufficiently stirred for Glass rod, stand so that it is layering；Pouring out phase after system balance, the part being layered mutually up and down utilizes separatory funnel will separate mutually up and down after standing；Choosing phase for fixing phase, lower phase is mobile phase, and ultrasonic degassed 30min, carefully moves, in order to avoid being mixed into bubble respectively；
2. the preparation of high speed adverse current chromatogram
Open computer, circulator bath system, adverse current chromatogram main frame and detector successively；Ensureing that power supply, holding wire consolidate, pipeline is closed；Design temperature is 25 DEG C, and detection wavelength is 405nm；
Open pump, pump into fixing phase with the speed of 10mL/min, when fixing from pipeline go out post mouth flow out after termination of pumping；Switching pump head, is inserted in mobile phase, regulates adverse current chromatogram engine speed, selects FWD pattern, and rotating speed is slowly adjusted to 900r/min, after engine speed is stable, pumps into lower phase with the speed of 2mL/min；
3. the separation of sample and collection
After HSCCC is ready, the sample pigment powder 5mL that will separate dissolving with the mixed liquor of phase under 5mL mutually, ultrasonic assistant dissolves, to without obvious granule；If there being obvious granule, then by mixture with 0.45 μm of organic membrane filtration, load in clean 20mL syringe；
Mixed liquor in mouth and out mouth are inserted respectively sampling injector and empty syringe, termination of pumping；Six-way valve handle is quickly pushed load shelves；Promoting empty syringe, to sampling injector, liquid level rises, and does not have bubble, is drained by the gas of sample circle afterbody, pulls sky syringe, and the sample in sampling injector is sucked quantitative loop；
Six-way valve handle being allocated to inject gear, turn on pump, opens HSCCC computer software and automatic collector simultaneously, detection wavelength is 405nm, collects 0-200min eluent, obtains Monascin and ankaflavin.
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|---|---|---|---|---|
| CN106590020A (en) * | 2016-11-02 | 2017-04-26 | 华南理工大学 | Method of separating water soluble monascus pigment by the use of macroreticular resin and application thereof |
| CN110592158A (en) * | 2019-09-19 | 2019-12-20 | 长江大学 | Liquid fermentation method for improving purity of monascus yellow pigment Monascin and Ankaflavin |
| CN111171597A (en) * | 2020-01-15 | 2020-05-19 | 安徽农业大学 | Method for separating and purifying rubropunctatin in red yeast glutinous rice |
| CN111304093A (en) * | 2019-12-23 | 2020-06-19 | 天津科技大学 | Monascus for preparing monascin C and method for preparing monascin C by using monascin C |
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| CN111304093A (en) * | 2019-12-23 | 2020-06-19 | 天津科技大学 | Monascus for preparing monascin C and method for preparing monascin C by using monascin C |
| CN111304093B (en) * | 2019-12-23 | 2021-12-28 | 天津科技大学 | Monascus for preparing monascin C and method for preparing monascin C by using monascin C |
| CN111171597A (en) * | 2020-01-15 | 2020-05-19 | 安徽农业大学 | Method for separating and purifying rubropunctatin in red yeast glutinous rice |
| CN113201230A (en) * | 2020-12-23 | 2021-08-03 | 天津科技大学 | Monascus pigment product and preparation method thereof |
| CN113201230B (en) * | 2020-12-23 | 2023-02-10 | 天津科技大学 | Monascus pigment product and preparation method thereof |
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