CN106267881A - Pure material extracting process - Google Patents
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Abstract
The open a kind of pure material extracting process of the present invention.Replacing the usual organic extract liquid of existing pure material extracting process and col-umn chromatography, the method for the present invention simply uses drinks solution and extracts the precipitate containing Monascin and ankaflavin in a red koji fermentation product；Additionally, the precipitate of the pure material obtained via the method for the present invention, its need not move through concentrating under reduced pressure step the most naturally contain recovery of extraction be up to 97.35% Monascin and recovery of extraction be up to 95.16% ankaflavin.Therefore, it can to confirm the pure material extracting process of the present invention possessed that rate of extraction is fast, high efficiency and reduce advantages such as using organic solvent.
Description
Technical field
The present invention relates to the extraction of material and the technical field of purification, the purification extracting process of the secondary metabolic product of a kind of red koji fermentation product.
Background technology
NTU 568 Monascus Strains (Monascus mutant, Monascus purpureus NTU 568), by the microorganism of TaiWan, China university and excellent native country, the Taiwan Monascus Strains that biochemical institute Pan Zi penetrating judgment is awarded and R&D team is researched and developed.This NTU 568 Monascus anka Nakazawa et sato has that quickly growth, Starch Hydrolysis ability be strong and secondary metabolism (metabolites production) feature that ability is strong.Additionally, the optimal medium of NTU 568 Monascus anka Nakazawa et sato must containing the rice flour (rice powder) of 2%, and its optimum culturing temperature be 30 DEG C, optimal incubation time be 48 hours, optimal pressure of cultivating be 1 atmospheric pressure.
In order to confirm the survival ability (viability) of this NTU 568 Monascus anka Nakazawa et sato, can by NTU 568 Monascus anka Nakazawa et sato from inclined tube (slant tube) transfer be moved from a potato dextrose agar (potato dextrose agar, PDA) on carry out the cultivation of 15 days；Then, the mycelium of 3 pieces of 1cm3 is dug on PDA, and mycelium is inserted on the culture fluid containing 2% rice flour (rice powder), after 48 hours, find that culture fluid presents redness, which show NTU 568 Monascus anka Nakazawa et sato and there is suitable survival ability.Further it is necessary to supplementary notes, the storing mode of NTU 568 Monascus anka Nakazawa et sato is to deposit in the environment of 4 DEG C in the inclined tube with PDA culture medium, and must cultivate (sub-cultured) once every 3 months times.
Health food is flourish in recent years, and the functional fermented product Monas cuspurpureus Went with many effects gradually comes into one's own.In Asia, Monascus anka Nakazawa et sato (Monascus species) is in diet, application pharmaceutically, the history of existing thousand.Wherein, the secondary metabolism product that Monascus anka Nakazawa et sato is important comprises following four kinds:
(1) pigment group, including: red pigments (rubropunctamine, monascorubramine), xanthein (ankaflavin, monascin) and orange pigment (rubropunctanin, monascorubrin)；
(2) cholesterol reducing material: such as monacolin K；
(3) material for lowering blood pressure: such as gaba (γ-aminobutyric acid, GABA)；
(4) antioxidant, including: dimerumic acid and 3-hydoxy-4-methoxy-benzoic acid.
In early days, in order to confirm the chemical constitution of Monascin (Monascin) and ankaflavin (Ankaflavin), research worker utilizes thin layer chromatography separation method (Thin layer chromatography) to isolate Monascin and ankaflavin in Monascus anka Nakazawa et sato filament.Wherein, thin layer chromatography separation method in early days comprises the following steps:
Step (S01a): collect mycelium and clean with water, and dried by its grinds at room temperature；
Step (S02a): with the mycelium of n-hexane extraction powdery, it is thus achieved that extract；
Step (S03a): concentrated extract, and at 0 DEG C, deposit 2 days；
Step (S04a): extracted by filtration liquid is to obtain a filtrate；
Step (S05a): get normal hexane and cellulose chromatography tubing string ready, and then through chromatography (chromatography), this filtrate is purified；
Step (S06a): get the silica gel thin layer chromatography board (TLC of 20 centimeter square ready, Thin Layer Chromatography) and developping solution (containing the benzene of 25% ether), and then separate the Monascin among the yellow layering that abovementioned steps (S05a) is obtained and ankaflavin, and scrape ankaflavin layering；
Step (S07a): extract ankaflavin layering with dichloromethane and ethyl acetate；
Step (S08a): crystallized with ethanol after being dried the extract of abovementioned steps (S07a), and then obtain a yellow prism (yellow prism)；Wherein, containing the ankaflavin of 74 milligrams among each gram of yellow prism.
It is well known that thin layer chromatography separation method (Thin layer chromatography) is mainly used in the isolated and purified of trace pure material, and the isolated and purified of a large amount of pure material cannot be applied to.As a example by thin layer chromatography separation method, it is necessary to use the ankaflavin that the silica gel thin layer chromatography board of 20 to 100 can isolate 1 gram among yellow layering simultaneously.
Because traditional thin layer chromatography separation method cannot be applied to the isolated and purified of a large amount of pure material, a large amount of extracting process of a kind of pure material are suggested among document one.In this, document one refers to Toshihiro et.al, " Azaphilones; Furanoisophthalides; and Amino Acids from the Extracts of Monascus pilosus-Fermented Rice (Red-Mold Rice) and Their Chemopreventive Effects ", J.Agric.Food Chem., 2005, Vol.53, pp.562-565.Document one describes monascus yellow pigment: Monascin (Monascin) and the purification extracting process of ankaflavin (Ankaflavin), comprises the following steps:
Step (S01b): extract the Monas cuspurpureus Went 30 minutes of 1.5 kilograms with the ethanol of 12 liters (70%)；
Step (S02b): filter the product of abovementioned steps (S01b), and with ethanol (70%) the cleaned screening of 3 liters；
Step (S03b): at 50 DEG C, the filtrate of abovementioned steps (S02b) and cleanout fluid are carried out (vacuum) concentrating under reduced pressure；
Step (S04b): the ethanol in the product of abovementioned steps (S03b) is removed, and then obtain the first thick extract of 152.9 grams；
Step (S05b): be placed in 1 liters of water by this thick extract, then the ethyl acetate with 0.5 liter carries out the liquid-liquid extraction of five times；
Step (S06b): at 50 DEG C, the acetic acid ethyl acetate extract of abovementioned steps (S05b) gained is carried out (vacuum) concentrating under reduced pressure；
Step (S07b): the ethyl acetate in the product of abovementioned steps (S06b) is removed, and then obtain the second thick extract of 26.1 grams；
Step (S08b): get silica gel column chromatography tubing string ready and purge with solvent, and then through chromatography (chromatography), the second thick extract of 11.9 grams is purified；Wherein, the described solvent that purges with is 11 kinds, it is respectively n-hexane/ethyl acetate=1/0, n-hexane/ethyl acetate=9:1, n-hexane/ethyl acetate=4:1, n-hexane/ethyl acetate=1:1, n-hexane/ethyl acetate=3:7, n-hexane/ethyl acetate=1:9, n-hexane/ethyl acetate=0/1, ethyl acetate/methanol=9:1, ethyl acetate/methanol=1:1, ethyl acetate/methanol=0/1 (v/v, volume ratio)；
Step (S09b): take out the C layering of 250 Bos gram among 11 layerings (A～K) that abovementioned steps (S08b) is obtained；
Step (S10b): get ready and purge with liquid, and then the C layering of 250 Bos gram is purified with the flow velocity that purges with of 2.5mL/min through high performance liquid chromatography (HPLC) (high performance liquid chromatography, HPLC)；So, collection 27min purges with liquid and obtains Monascin 14 Bo gram, and collection 40min purges with liquid and obtains ankaflavin 4 Bo gram；Wherein, purging with liquid described in is methanol/water/acetic acid=75:25:3.
The engineering staff being familiar with pure material abstraction technique can be found by Practical Operation, the purification extracting process of document one cannot be applied to extract substantial amounts of monascus yellow pigment in the Monas cuspurpureus Went (Red Mold Rice) that ferments, reason is as follows: owing to the finally obtained monascus yellow pigment of purification extracting process of document one is 18 Bos gram (14 Bo gram monascin and 4 Bo gram ankaflavin), and people can deduce that the C of 250 Bos gram only contains only the flavochrome of 7.2% (18/250) in being layered still；That is, the technology that the step (S01b) of the purification extracting process of document one～step (S09b) are used, it should be not best suited for the technology of separating yellow layering.
Because the purification extracting process of document one cannot extract monascin and nkaflavin expeditiously among fermentation Monas cuspurpureus Went, a kind of high performance liquid chromatography (HPLC) (Liquid Chromatograph) is suggested among document two.In this, document two refers to Chinese Patent No. CN101255168B.High performance liquid chromatography (HPLC) described in document two, comprises the following steps:
Step (S01c): extract the Monas cuspurpureus Went powder of 10 grams using normal hexane as extracting solution, until extracting liquid colourless；
Step (S02c): collect extracting solution and filter, obtaining a filtrate；
Step (S03c): this filtrate is carried out (vacuum) concentrating under reduced pressure, obtains a crude extract；
Step (S04c): be dissolved among methanol by this crude extract, obtains the methanol solution of 50mL；
Step (S05c): filter the methanol solution of abovementioned steps (S04c) gained, obtain a sample solution；
Step (S06c): get ready and purge with liquid, and then by high performance liquid chromatography (HPLC) (high performance liquid chromatography, HPLC) with the flow velocity that purges with of 5mL/min n, this sample solution is purified, and collection purges with liquid；Wherein, purging with liquid described in is methanol/water=8:2.
Although document two points out that its high performance liquid chromatography (HPLC) can prepare Monascin sterling (Monascin) and the purity 99.234% Monascus anka flavine pure product (Akaflavin) of purity 96.761%；But, the engineering staff being familiar with pure material abstraction technique can be found by Practical Operation, and the high performance liquid chromatography (HPLC) of document two still demonstrates following shortcoming:
The sample solution of 1 milliliter within 80 minutes, could be separated owing to each HPLC must last, therefore the sample solution of 50 milliliters must carry out the HPLC (lasting 4000 minutes altogether) of 50 times can complete；Further, obtain the Monascin of 30 milligrams owing to the Monas cuspurpureus Went powder of 10 grams are only capable of after described high-speed countercurrent chromatography technique, therefore can deduce that the HPLC of each 80 minutes is merely able to obtain the Monascin of 0.6 milligram.
Because the purification extracting process of document two cannot extract Monascin and ankaflavin rapidly in fermentation Monas cuspurpureus Went, a kind of high-speed countercurrent chromatography method (High Speed Counter Current Chromatograph, HSCCC) is suggested among document three.In this, document three refers to Chinese Patent No. CN101864191 B.High-speed countercurrent chromatography method described in document three, comprises the following steps:
Step (S01d): under the water-bath of 60 DEG C, uses methanol solution extraction powdery Monas cuspurpureus Went；Wherein, powdery Monas cuspurpureus Went is 1:15 with the solid-to-liquid ratio of methanol solution；
Step (S02d): the product vacuum that abovementioned steps (S01d) is obtained is concentrated into the 1/3 of original volume, and then acquisition one slightly extracts thing；
Step (S03d): that gets 800mg ready slightly extracts thing, then with the fixing phased soln of 50mL；
Step (S04d): get high-speed countercurrent chromatography system ready and purge with solvent (that is, flowing phase), and then the product obtained abovementioned steps (S03d) by high-speed countercurrent chromatography (HSCCC) is purified；Wherein, solvent petrol ether/ethyl acetate/ethanol/water=2.5:7.5:5:5 (v:v:v:v) is purged with described in；
Step (S05d): be concentrated in vacuo to dry by the separation liquid collected by this high-speed countercurrent chromatography system under 60 DEG C, then collects xanthein purified components, and then dissolves this xanthein purified components with 85% acetone soln；
Step (S06d): under 60 DEG C, the product vacuum of abovementioned steps (S07d) is concentrated into supersaturation, then slow cooling is to 15 DEG C, then obtains Monascin and ankaflavin crystal.
But, the engineering staff being familiar with pure material abstraction technique can be found by Practical Operation, and the HSCCC of document three still demonstrates following shortcoming:
(1) HSCCC need to use multi-solvents single treatment 800mg Monas cuspurpureus Went extract simultaneously；Wherein, 800mg Monas cuspurpureus Went extract is obtained by extraction among the Monas cuspurpureus Went of about 12 grams.
(2) in addition, although the capacity of the HSCCC equipment of preparative is 200mL (=50mLx4) up to 4800mL and each amount processing sample solution at present, uses the HSCCC equipment of preparative that the Monas cuspurpureus Went powder of 48 grams (=12 grams of x4) is carried out extraction and be still required for lasting 133 minutes.More additionally it has to be considered that the price of the HSCCC equipment of preparative is much more expensive, its price about New Taiwan Dollar 14,000,000 yuan.
Therefore, because the extracting process of existing pure material, liquid chromatography and high-speed countercurrent chromatography method all manifest many defects in practice application, continual exploitation one novelty pure material extracting process, is suitable to extract at least one secondary metabolic product among red koji fermentation product.
Summary of the invention
The main object of the present invention, is to provide a kind of pure material extracting process so that it is possess the advantage that rate of extraction is fast and reduces use organic solvent when extraction Monascin and ankaflavin.
In order to reach above-mentioned purpose, the present invention proposes a kind of pure material extracting process, is suitable to extract at least one secondary metabolic product in red koji fermentation product, and the method comprises the following steps:
(1) get a red koji fermentation product of a specified weight ready, and at a temperature of one first, this red koji fermentation product is extracted with an extracting solution of one first volume, last one first extraction time；
(2) collect one first extract of abovementioned steps (1) and filter, and then obtaining one first extracted by filtration liquid and this red koji fermentation product through single extraction；
(3) this red koji fermentation product through single extraction is extracted again at a temperature of first in this with this extracting solution of one second volume, last one second extraction time；
(4) collect one second extract of abovementioned steps (3), and filter, and then obtain one second extracted by filtration liquid；And
(5) collect this first extracted by filtration liquid and this second extracted by filtration liquid, and then be processed to become a pure material.
In above-mentioned pure material extracting process, it is preferred that this step (5) includes following thin portion step:
(51) mix this first extracted by filtration liquid with this second extracted by filtration liquid to obtain a hybrid extraction liquid, and this hybrid extraction liquid is concentrated into thick；
(52) solvent of a third volume is added in this hybrid extraction liquid, to obtain a sample solution；
(53) under the water-bath of one second temperature, stir this sample solution, last one first mixing time；
(54) it is subsequently added into the water of a fourth volume in this sample solution, is stirred for this sample solution and lasts one second mixing time；
(55) with rotating speed 2000rpm/min, this sample solution of abovementioned steps (54) gained is centrifuged step, lasts one first centrifugation time；And
(56) complete centrifugation step, then collect a precipitate of this pure material.
In above-mentioned pure material extracting process, it is preferred that described red koji fermentation product can be lower any one: Monas cuspurpureus Went or red yeast yam.
In above-mentioned pure material extracting process, it is preferred that this specified weight of red koji fermentation product is at least 60 grams, and described red koji fermentation product can be lower any one: Monas cuspurpureus Went or red yeast yam.
In above-mentioned pure material extracting process, it is preferred that the ethanol that this extracting solution is concentration 95%.
In above-mentioned pure material extracting process, it is preferred that this first volume and this second volume are between 250 milliliters to 320 milliliters, and this fourth volume is 8～12 milliliters.
In above-mentioned pure material extracting process, it is preferred that this first temperature is between 50 DEG C to 65 DEG C, and this second temperature is between 55 DEG C to 75 DEG C.
In above-mentioned pure material extracting process, it is preferred that this first extraction time is 1～3 hour, and this second extraction time is 1～3 hour.
In above-mentioned pure material extracting process, it is preferred that the ethanol that this solvent is concentration 95%, and this third volume of this solvent is 8～12 milliliters.
In above-mentioned pure material extracting process, it is preferred that this first mixing time is 3～7 minutes, and this second mixing time is 3～7 minutes.
In above-mentioned pure material extracting process, it is preferred that this first centrifugation time is 8～12 minutes.
In above-mentioned pure material extracting process, it is preferred that this precipitate of each gram contains the Monascin (Monascin) of at least 22.5 milligrams and the ankaflavin (Ankaflavin) of at least 16.83 milligrams.
In above-mentioned pure material extracting process, it is preferred that the percentage by weight that this precipitate accounts for this sample solution is at least 25%.
In above-mentioned pure material extracting process, it is preferred that the recovery of extraction of described Monascin (Monascin) is at least 97.35%, and the recovery of extraction of described ankaflavin (Ankaflavin) is at least 95.16%.
Further, for above-mentioned purpose, the present invention also proposes another kind of pure material extracting process, is suitable to extract at least one secondary metabolic product in red koji fermentation product, comprises the following steps:
(1 ') gets a red koji fermentation product of a specified weight ready, and extracts this red koji fermentation product at a temperature of one first with an extracting solution of one first volume, lasts one first extraction time；
(2 ') collect one first extract of abovementioned steps (1 '), and filter, and then obtain one first extracted by filtration liquid and this red koji fermentation product through single extraction；
This red koji fermentation product through single extraction is again extracted at a temperature of first in this by (3 ') with this extracting solution of one second volume, lasts one second extraction time；
(4 ') collect one second extract of abovementioned steps (3 '), and filter, and then obtain one second extracted by filtration liquid；And
(5 ') collect this first extracted by filtration liquid and this second extracted by filtration liquid, and then are processed to become a pure material.
In above-mentioned pure material extracting process, it is preferred that this step (5 ') includes following thin portion step:
(51 ') mix this first extracted by filtration liquid and this second extracted by filtration liquid, obtain a hybrid extraction liquid；
This hybrid extraction liquid is concentrated into a third volume by (52 '), is then incorporated in by the water of a fourth volume in this hybrid extraction liquid, to obtain one first sample solution；
(53 ') are centrifuged step with rotating speed 2000rpm/min to this first sample solution of abovementioned steps (52 ') gained, last one first centrifugation time；
After (54 ') complete centrifugation step, then collect one first precipitate；
This first precipitate is dissolved in a solvent of one the 5th volume by (55 '), to obtain one second sample solution；
(56 ') stir this second sample solution under the water-bath of one second temperature, last one first mixing time；
The water that one hexasomic is long-pending is then added in this second sample solution by (57 '), is stirred for this second sample solution and lasts one second mixing time；
(58 ') are centrifuged step with rotating speed 2000rpm/min to this second sample solution of abovementioned steps (57 ') gained, last one second centrifugation time
After (59 ') complete centrifugation step, then collect one second precipitate, for this pure material.
In another embodiment of above-mentioned pure material extracting process, it is preferred that this specified weight of described red koji fermentation product is at least 20 kilograms, and described red koji fermentation product can be lower any one: Monas cuspurpureus Went or red yeast yam.
In another embodiment of above-mentioned pure material extracting process, it is preferred that the ethanol that this extracting solution is concentration 95%, and this first volume is at least 60 liters with this second volume.
In another embodiment of above-mentioned pure material extracting process, it is preferred that this first temperature is between 50 DEG C to 65 DEG C, and this this second temperature is between 55 DEG C to 75 DEG C.
In another embodiment of above-mentioned pure material extracting process, it is preferred that this first extraction time is 1～3 hour, and this second extraction time is 1～3 hour.
In another embodiment of above-mentioned pure material extracting process, it is preferred that this third volume is at least 4 liters, this fourth volume is at least 4 liters, and the 5th volume is 1～2 liter, and this hexasomic to amass be 1.5～2.5 liters.
In another embodiment of above-mentioned pure material extracting process, it is preferred that this first centrifugation time is 8～12 minutes, and this second centrifugation time is 8～12 minutes.
In another embodiment of above-mentioned pure material extracting process, it is preferred that the ethanol that this solvent is concentration 95%.
In another embodiment of above-mentioned pure material extracting process, it is preferred that this first mixing time is 10～20 minutes, and this second mixing time is 3～7 minutes.
In another embodiment of above-mentioned pure material extracting process, it is preferred that this second precipitate of each gram contains the Monascin (Monascin) of at least 22.5 milligrams and the ankaflavin (Ankaflavin) of at least 16.83 milligrams.
In another embodiment of above-mentioned pure material extracting process, it is preferred that the percentage by weight that this second precipitate accounts for this second sample solution is at least 25%.
In another embodiment of above-mentioned pure material extracting process, it is preferred that the recovery of extraction of described Monascin (Monascin) is at least 98%, and the recovery of extraction of described ankaflavin (Ankaflavin) is at least 98%.
The present invention provides a kind of novel pure material extracting process being different from existing pure material extracting process.Replace existing organic extract liquid and col-umn chromatography, the method of the present invention only extracts containing Monascin (Monascin) and the precipitate of ankaflavin (Ankaflavin) with drinks solution in a red koji fermentation product, has therefore possessed the advantage that rate of extraction is fast and reduces use organic solvent.In addition, the precipitate obtained via the method for the present invention, its need not move through concentrating under reduced pressure step the most naturally contain recovery of extraction be up to 97.35% Monascin (Monascin) and recovery of extraction be up to 95.16% ankaflavin (Ankaflavin).
Describe the present invention below in conjunction with the drawings and specific embodiments, but not as a limitation of the invention.
Accompanying drawing explanation
Figure 1A Yu Figure 1B is the flow chart of steps of the first embodiment of a kind of pure material extracting process proposed by the invention；
Fig. 2 A and Fig. 2 B is the flow chart of steps of the second embodiment of a kind of pure material extracting process proposed by the invention.
Wherein, reference:
S01～S04 method step
S05～S08 method step
S01 '～S05 ' method step
S06 '～S10 ' method step
Detailed description of the invention
In order to more clearly describe a kind of novel pure material extracting process proposed by the invention, below in conjunction with graphic, elaborate presently preferred embodiments of the present invention.
Refer to Figure 1A and Figure 1B, for the flow chart of steps of the first embodiment of a kind of pure material extracting process proposed by the invention.As shown in Figure 1A Yu Figure 1B, the first embodiment of the pure material extracting process of the present invention comprises the following steps:
Step (S01): get the fermentation Monas cuspurpureus Went of 60 grams ready and using the ethanol (95%) of 300 milliliters as extracting solution, then at 60 DEG C, this fermentation Monas cuspurpureus Went is extracted, last 2 hours；
Step (S02): collect one first extract of abovementioned steps (S01) and filter, and then obtaining one first extracted by filtration liquid and this fermentation Monas cuspurpureus Went through single extraction；
Step (S03): with the ethanol of 300 milliliters (95%) as extracting solution, then again extracts this fermentation Monas cuspurpureus Went at 60 DEG C, lasts 2 hours；
Step (S04): collect one second extract of abovementioned steps (S03) and filter, and then obtaining one second extracted by filtration liquid；
Step (S05): mix this first extracted by filtration liquid and this second extracted by filtration liquid, obtain a hybrid extraction liquid, and this hybrid extraction liquid is concentrated into thick；
Step (S06): the ethanol (95%) of addition 10mL is in this hybrid extraction liquid, to obtain a sample solution；
Step (S07): stir this sample solution 5 minutes under the water-baths of 60 DEG C (water bath), is subsequently added into the water of 10mL in this sample solution, is then again stirring for this sample solution 5 minutes；
Step (S08): with rotating speed 2000rpm/min, this sample solution of abovementioned steps (S07) gained is carried out the centrifugation step of 10 minutes, then collects a precipitate.
In this, must be supplemented with explanation be, although fermentation Monas cuspurpureus Went is extracted by step (S01) with ethanol, but, owing to the pure material extracting process of the present invention is in order to extract pure material in red koji fermentation product, therefore step (S01) can also use ethanol to extract red yeast yam.Certainly, although step (S01) illustrates that weighed fermentation Monas cuspurpureus Went is 60 grams, the most not so limited.Further, the present invention is also not particularly limited the volume of the ethanol extract that step (S01) is used with step (S03) and is necessary for 300 milliliters, and in Practical Operation, the volume of this extracting solution is between 250 milliliters to 320 milliliters.It addition, Extracting temperature (30 DEG C) can optionally adjust between 50 DEG C to 65 DEG C, and the bath temperature of 60 DEG C also can optionally adjust between 55 DEG C to 75 DEG C.
Must especially it is emphasised that, the volume of the alcohol solvent described in step (S05) is (i.e., 10 milliliters) and step (S06) described in water volume (i.e., 10 milliliters) must suitably allocate, the size of the rwo accounts for a percentage by weight and the recovery of extraction of monascus yellow pigment (ankaflavin ankaflavin and Monascin monascin) of sample solution by affecting precipitate.Lower list one describes the impact for weight of precipitate Yu monascus yellow pigment recovery of extraction of different alcohol solvent volumes and water volume.
Table one
By table one, it may be found that, ethanol volume described in step (S05) and the water volume described in step (S06) are suitably deployed into 8 milliliters and 12 milliliters, then obtained precipitate demonstrates the percentage by weight (43.24%) of maximum；Meanwhile, the recovery of extraction of monascus yellow pigment (ankaflavin ankaflavin with Monascin monascin) also reaches the highest by 99.8% and 99.79%.
The extracting process of aforementioned first embodiment belongs to the extracting process of laboratory level, and it the most only just can extract ankaflavin (ankaflavin) and Monascin (monascin) in the secondary metabolic product of red koji fermentation product using drinks solution as extracting solution.Further, below by going on to say the second embodiment of pure material extracting process proposed by the invention, the extracting process of volume production level is belonged to.Refer to Fig. 2 A and Fig. 2 B, for the flow chart of steps of the second embodiment of a kind of pure material extracting process proposed by the invention.As shown in Fig. 2 A and Fig. 2 B, the second embodiment of the pure material extracting process of the present invention comprises the following steps:
Step (S01 '): get the fermentation Monas cuspurpureus Went of 20 kilograms ready and using the ethanol (95%) of 60 liters as extracting solution, then at 60 DEG C, this fermentation Monas cuspurpureus Went is extracted, last 2 hours；
Step (S02 '): collect one first extract of abovementioned steps (S01 '), and filter, and then obtain one first extracted by filtration liquid and this fermentation Monas cuspurpureus Went through single extraction；
Step (S03 '): with the ethanol of 60 liters (95%) as extracting solution, then at 60 DEG C, this fermentation Monas cuspurpureus Went is extracted again, last 2 hours；
Step (S04 '): collect one second extract of abovementioned steps (S03 '), and filter, and then obtain one second extracted by filtration liquid；
Step (S05 '): mix this first extracted by filtration liquid and this second extracted by filtration liquid, obtain a hybrid extraction liquid；
Step (S06 '): this hybrid extraction liquid is concentrated into 4 liters, during then the water of 4 liters adds this hybrid extraction liquid, to obtain one first sample solution；
Step (S07 '): with rotating speed 2000rpm/min, this first sample solution of abovementioned steps (S06 ') gained is carried out the centrifugation step of 10 minutes, then collects one first precipitate；
Step (S08 '): this first precipitate is dissolved in the ethanol (95%) of 1.5 liters, to obtain one second sample solution；
Step (S09 '): stir this second sample solution under the water-bath of 60 DEG C 15 minutes, is subsequently added into the water of 2 liters in this second sample solution, is stirred for this second sample solution afterwards 5 minutes；
Step (S10 '): with rotating speed 2000rpm/min, this second sample solution of abovementioned steps (S09 ') gained is carried out the centrifugation step of 10 minutes, then collects one second precipitate；Wherein, this second precipitate comprises recovery of extraction reach 98.59% Monascin and recovery of extraction reach 98.51% ankaflavin.
In order to confirm that the second precipitate of above-mentioned steps (S10 ') gained contains ankaflavin and Monascin really, the present invention further with following pure material separating step to separate the ankaflavin in the second precipitate and Monascin.
Step (S11 '): standby xerophilous second precipitate totally 662 grams with purge with liquid, followed by medium pressure liquid chromatography (Medium Pressure Liquid Chromatography, MPLC), this second precipitate is purified；Wherein, described in purge with solvent be 5 kinds, respectively n-hexane/ethyl acetate=1/0, n-hexane/ethyl acetate=9:1, n-hexane/ethyl acetate=8:2, n-hexane/ethyl acetate=7:3, n-hexane/ethyl acetate=6:4 (v/v, volume ratio)；
Step (S12 '): take out flavochrome layering in 5 layerings that abovementioned steps (S11 ') is obtained；
Step (S13 '): the layering of this flavochrome is carried out (vacuum) concentrating under reduced pressure, obtains a drying sample；
Step (S14 '): the drying sample of 1 gram is dissolved in the methanol of 50 milliliters, this methanol solution is purified with the flow velocity that purges with of 15mL/min by high performance liquid chromatography (HPLC) (high performance liquid chromatography, HPLC)；So, collection 13.51min purges with liquid and obtains Monascin 22.5 Bo gram (purity=99.1%), and collection 18.78min purges with liquid and obtains ankaflavin 16.83 Bo gram (purity=99.0%)；Wherein, purging with liquid described in is methanol/water=87:13.
So, above-mentioned steps (S11 ')～step (S14 ') confirm by step (S10 ') gained second sedimentary in really contain highly purified ankaflavin and Monascin；Further, described second precipitate is after medium pressure liquid chromatography, and it contains ankaflavin and the Monascin of at least 57.5% via the flavochrome layering of chromatography gained.Predictably, if with the ankaflavin among the layering of preparation HPLC equipment separating yellow element and Monascin, separate (every 20 minutes) the most every time and can obtain the Monascin of 22.5 milligrams and the ankaflavin of 16.83 milligrams.Further, if with the ankaflavin among the layering of industrial hplc device separating yellow element and Monascin, then mass producible ankaflavin and Monascin.
So, described above is complete and clearly demonstrates the detailed step of pure material extracting process and the technical characteristic of the present invention；Further, have the advantage that via the pure material extracting process of the above-mentioned present invention of being appreciated that
(1) being different from existing pure material extracting process, the novel pure material extracting process of the present invention replaces existing extract and col-umn chromatography with drinks solution, has therefore possessed the advantage that rate of extraction is fast and reduces use organic solvent.
(2) additionally, the precipitate obtained via the method for the present invention, its need not move through concentrating under reduced pressure step the most naturally contain recovery of extraction be up to 97.35% Monascin and recovery of extraction be up to 95.16% ankaflavin.
(3) furthermore, it is different from existing high-speed countercurrent chromatography method, the precipitate obtained via the method for the present invention, it is only necessary to just (every 20 minutes) Monascin of 22.5 milligrams and the ankaflavin of 16.83 milligrams can be obtained in each separating step by preparation HPLC equipment；It is apparent that the method for the present invention demonstrates low cost, the advantage of high volume production efficiency.
Certainly; the present invention also can have other various embodiments; in the case of without departing substantially from present invention spirit and essence thereof, those of ordinary skill in the art can make various corresponding change and deformation according to the present invention, but these change accordingly and deform the protection domain that all should belong to the claims in the present invention.
Claims (24)
1. a pure material extracting process, is suitable to extract at least one two second generation in a red koji fermentation product
Thank to product, it is characterised in that this pure material extracting process comprises the following steps:
(1) red koji fermentation product of a specified weight is got ready, and with an extracting solution of one first volume in one
At a temperature of first, this red koji fermentation product is extracted, last one first extraction time；
(2) collect one first extract of abovementioned steps (1) and filter, and then obtaining one first extracted by filtration liquid
And this red koji fermentation product through single extraction；
(3) with this extracting solution of one second volume in this at a temperature of first to this Monas cuspurpureus Went through single extraction
Fermented product extracts again, lasts one second extraction time；
(4) collect one second extract of abovementioned steps (3), and filter, and then obtain one second extracted by filtration
Liquid；And
(5) collect this first extracted by filtration liquid and this second extracted by filtration liquid, and then it is pure to be processed to become one
Material.
Pure material extracting process the most according to claim 1, it is characterised in that this step (5) include with
The thinnest portion step:
(51) this first extracted by filtration liquid is mixed with this second extracted by filtration liquid to obtain a hybrid extraction liquid,
And this hybrid extraction liquid is concentrated into thick；
(52) solvent of a third volume is added in this hybrid extraction liquid, to obtain a sample solution；
(53) under the water-bath of one second temperature, stir this sample solution, last one first mixing time；
(54) it is subsequently added into the water of a fourth volume in this sample solution, is stirred for this sample solution and lasts
One second mixing time；
(55) it is centrifuged walking to this sample solution of abovementioned steps (54) gained with rotating speed 2000rpm/min
Suddenly, one first centrifugation time is lasted；And
(56) complete centrifugation step, then collect a precipitate of this pure material.
Pure material extracting process the most according to claim 1, it is characterised in that described red koji fermentation produces
This specified weight of product is at least 60 grams, and described red koji fermentation product can be lower any one: Monas cuspurpureus Went or
Red yeast yam.
Pure material extracting process the most according to claim 1, it is characterised in that this extracting solution is concentration
The ethanol of 95%.
Pure material extracting process the most according to claim 2, it is characterised in that this first volume with should
Second volume is between 250 milliliters to 320 milliliters, and this fourth volume is 8～12 milliliters.
Pure material extracting process the most according to claim 2, it is characterised in that this first temperature between
Between 50 DEG C to 65 DEG C, and this second temperature is between 55 DEG C to 75 DEG C.
Pure material extracting process the most according to claim 1, it is characterised in that this first extraction time
It is 1～3 hour, and this second extraction time is 1～3 hour.
Pure material extracting process the most according to claim 2, it is characterised in that this solvent is concentration 95%
Ethanol, and this third volume of this solvent is 8～12 milliliters.
Pure material extracting process the most according to claim 2, it is characterised in that this first mixing time
It is 3～7 minutes, and this second mixing time is 3～7 minutes.
Pure material extracting process the most according to claim 2, it is characterised in that this of each gram sinks
Shallow lake thing contains the Monascin of at least 22.5 milligrams and the ankaflavin of at least 16.83 milligrams.
11. pure material extracting process according to claim 2, it is characterised in that this precipitate accounts for this
One percentage by weight of sample solution is at least 25%.
12. pure material extracting process according to claim 11, it is characterised in that described Monascin
Recovery of extraction is at least 97.35%, and the recovery of extraction of described ankaflavin is at least 95.16%.
13. pure material extracting process according to claim 1, it is characterised in that this step (5 ') is wrapped
Include following thin portion step:
(51 ') mix this first extracted by filtration liquid and this second extracted by filtration liquid, obtain a hybrid extraction liquid；
This hybrid extraction liquid is concentrated into a third volume by (52 '), is then added by the water of a fourth volume
In this hybrid extraction liquid, to obtain one first sample solution；
This first sample solution of abovementioned steps (52 ') gained is carried out by (53 ') with rotating speed 2000rpm/min
Centrifugation step, lasts one first centrifugation time；
After (54 ') complete centrifugation step, then collect one first precipitate；
This first precipitate is dissolved in a solvent of one the 5th volume by (55 '), to obtain one second sample
Solution；
(56 ') stir this second sample solution under the water-bath of one second temperature, when lasting one first stirring
Between；
The water that one hexasomic is long-pending is then added in this second sample solution by (57 '), is stirred for this second sample
Product solution lasts one second mixing time；
This second sample solution of abovementioned steps (57 ') gained is carried out by (58 ') with rotating speed 2000rpm/min
Centrifugation step, lasts one second centrifugation time；
After (59 ') complete centrifugation step, then collect one second precipitate, for this pure material.
14. pure material extracting process according to claim 1, it is characterised in that described red koji fermentation
This specified weight of product is at least 20 kilograms, and described red koji fermentation product can be lower any one: Monas cuspurpureus Went
Rice or red yeast yam.
15. pure material extracting process according to claim 1, it is characterised in that this extracting solution is dense
Spend the ethanol of 95%, and this first volume is at least 60 liters with this second volume.
16. pure material extracting process according to claim 13, it is characterised in that this first temperature is situated between
Between 50 DEG C to 65 DEG C, and this this second temperature is between 55 DEG C to 75 DEG C.
17. pure material extracting process according to claim 13, wherein, this first extraction time is 1～3
Hour, and this second extraction time is 1～3 hour.
18. pure material extracting process according to claim 13, it is characterised in that this third volume is extremely
Being 4 liters less, this fourth volume is at least 4 liters, and the 5th volume is 1～2 liter, and this hexasomic
Amass is 1.5～2.5 liters.
19. pure material extracting process according to claim 13, it is characterised in that this is first when being centrifuged
Between be 8～12 minutes, and this second centrifugation time is 8～12 minutes.
20. pure material extracting process according to claim 13, it is characterised in that this solvent is concentration
The ethanol of 95%.
21. pure material extracting process according to claim 13, it is characterised in that during this first stirring
Between be 10～20 minutes, and this second mixing time is 3～7 minutes.
22. pure material extracting process according to claim 13, it is characterised in that each gram this
Two precipitate contain the Monascin of at least 22.5 milligrams and the ankaflavin of at least 16.83 milligrams.
23. pure material extracting process according to claim 13, it is characterised in that this second precipitate
The percentage by weight accounting for this second sample solution is at least 25%.
24. pure material extracting process according to claim 23, it is characterised in that described Monascin
Recovery of extraction is at least 98%, and the recovery of extraction of described ankaflavin is at least 98%.
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| CN113521795A (en) * | 2020-04-17 | 2021-10-22 | 郑长义 | Extraction method and application of purple yam active substances |
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