CN114836485B - Method for extracting and separating ajoene from garlic - Google Patents
Method for extracting and separating ajoene from garlic Download PDFInfo
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- 235000004611 garlic Nutrition 0.000 title claims abstract description 147
- IXELFRRANAOWSF-FNORWQNLSA-N (E)-Ajoene Chemical compound C=CCSS\C=C\CS(=O)CC=C IXELFRRANAOWSF-FNORWQNLSA-N 0.000 title claims abstract description 83
- IXELFRRANAOWSF-CYBMUJFWSA-N ajoene Natural products C=CCSSC=CC[S@](=O)CC=C IXELFRRANAOWSF-CYBMUJFWSA-N 0.000 title claims abstract description 83
- IXELFRRANAOWSF-UHFFFAOYSA-N cis-ajoene Natural products C=CCSSC=CCS(=O)CC=C IXELFRRANAOWSF-UHFFFAOYSA-N 0.000 title claims abstract description 83
- 238000000034 method Methods 0.000 title claims abstract description 32
- 244000245420 ail Species 0.000 title 1
- 240000002234 Allium sativum Species 0.000 claims abstract description 146
- 244000223014 Syzygium aromaticum Species 0.000 claims abstract description 103
- 235000016639 Syzygium aromaticum Nutrition 0.000 claims abstract description 103
- 238000000605 extraction Methods 0.000 claims abstract description 59
- 239000010647 garlic oil Substances 0.000 claims abstract description 53
- 238000000199 molecular distillation Methods 0.000 claims abstract description 29
- 239000012530 fluid Substances 0.000 claims abstract description 18
- 239000007791 liquid phase Substances 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 14
- 238000006911 enzymatic reaction Methods 0.000 claims abstract description 9
- 238000000746 purification Methods 0.000 claims abstract description 8
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 25
- 239000001273 butane Substances 0.000 claims description 22
- IJDNQMDRQITEOD-UHFFFAOYSA-N n-butane Chemical compound CCCC IJDNQMDRQITEOD-UHFFFAOYSA-N 0.000 claims description 22
- OFBQJSOFQDEBGM-UHFFFAOYSA-N n-pentane Natural products CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 claims description 22
- 238000001914 filtration Methods 0.000 claims description 12
- 239000003480 eluent Substances 0.000 claims description 11
- 239000000945 filler Substances 0.000 claims description 11
- 238000004587 chromatography analysis Methods 0.000 claims 1
- 238000004237 preparative chromatography Methods 0.000 claims 1
- 108010092760 Alliin lyase Proteins 0.000 abstract description 31
- XUHLIQGRKRUKPH-GCXOYZPQSA-N Alliin Natural products N[C@H](C[S@@](=O)CC=C)C(O)=O XUHLIQGRKRUKPH-GCXOYZPQSA-N 0.000 abstract description 13
- XUHLIQGRKRUKPH-UHFFFAOYSA-N S-allyl-L-cysteine sulfoxide Natural products OC(=O)C(N)CS(=O)CC=C XUHLIQGRKRUKPH-UHFFFAOYSA-N 0.000 abstract description 13
- XUHLIQGRKRUKPH-DYEAUMGKSA-N alliin Chemical compound OC(=O)[C@@H](N)C[S@@](=O)CC=C XUHLIQGRKRUKPH-DYEAUMGKSA-N 0.000 abstract description 13
- 235000015295 alliin Nutrition 0.000 abstract description 13
- JDLKFOPOAOFWQN-VIFPVBQESA-N Allicin Natural products C=CCS[S@](=O)CC=C JDLKFOPOAOFWQN-VIFPVBQESA-N 0.000 abstract description 4
- JDLKFOPOAOFWQN-UHFFFAOYSA-N allicin Chemical compound C=CCSS(=O)CC=C JDLKFOPOAOFWQN-UHFFFAOYSA-N 0.000 abstract description 4
- 235000010081 allicin Nutrition 0.000 abstract description 4
- 238000012545 processing Methods 0.000 abstract description 3
- 230000003213 activating effect Effects 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 description 18
- 238000005303 weighing Methods 0.000 description 17
- 230000000694 effects Effects 0.000 description 10
- 238000002161 passivation Methods 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 9
- 238000006555 catalytic reaction Methods 0.000 description 9
- 238000011049 filling Methods 0.000 description 9
- 229940029982 garlic powder Drugs 0.000 description 9
- 238000010438 heat treatment Methods 0.000 description 9
- 238000011068 loading method Methods 0.000 description 9
- 239000000463 material Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 8
- 238000007789 sealing Methods 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000004821 distillation Methods 0.000 description 3
- 230000000415 inactivating effect Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000002253 acid Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- KZJWDPNRJALLNS-VPUBHVLGSA-N (-)-beta-Sitosterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]([C@H](CC[C@@H](C(C)C)CC)C)CC4)CC3)CC=2)CC1 KZJWDPNRJALLNS-VPUBHVLGSA-N 0.000 description 1
- CSVWWLUMXNHWSU-UHFFFAOYSA-N (22E)-(24xi)-24-ethyl-5alpha-cholest-22-en-3beta-ol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(CC)C(C)C)C1(C)CC2 CSVWWLUMXNHWSU-UHFFFAOYSA-N 0.000 description 1
- KLEXDBGYSOIREE-UHFFFAOYSA-N 24xi-n-propylcholesterol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CCC)C(C)C)C1(C)CC2 KLEXDBGYSOIREE-UHFFFAOYSA-N 0.000 description 1
- 241000383638 Allium nigrum Species 0.000 description 1
- LPZCCMIISIBREI-MTFRKTCUSA-N Citrostadienol Natural products CC=C(CC[C@@H](C)[C@H]1CC[C@H]2C3=CC[C@H]4[C@H](C)[C@@H](O)CC[C@]4(C)[C@H]3CC[C@]12C)C(C)C LPZCCMIISIBREI-MTFRKTCUSA-N 0.000 description 1
- ARVGMISWLZPBCH-UHFFFAOYSA-N Dehydro-beta-sitosterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)CCC(CC)C(C)C)CCC33)C)C3=CC=C21 ARVGMISWLZPBCH-UHFFFAOYSA-N 0.000 description 1
- UQBOJOOOTLPNST-UHFFFAOYSA-N Dehydroalanine Chemical compound NC(=C)C(O)=O UQBOJOOOTLPNST-UHFFFAOYSA-N 0.000 description 1
- UBJVUCKUDDKUJF-UHFFFAOYSA-N Diallyl sulfide Chemical class C=CCSCC=C UBJVUCKUDDKUJF-UHFFFAOYSA-N 0.000 description 1
- 239000006000 Garlic extract Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- MJVXAPPOFPTTCA-UHFFFAOYSA-N beta-Sistosterol Natural products CCC(CCC(C)C1CCC2C3CC=C4C(C)C(O)CCC4(C)C3CCC12C)C(C)C MJVXAPPOFPTTCA-UHFFFAOYSA-N 0.000 description 1
- LGJMUZUPVCAVPU-UHFFFAOYSA-N beta-Sitostanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 LGJMUZUPVCAVPU-UHFFFAOYSA-N 0.000 description 1
- NJKOMDUNNDKEAI-UHFFFAOYSA-N beta-sitosterol Natural products CCC(CCC(C)C1CCC2(C)C3CC=C4CC(O)CCC4C3CCC12C)C(C)C NJKOMDUNNDKEAI-UHFFFAOYSA-N 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000007323 disproportionation reaction Methods 0.000 description 1
- RIYVKHUVXPAOPS-UHFFFAOYSA-N dithiine Chemical compound S1SC=CC=C1 RIYVKHUVXPAOPS-UHFFFAOYSA-N 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 235000020706 garlic extract Nutrition 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 description 1
- 229950005143 sitosterol Drugs 0.000 description 1
- NLQLSVXGSXCXFE-UHFFFAOYSA-N sitosterol Natural products CC=C(/CCC(C)C1CC2C3=CCC4C(C)C(O)CCC4(C)C3CCC2(C)C1)C(C)C NLQLSVXGSXCXFE-UHFFFAOYSA-N 0.000 description 1
- 235000015500 sitosterol Nutrition 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P11/00—Preparation of sulfur-containing organic compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C319/00—Preparation of thiols, sulfides, hydropolysulfides or polysulfides
- C07C319/26—Separation; Purification; Stabilisation; Use of additives
- C07C319/28—Separation; Purification
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for extracting and separating ajoene from garlic, belonging to the technical field of agricultural product deep processing. The method of the invention comprises the following steps: pretreating garlic cloves under the following conditions: vacuum, pressure of 400MPa or more and time of 10min or more; crushing the pretreated garlic cloves at the temperature below-4 ℃ and then carrying out enzymatic reaction under the conditions of 100-200MPa and 25-35 ℃ to obtain the minced garlic; extracting the minced garlic by subcritical fluid to obtain garlic oil; molecular distillation and liquid phase purification are sequentially carried out on the garlic oil to obtain the ajoene. The method comprises the steps of firstly passivating alliinase to extract alliin by using ultrahigh pressure, preparing alliinase liquid, then activating alliinase in garlic by using ultrahigh pressure, accelerating enzymatic reaction, generating more allicin and further generating higher-content ajoene, and finally obtaining high-yield and high-purity ajoene products by ultrahigh pressure regulation, subcritical fluid extraction, molecular distillation and liquid phase purification.
Description
Technical Field
The invention relates to a method for extracting and separating ajoene from garlic, belonging to the technical field of agricultural product deep processing.
Background
Garlic contains alliin and alliinase, but alliin and alliinase are present in the cytoplasm and vacuole, respectively, and alliinase cannot act on alliin. When garlic is broken, alliin is converted into allylsulfenac acid and dehydroalanine under the action of allinase, and the allylsulfenac acid generates allicin through disproportionation reaction, and the allicin is extremely unstable and can be further decomposed into diallyl sulfide compounds, dithiine and ajoene, so that the ajoene can be said to be a second degradation product of alliin. Under natural conditions, the yield of ajoene in garlic is very small, and the stability of ajoene is poor, so that the extraction of ajoene is the focus of research.
Chinese patent CN1324142C discloses a defatted garlic extract containing ajoene from which other fat-soluble substances such as sitosterol are removed, and its preparation method and application. Chinese patent CN104974068 discloses a preparation method of ajoene, which comprises crushing garlic, mixing with extractive solution, processing to obtain product, filtering and/or centrifuging, removing solvent from the obtained product, mixing with vegetable oil, adding adsorbent into the obtained product, and eluting to obtain high concentration ajoene product. However, the two methods have the problems of low ajoene preparation rate and low purity.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for extracting and separating ajoene from garlic.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for extracting and separating ajoene from garlic, comprising the following steps:
pretreating garlic cloves under the following conditions: vacuum, pressure of 400MPa or more and time of 10min or more;
crushing the pretreated garlic cloves at the temperature below-4 ℃ and then carrying out enzymatic reaction under the conditions of 100-200MPa and 25-35 ℃ to obtain the minced garlic;
extracting the minced garlic by subcritical fluid to obtain garlic oil;
molecular distillation and liquid phase purification are sequentially carried out on the garlic oil to obtain the ajoene.
The invention is found by experimental study: the extraction and separation of ajoene Huo Xishi from garlic is unstable and results in low ajoene yield. The method can inactivate alliinase in garlic by pretreating the garlic cloves; therefore, during the process of crushing garlic cloves and releasing alliin, alliinase does not react with the alliin and cannot cause the loss of ajoene, so that the yield of ajoene is obviously improved.
The method comprises the steps of firstly passivating alliinase to extract alliin by using ultrahigh pressure, preparing alliinase liquid, then activating alliinase in garlic by using ultrahigh pressure, accelerating enzymatic reaction, generating more allicin and further generating higher-content ajoene, and finally obtaining a high-purity ajoene product by ultrahigh pressure regulation, subcritical fluid extraction, molecular distillation and liquid phase preparation purification.
The method of the invention can be used for extracting and separating ajoene from garlic, and can also be used for extracting ajoene from black garlic.
When the garlic cloves are pretreated, the pressure reaches 400MPa, so that the allinase in the garlic can be passivated. The alliinase passivation effect becomes better with further increase of the pressure; however, the more the pressure is not, the better the alliinase inactivating effect is. Experimental study proves that alliinase passivation effect is obviously reduced when the pressure exceeds 650 MPa. Therefore, the present invention limits the pressure conditions to 400-600MPa; in the pressure condition, alliinase deactivation effect is better when the pressure is 500-600. Specifically, the pressure conditions may be 520 to 550MPa or 550 to 580MPa.
When the garlic cloves are pretreated, the time reaches 10min, and the effect of passivating the allinase in the garlic can be achieved. The alliinase passivation effect becomes better with further extension of time; however, the more time it is not, the better the alliinase inactivating effect. Experimental study proves that alliinase passivation effect is obviously reduced when time exceeds 35 min. Therefore, the time is limited to 10-30min, and the allinase passivation effect is better when the time is 15-20min in the time range.
Wherein the alliinase passivation effect is measured in terms of the yield of garlic oil; the higher the yield of garlic oil, the better the alliinase inactivating effect.
When the pretreated garlic cloves are crushed, the yield of garlic oil is obviously improved when the temperature reaches-4 ℃; along with the decrease of the temperature, the yield of the garlic oil is increased; however, when the temperature is lower than-20 ℃, the yield of garlic oil is rather remarkably reduced. The possible reason is that the alliinase can maintain a good passivation state in a temperature range of-4 ℃ to-20 ℃, the alliinase can not be activated in the crushing process, the alliinase can not react with alliin, and the ajoene loss can not be caused. When the temperature is higher than-4 ℃, the alliinase cannot be kept in a good passivation state, and the alliinase is activated in the crushing process and reacts with alliin, so that ajoene is lost. When the temperature is lower than-20 ℃, alliinase can maintain a good passivation state, but the effect is not ideal and the cost is high. Therefore, the temperature at which the pretreated garlic cloves are crushed can be limited to-4℃to-20 ℃.
The crushed garlic cloves are heated to 25-35 ℃ and the pressure is raised to 100-200MPa, at this time, the alliinase in a passivation state is activated and reacts with the alliin released in the crushing process rapidly, namely, enzymatic reaction is carried out. Wherein, the temperature of 25-35 ℃ is the activity temperature of alliinase; the aim of raising the pressure to 100-200MPa is to activate the inactivated allinase and to increase the yield of garlic oil. The possible reason is that the pressure condition can increase the reaction rate of alliinase and alliin, so that the ajoene decomposition loss can be remarkably reduced. The yield of the garlic oil is higher when the pressure is 150-180MPa.
The reaction time of the enzymatic reaction is preferably the complete reaction, i.e., the complete enzymatic reaction of alliinase.
The pretreated garlic cloves can be frozen at-4 ℃ for later use if the garlic cloves cannot be processed in time for the next step. In order to ensure the inactivation of alliinase in the pretreated garlic cloves, the freezing storage temperature can be between-4 and-20 ℃.
The extraction of garlic oil from the minced garlic by subcritical fluid means that: extracting butane as extractant at-0.1 Mpa and 20-50deg.C, and then reducing the pressure of the extractive solution to-0.1 Mpa, separating butane from oleum Bulbus Allii to obtain oleum Bulbus Allii;
the conditions of the molecular distillation are as follows: the dripping speed of the garlic oil is 1-3 drops/3 seconds at 50-100 ℃ and the vacuum degree is 30-35Pa,
the liquid phase purification refers to: dissolving ajoene fraction collected by molecular distillation with 5-8 times of 50-80% ethanol, filtering, purifying with Innoval ODS-2 as filler and 90-95% ethanol as eluent, eluting at 10-30mL/min, collecting ajoene separating liquid, and concentrating under reduced pressure at 50-70deg.C to obtain ajoene.
Other operation steps, conditions and parameters in subcritical fluid extraction, molecular distillation and liquid phase purification are all in the prior art; the present invention is not described in detail.
The beneficial effects of the invention are as follows:
the invention adopts the method of extracting and separating ajoene from garlic by ultra-high pressure subcritical fluid extraction, molecular distillation separation and preparation chromatography purification as a core complete technology for the first time, improves the yield and purity of ajoene, has reasonable and feasible process, simple process, small solvent toxicity and convenient recovery of solvent, can realize large-scale industrialized production, and has strong practical value.
In the present invention, the percentages are mass percentages unless otherwise indicated.
Drawings
FIG. 1 is a liquid chromatograph of the determination of the ajoene content of the product of step (4) in an embodiment of the present invention;
FIG. 2 is a graph showing the measurement of ajoene content using liquid chromatography on the product of step (5) in an embodiment of the present invention.
Detailed Description
The invention will be further described with reference to the drawings and examples.
The structures, proportions, sizes, etc. shown in the drawings are shown only in connection with the present disclosure, and therefore should not be construed as limiting the invention, but rather as limiting the scope of the invention, so that any structural modifications, proportional changes, or dimensional adjustments should fall within the scope of the invention without affecting the efficacy or achievement thereof. Also, the terms such as "upper," "lower," "left," "right," "middle," and "a" and the like recited in the present specification are merely for descriptive purposes and are not intended to limit the scope of the invention, but are intended to provide relative positional changes or modifications without materially altering the technical context in which the invention may be practiced.
Example 1
A method for extracting and separating ajoene from Bulbus Allii comprises the following steps:
(1) Pretreating garlic: weighing 100g of garlic cloves, placing the garlic cloves into an ultrahigh-pressure special bag, vacuum-sealing the garlic cloves, placing the garlic cloves into a pressure cavity, pretreating the garlic cloves for 10min under the pressure of 500MPa, and taking the garlic cloves out after the garlic cloves are finished and placing the garlic cloves in an environment of-4 to-20 ℃ for standby.
(2) Crushing the pretreated garlic cloves at the temperature of between-4 ℃ and-20 ℃, then placing the crushed garlic cloves into an ultrahigh pressure cavity, and carrying out enzyme catalytic reaction for 30min in a pressure cavity with the pressure of 100MPa and the temperature of 25 ℃; obtaining the minced garlic.
(3) Subcritical fluid extraction for preparing garlic oil: weighing 150g of garlic powder, filling into a material bag, placing into a subcritical extraction tank, and vacuumizing the extraction tank to a vacuum degree of-0.1 Mpa; adding 600mL of butane, extracting at 30 ℃ for 120min, and extracting for 3 times; after the extraction was completed, the extract was introduced into a separation tank, the pressure was reduced to-0.1 Mpa, butane was separated from garlic oil, 0.82g of garlic oil was obtained, and the garlic oil extraction rate was 0.55%.
(4) Molecular distillation separation: loading oleum Bulbus Allii into a molecular distillation device, heating to 50deg.C, controlling vacuum degree to 30Pa, dripping at 1 drop/3 sec, and collecting ajoene fraction.
(5) Preparing a liquid phase and purifying: dissolving ajoene fraction with 50% ethanol of 5 times volume, filtering, purifying with Innoval ODS-2 as filler and 20% ethyl acetate as eluent, eluting at 15mL/min, collecting ajoene separating liquid, concentrating under reduced pressure at 60deg.C to obtain ajoene with purity of 97.7%.
Example 2
A method for extracting and separating ajoene from Bulbus Allii comprises the following steps:
(1) Pretreating garlic: weighing 150g of garlic cloves, placing the garlic cloves into an ultrahigh-pressure special bag, vacuum-sealing the garlic cloves, placing the garlic cloves into a pressure cavity, pretreating the garlic cloves for 15min under the pressure of 520MPa, and taking the garlic cloves out after the garlic cloves are finished and placing the garlic cloves in an environment of-4 to-20 ℃ for standby.
(2) Crushing the pretreated garlic cloves at the temperature of between 4 ℃ below zero and 20 ℃ below zero, then placing the crushed garlic cloves into an ultrahigh pressure cavity, and carrying out enzyme catalytic reaction for 30min in a pressure cavity with the pressure of 100MPa and the temperature of 35 ℃; obtaining the minced garlic.
(3) Subcritical fluid extraction for preparing garlic oil: weighing 200g of garlic powder, filling into a material bag, placing into a subcritical extraction tank, and vacuumizing the extraction tank to a vacuum degree of-0.1 Mpa; adding 900mL of butane, extracting at 40 ℃ for 120min, and extracting for 4 times; after the extraction was completed, the extract was introduced into a separation tank, the pressure was reduced to-0.1 Mpa, butane was separated from garlic oil to obtain 1.28g of garlic oil, and the garlic oil extraction rate was 0.64%.
(4) Molecular distillation separation: loading oleum Bulbus Allii into a molecular distillation device constant temperature tank, heating to 70deg.C with a hot oil machine, controlling vacuum degree to 35Pa, dropping at 2 drop/3 sec, starting distillation process, and collecting ajoene fraction;
(5) Preparing a liquid phase and purifying: dissolving ajoene fraction with 5 times volume of 60% ethanol, filtering, purifying with Innoval ODS-2 as filler and 20% ethyl acetate as eluent, eluting at 15mL/min, collecting ajoene separating liquid, and concentrating under reduced pressure at 60deg.C to obtain ajoene with purity of 98.1%.
Example 3
A method for extracting and separating ajoene from Bulbus Allii comprises the following steps:
(1) Pretreating garlic: weighing 150g of garlic cloves, placing the garlic cloves into an ultrahigh pressure special bag, vacuum-sealing the garlic cloves, placing the garlic cloves into a pressure cavity, pretreating the garlic cloves for 20 minutes under the pressure of 550MPa, and taking the garlic cloves out after the garlic cloves are finished and placing the garlic cloves in an environment of-4 to-20 ℃ for standby.
(2) Crushing the pretreated garlic cloves at the temperature of between-4 and-20 ℃, then placing the crushed garlic cloves into an ultrahigh pressure cavity, and carrying out enzyme catalytic reaction for 35min in a pressure cavity with the pressure of 180MPa and the temperature of 35 ℃; obtaining the minced garlic.
(3) Subcritical fluid extraction for preparing garlic oil: weighing 200g of garlic powder, filling into a material bag, placing into a subcritical extraction tank, and vacuumizing the extraction tank to a vacuum degree of-0.1 Mpa; butane 1200mL is added, the extraction temperature is 50 ℃, the time is 120min, and the extraction is carried out for 4 times; after the extraction is finished, the extract is introduced into a separating tank, the pressure is reduced to-0.1 Mpa, butane is separated from garlic oil, 1.20g of garlic oil is obtained, and the extraction rate of the garlic oil is 0.60%.
(4) Molecular distillation separation: loading oleum Bulbus Allii into a molecular distillation device constant temperature tank, heating to 80deg.C, controlling vacuum degree to 35Pa, dropping for 2 drops/3 seconds, starting distillation process, and collecting ajoene fraction;
(5) Preparing a liquid phase and purifying: dissolving ajoene fraction with 60% ethanol of 6 times volume, filtering, purifying with Innoval ODS-2 as filler and 20% ethyl acetate as eluent, eluting at 10mL/min, collecting ajoene separating liquid, concentrating under reduced pressure at 60deg.C to obtain ajoene with purity of 98.4%.
Example 4
A method for extracting and separating ajoene from Bulbus Allii comprises the following steps:
(1) Pretreating garlic: weighing 200g of garlic cloves, placing the garlic cloves into an ultrahigh-pressure special bag, vacuum-sealing the garlic cloves, placing the garlic cloves into a pressure cavity, pretreating the garlic cloves for 20min under the pressure of 580MPa, and taking the garlic cloves out after the garlic cloves are finished and placing the garlic cloves in an environment of-4 ℃ to-20 ℃ for standby.
(2) Crushing the pretreated garlic cloves at the temperature of between-4 and-20 ℃, then placing the crushed garlic cloves into an ultrahigh pressure cavity, and carrying out enzyme catalytic reaction for 30min in a pressure cavity with the pressure of 150MPa and the temperature of 40 ℃; obtaining the minced garlic.
(3) Subcritical fluid extraction for preparing garlic oil: weighing 300g of garlic powder, filling into a material bag, placing into a subcritical extraction tank, and vacuumizing the extraction tank to a vacuum degree of-0.1 Mpa; butane 2000mL is added, the extraction temperature is 40 ℃, the time is 150min, and the extraction is carried out for 4 times; after the extraction was completed, the extract was introduced into a separation tank, the pressure was reduced to-0.1 Mpa, butane was separated from garlic oil to obtain 1.82g of garlic oil, and the garlic oil extraction rate was 0.60%.
(4) Molecular distillation separation: loading oleum Bulbus Allii into a molecular distillation device constant temperature tank, heating to 80deg.C, controlling vacuum degree to 35Pa, dropping for 3 drops/3 seconds, starting distillation process, and collecting ajoene fraction;
(5) Preparing a liquid phase and purifying: dissolving ajoene fraction with 80% ethanol of 5 times volume, filtering, purifying with Flash silica as filler and 20% ethyl acetate as eluent, eluting at 20mL/min, collecting ajoene separating liquid, concentrating under reduced pressure at 60deg.C to obtain ajoene with purity of 98.8%.
Example 5
A method for extracting and separating ajoene from Bulbus Allii comprises the following steps:
(1) Pretreating garlic: weighing 250g of garlic cloves, placing the garlic cloves into an ultrahigh-pressure special bag, vacuum-sealing the garlic cloves, placing the garlic cloves into a pressure cavity, pretreating the garlic cloves for 30 minutes under the pressure of 600MPa, and taking the garlic cloves out after the garlic cloves are finished and placing the garlic cloves in an environment of-4 to-20 ℃ for standby.
(2) Crushing the pretreated garlic cloves at the temperature of between-4 and-20 ℃, then placing the crushed garlic cloves into an ultrahigh pressure cavity, and carrying out enzyme catalytic reaction for 30min in a pressure cavity with the pressure of 200MPa and the temperature of 35 ℃; obtaining the minced garlic.
(3) Subcritical fluid extraction for preparing garlic oil: weighing 300g of garlic powder, filling into a material bag, placing into a subcritical extraction tank, and vacuumizing the extraction tank to a vacuum degree of-0.1 Mpa; butane 2000mL is added, the extraction temperature is 50 ℃, the time is 120min, and the extraction is carried out for 5 times; after the extraction was completed, the extract was introduced into a separation tank, the pressure was reduced to-0.1 Mpa, butane was separated from garlic oil to obtain 1.74g of garlic oil, and the garlic oil extraction rate was 0.58%.
(4) Molecular distillation separation: loading oleum Bulbus Allii into a molecular distillation device, heating to 100deg.C, controlling vacuum degree to 35Pa, dripping at 1 drop/3 sec, and collecting ajoene fraction.
(5) Preparing a liquid phase and purifying: dissolving ajoene fraction with 5 times volume of absolute ethanol, filtering, purifying with Flash silica as filler and 20% ethyl acetate as eluent, eluting at 30mL/min, collecting ajoene separating liquid, concentrating under reduced pressure at 50deg.C to obtain ajoene with purity of 99.1%.
Comparative example 1
(1) Pretreating garlic: weighing 100g of garlic cloves, placing the garlic cloves into an ultrahigh-pressure special bag, vacuum-sealing the garlic cloves, placing the garlic cloves into a pressure cavity, pretreating the garlic cloves for 35 minutes under the pressure of 500MPa, and taking the garlic cloves out after the garlic cloves are finished and placing the garlic cloves in an environment of-4 to-20 ℃ for standby.
(2) Crushing the pretreated garlic cloves at the temperature of between-4 ℃ and-20 ℃, then placing the crushed garlic cloves into an ultrahigh pressure cavity, and carrying out enzyme catalytic reaction for 30min in a pressure cavity with the pressure of 100MPa and the temperature of 25 ℃; obtaining the minced garlic.
(3) Subcritical fluid extraction for preparing garlic oil: weighing 150g of garlic powder, filling into a material bag, placing into a subcritical extraction tank, and vacuumizing the extraction tank to a vacuum degree of-0.1 Mpa; adding 600mL of butane, extracting at 30 ℃ for 120min, and extracting for 3 times; after the extraction is finished, the extract is introduced into a separating tank, the pressure is reduced to-0.1 Mpa, butane is separated from garlic oil, 0.60g of garlic oil is obtained, and the extraction rate of the garlic oil is 0.40%.
(4) Molecular distillation separation: loading oleum Bulbus Allii into a molecular distillation device, heating to 50deg.C, controlling vacuum degree to 30Pa, dripping at 1 drop/3 sec, and collecting ajoene fraction.
(5) Preparing a liquid phase and purifying: dissolving ajoene fraction with 50% ethanol of 5 times volume, filtering, purifying with Innoval ODS-2 as filler and 20% ethyl acetate as eluent, eluting at 15mL/min, collecting ajoene separating liquid, concentrating under reduced pressure at 60deg.C to obtain ajoene with purity of 97.1%.
Comparative example 2
(1) Pretreating garlic: weighing 100g of garlic cloves, placing the garlic cloves into an ultrahigh-pressure special bag, vacuum-sealing the garlic cloves, placing the garlic cloves into a pressure cavity, pretreating the garlic cloves for 10min under the pressure of 650MPa, and taking the garlic cloves out after the garlic cloves are finished and placing the garlic cloves in an environment of-4 to-20 ℃ for standby.
(2) Crushing the pretreated garlic cloves at the temperature of between-4 ℃ and-20 ℃, then placing the crushed garlic cloves into an ultrahigh pressure cavity, and carrying out enzyme catalytic reaction for 30min in a pressure cavity with the pressure of 100MPa and the temperature of 25 ℃; obtaining the minced garlic.
(3) Subcritical fluid extraction for preparing garlic oil: weighing 150g of garlic powder, filling into a material bag, placing into a subcritical extraction tank, and vacuumizing the extraction tank to a vacuum degree of-0.1 Mpa; adding 600mL of butane, extracting at 30 ℃ for 120min, and extracting for 3 times; after the extraction was completed, the extract was introduced into a separation tank, the pressure was reduced to-0.1 Mpa, butane was separated from garlic oil, 0.54g of garlic oil was obtained, and the garlic oil extraction rate was 0.36%.
(4) Molecular distillation separation: loading oleum Bulbus Allii into a molecular distillation device, heating to 50deg.C, controlling vacuum degree to 30Pa, dripping at 1 drop/3 sec, and collecting ajoene fraction.
(5) Preparing a liquid phase and purifying: dissolving ajoene fraction with 50% ethanol of 5 times volume, filtering, purifying with Innoval ODS-2 as filler and 20% ethyl acetate as eluent, eluting at 15mL/min, collecting ajoene separating liquid, concentrating under reduced pressure at 60deg.C to obtain ajoene with purity of 96.8%.
Comparative example 3
(1) Pretreating garlic: weighing 100g of garlic cloves, placing the garlic cloves into an ultrahigh-pressure special bag, vacuum-sealing the garlic cloves, placing the garlic cloves into a pressure cavity, pretreating the garlic cloves for 10min under the pressure of 500MPa, and taking the garlic cloves out after the garlic cloves are finished and placing the garlic cloves in an environment of-4 to-20 ℃ for standby.
(2) Crushing the pretreated garlic cloves at the temperature of between-4 and-20 ℃, then placing the crushed garlic cloves into an ultrahigh pressure cavity, and carrying out enzyme catalytic reaction for 30min in a pressure cavity with normal pressure and the temperature of 25 ℃; obtaining the minced garlic.
(3) Subcritical fluid extraction for preparing garlic oil: weighing 150g of garlic powder, filling into a material bag, placing into a subcritical extraction tank, and vacuumizing the extraction tank to a vacuum degree of-0.1 Mpa; adding 600mL of butane, extracting at 30 ℃ for 120min, and extracting for 3 times; after the extraction was completed, the extract was introduced into a separation tank, the pressure was reduced to-0.1 Mpa, butane was separated from garlic oil, 0.53g of garlic oil was obtained, and the garlic oil extraction rate was 0.35%.
(4) Molecular distillation separation: loading oleum Bulbus Allii into a molecular distillation device, heating to 50deg.C, controlling vacuum degree to 30Pa, dripping at 1 drop/3 sec, and collecting ajoene fraction.
(5) Preparing a liquid phase and purifying: dissolving ajoene fraction with 50% ethanol of 5 times volume, filtering, purifying with Innoval ODS-2 as filler and 20% ethyl acetate as eluent, eluting at 15mL/min, collecting ajoene separating liquid, concentrating under reduced pressure at 60deg.C to obtain ajoene with purity of 97.4%.
Comparative example 4
(1) Crushing garlic cloves at the temperature of 0 ℃, then placing the crushed garlic cloves into an ultrahigh pressure cavity, and carrying out enzyme catalytic reaction for 30min in a pressure cavity with the pressure of 100MPa and the temperature of 25 ℃; obtaining the minced garlic.
(2) Subcritical fluid extraction for preparing garlic oil: weighing 150g of garlic powder, filling into a material bag, placing into a subcritical extraction tank, and vacuumizing the extraction tank to a vacuum degree of-0.1 Mpa; adding 600mL of butane, extracting at 30 ℃ for 150min, and extracting for 3 times; after the extraction was completed, the extract was introduced into a separation tank, the pressure was reduced to-0.1 Mpa, butane was separated from garlic oil, 0.36g of garlic oil was obtained, and the garlic oil extraction rate was 0.24%.
(3) Molecular distillation separation: loading oleum Bulbus Allii into a molecular distillation device, heating to 50deg.C, controlling vacuum degree to 30Pa, dripping at 1 drop/3 sec, and collecting ajoene fraction.
(4) Preparing a liquid phase and purifying: dissolving ajoene fraction with 50% ethanol of 5 times volume, filtering, purifying with Innoval ODS-2 as filler and 90% ethanol as eluent, eluting at 15mL/min, collecting ajoene separating liquid, concentrating under reduced pressure at 60deg.C to obtain ajoene with purity of 97.6%.
While the foregoing description of the embodiments of the present invention has been presented in conjunction with the drawings, it should be understood that it is not intended to limit the scope of the invention, but rather, it is intended to cover all modifications or variations within the scope of the invention as defined by the claims of the present invention.
Claims (8)
1. A method for extracting and separating ajoene from garlic, which is characterized by comprising the following steps:
pretreating garlic cloves under the following conditions: vacuum, pressure of 500-600MPa, and time of 10-30 min;
crushing the pretreated garlic cloves at the temperature below-4 ℃ and then carrying out enzymatic reaction under the conditions of 100-200MPa and 25-35 ℃ to obtain the minced garlic;
extracting the minced garlic by subcritical fluid to obtain garlic oil;
molecular distillation and liquid phase purification are sequentially carried out on the garlic oil to obtain ajoene;
the extraction of garlic oil from the minced garlic by subcritical fluid means that: extracting butane as extractant at-0.1 Mpa and 20-50deg.C, and then reducing the pressure of the extractive solution to-0.1 Mpa, separating butane from oleum Bulbus Allii to obtain oleum Bulbus Allii;
the conditions of the molecular distillation are as follows: the dripping speed of the garlic oil is 1-3 drops/3 seconds at 50-100 ℃ and the vacuum degree is 30-35Pa,
the liquid phase purification refers to: dissolving ajoene fraction collected by molecular distillation with 5-8 times of ethanol, filtering, purifying with chromatography, eluting with 20% ethyl acetate as eluent at 10-30mL/min, collecting ajoene separating liquid, and concentrating under reduced pressure at 50-70deg.C to obtain ajoene.
2. The method according to claim 1, wherein the garlic clove is pretreated at a pressure of 520-550MPa.
3. The method according to claim 1, wherein the garlic clove is pretreated at a pressure of 550-580MPa.
4. A method according to any one of claims 1-3, characterized in that the garlic cloves are pre-treated for a period of 15-20min.
5. A method according to any one of claims 1-3, characterized in that the pressure conditions of the enzymatic reaction are 150-180MPa.
6. A method according to any one of claims 1-3, characterized in that the pre-treated garlic cloves are placed below-4 ℃ for use before they are crushed.
7. A method according to any one of claims 1-3, characterized in that the pre-treated garlic cloves are crushed at a temperature of-4 ℃ to-20 ℃.
8. The method according to claim 1, wherein Innoval ODS-2 is used as a filler in the preparative chromatography.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1320387A (en) * | 2001-04-25 | 2001-11-07 | 潘见 | Process for preparing deodourized garlic cloves |
| CN101591587A (en) * | 2009-04-28 | 2009-12-02 | 江苏大学 | A kind of method of extracting onion essential oil through ultrahigh pressure regulation of alliinase reaction |
| CN106518739A (en) * | 2016-10-31 | 2017-03-22 | 中山大学 | Method for extracting and separating ajoene from black garlic |
| CN107183596A (en) * | 2017-06-07 | 2017-09-22 | 江南大学 | The processing method of allinnase in a kind of radio frequency passivation garlic |
| CN107473996A (en) * | 2017-08-21 | 2017-12-15 | 深圳市阿霍烯生物科技有限公司 | The extraction separation method of 4,5,9-trithiadodeca-1,6,11-triene 9-oxide in a kind of garlic |
| CN107501146A (en) * | 2017-08-21 | 2017-12-22 | 深圳市阿霍烯生物科技有限公司 | A kind of method that molecular distillation isolates and purifies 4,5,9-trithiadodeca-1,6,11-triene 9-oxide in garlic P.E |
| CN114133346A (en) * | 2021-12-06 | 2022-03-04 | 无锡江大百泰科技有限公司 | Alliin and allicin combined extraction and preparation method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2377219A (en) * | 2001-07-04 | 2003-01-08 | Neem Biotech Ltd | Preparation of allicin |
-
2022
- 2022-04-19 CN CN202210412124.5A patent/CN114836485B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1320387A (en) * | 2001-04-25 | 2001-11-07 | 潘见 | Process for preparing deodourized garlic cloves |
| CN101591587A (en) * | 2009-04-28 | 2009-12-02 | 江苏大学 | A kind of method of extracting onion essential oil through ultrahigh pressure regulation of alliinase reaction |
| CN106518739A (en) * | 2016-10-31 | 2017-03-22 | 中山大学 | Method for extracting and separating ajoene from black garlic |
| CN107183596A (en) * | 2017-06-07 | 2017-09-22 | 江南大学 | The processing method of allinnase in a kind of radio frequency passivation garlic |
| CN107473996A (en) * | 2017-08-21 | 2017-12-15 | 深圳市阿霍烯生物科技有限公司 | The extraction separation method of 4,5,9-trithiadodeca-1,6,11-triene 9-oxide in a kind of garlic |
| CN107501146A (en) * | 2017-08-21 | 2017-12-22 | 深圳市阿霍烯生物科技有限公司 | A kind of method that molecular distillation isolates and purifies 4,5,9-trithiadodeca-1,6,11-triene 9-oxide in garlic P.E |
| CN114133346A (en) * | 2021-12-06 | 2022-03-04 | 无锡江大百泰科技有限公司 | Alliin and allicin combined extraction and preparation method |
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Address after: 250353 University Road, Changqing District, Ji'nan, Shandong Province, No. 3501 Patentee after: Qilu University of Technology (Shandong Academy of Sciences) Country or region after: China Patentee after: Shandong garlic Yuan Tang Bio Technology Co.,Ltd. Address before: 250353 University Road, Changqing District, Ji'nan, Shandong Province, No. 3501 Patentee before: Qilu University of Technology Country or region before: China Patentee before: Shandong garlic Yuan Tang Bio Technology Co.,Ltd. |