CN105820097A - Method for extracting isoalliin in onion cells through nitrogen pressurization and ethanol substitution - Google Patents
Method for extracting isoalliin in onion cells through nitrogen pressurization and ethanol substitution Download PDFInfo
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- CN105820097A CN105820097A CN201610336758.1A CN201610336758A CN105820097A CN 105820097 A CN105820097 A CN 105820097A CN 201610336758 A CN201610336758 A CN 201610336758A CN 105820097 A CN105820097 A CN 105820097A
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Abstract
The invention belongs to the field of agricultural product deep processing and discloses a method for extracting isoalliin in onion cells through nitrogen pressurization and ethanol substitution .The method includes the sequential extraction steps that isoalliin is extracted through onion preprocessing, nitrogen pressurization and ethanol substitution, an extraction solution is concentrated through vacuum distillation under nitrogen protection, isoalliin is separated from polysaccharide through cation exchange resin, isoalliin is obtained through re-crystallization .The method is characterized in that onions are not smashed, high-temperature or microwave heating is not needed, enzymes in the onions are not inactivated, ethanol is pressed into onion cells under nitrogen protection to substitute for isoalliin in cytoplasm, enzyme in vacuole is not extracted, enzymatic hydrolysis of isoalliin does not occur, and pure isoalliin rather than enzymolysis product allicin (onion essence oil) can be obtained .The extraction rate of isoalliin can reach 0.426 mg/g and is 70% of the theoretical extraction rate, and isoalliin with the purity being 99% is obtained through re-crystallization.
Description
Technical field
The invention belongs to agricultural product intensive processing technology, be a kind of method extracting active ingredient from Bulbus Allii Cepae, be specifically related to a kind of nitrogen pressurization ethanol replacement and extract the method for different alliin in onion cells.
Background technology
Bulbus Allii Cepae, belongs to herbaceos perennial, is the one of Liliaceae allium, has the function of medicine-food two-purpose.Modern medicine study shows, Bulbus Allii Cepae has sterilization, Adjust-blood lipid, suppression platelet aggregation, blood pressure lowering, prevents and treats atherosclerosis, blood sugar lowering, the effect such as anticancer, especially has prevention cardiovascular disease and the biological activity of the aspect such as anticancer.
Main active ingredient in Bulbus Allii Cepae is different alliin, the most trans SACS, S-allyl-L-cysteinesulfoxide, i.e. alliumcepa.It has inhibitory or killing effect to multiple pathogen, it it is natural phytocide, to the most bacillary, fungoid and anti-protozoal infection, treatment and prevention are all had to be worth, there is in terms of biological medicine research and development the biggest effect, be referred to as " one of 21st century the greatest magical discovery in natural medicine field " by world medical circle.This polar monomer (different) alliin caused the great attention of scholars in recent years.
Those skilled in the art is known: different alliin is in the Cytoplasm of onion cells; allinase and tear-gas factor enzyme are in the vacuole of cell; in complete cell, both keep apart, only after cell breakage; enzyme r e lease in vacuole is out; the different alliin of allinase enzymolysis, generates a small amount of sulfur-containing compound, referred to as allicin (thiosulfinate; TS) and the tear-gas factor different alliin of enzyme enzymolysis generate a large amount of volatility tear-gas factor (LF), be a kind of measure of Bulbus Allii Cepae self-protection.Therefore, different alliin to be extracted, cannot contact with enzyme.
The extraction conditions of the different alliin of sterling is the harshest, technical difficulty is the biggest, its chemical property extremely unstable, easily changes into allicin and gaseous matter with the allinase in Bulbus Allii Cepae and tear-gas factor enzyme reaction in the case of smashing breakage to pieces, easily oxidation deterioration in air and high temperature.This makes the international market price of the different alliin of high-purity much more expensive, and the price of different alliin standard specimen is 1200 yuan/5mg.Therefore, high-purity different alliin goods and high-purity medicinal different alliin have high commercial value.
The extracting method of onion essential oil mainly has steam distillation, solvent extraction method, supercritical CO at present2Extraction and enzyme-deactivating method.
Steam distillation: the onion essential oil that water at atmospheric pressure vapor distillation method obtains is ripe onion flavors, Main Ingredients and Appearance is propyl group, acrylic disulfide, is not different alliin.Decompression method can prepare the quintessence oil allicin (TS) containing fresh onion flavor, but yield only reaches 0.113 ‰.
Solvent extraction method: with ether, alcohol solvent extraction time, it is thus achieved that be containing methyl, propyl disulfide compound, be not different alliin, during with dichloromethane for extraction, obtain methyl, acrylic allicin (TS), yield is about 3.5 times of steam distillation.
Supercritical CO2Extraction: since the nineties, this method is widely used in the extraction of onion essential oil, it is thus achieved that have the quintessence oil allicin (TS) of fresh onion flavor, and quintessence oil yield is 0.23 ‰～0.38 ‰.
Enzyme-deactivating method: first with boiling water inactivation allinase and tear-gas factor synzyme before Bulbus Allii Cepae is pulled an oar, again with the different alliin of dichloroethane extraction, then different alliin is decomposed with exogenous enzyme allinase, prepare allicin, avoid becoming the tear-gas factor to vapor away, the quintessence oil yield of report is 0.921 ‰, and wherein allicin (TS) content is 71.3%.
The technical scheme disclosed in application for a patent for invention (Application No. 201410622904.8, Publication No. CN104498351A, invention entitled low temperature isolation air allicin process units and using method thereof) that Xinjiang Ailexin Pharmacy Co., Ltd. submits to is to split conjunction biosynthesis allicin by allinase catalysis alliin, microfiltration and ultrafiltration purification allicin, cycling extraction and molecular distillation Liquid liquid Separation, physics is degerming and argon filling is pre-installed, isolation air, reduce synthesis temperature, it is to avoid decompose.
The application for a patent for invention of the Application No. 201410354130.5 that Yunnan Prov Agriculture University submits to, disclose a kind of method simultaneously preparing garlic polysaccharide and alliin, adopt the technical scheme that applied microwave inactivates allinase, the exchange of membrance separation, ion and crystallization, prepare garlic polysaccharide and alliin simultaneously.
The application for a patent for invention of the Application No. 201410296457.1 that Qilu University of Technology submits to, disclose a kind of method that ultrasonic wave added aqueous two-phase extracts alliin, adopt the technical scheme that and Bulbus Allii powder is added in aqueous two-phase, buffer solution and Small molecule organic solvents with inorganic salt prepare aqueous two-phase, regulation aqueous two-phase pH value is 2.0 ~ 4.0, supersound extraction, centrifugation, stratification, has alliin in two-phase.
The application for a patent for invention of Application No. 201410101556.X that BannerBio Nutraceutical Inc. submits to proposes a kind of method preparing alliin microemulsion, use is affected because alliin is insoluble in organic solvent, microemulsion prepared by proposition water, oil and surfactant, stablizes and can prevent air oxidation.
Patent No. 201210322853.8 patent of invention of Jiangsu University, discloses a kind of method for ultrasonic assisted extraction of alliin in garlic.Adopting the technical scheme that and Bulbus Allii is used heating in water bath enzyme denaturing, smash, impulse ultrasound extracts, and Ultra filtration membrane obtains alliin.
In recent years, it is also proposed that some new methods, such as ionic liquid applied research in onion essential oil is produced, the repeatedly rectification coupling extraction of onion essential oil, supercritical liquid extraction technique application in onion essential oil high efficiency extraction.In prepared onion essential oil, main matter is sulfur-containing compound, i.e. thiosulfinate TS rather than different alliin sterling.
In sum, the extraction of onion essential oil uses two class methods: enzymatic isolation method prepares allicin and enzyme denaturing method prepares different alliin.The shortcoming of enzymatic isolation method is to generate a large amount of volatility tear-gas factors, the different alliin that serious waste is expensive；The shortcoming of enzyme denaturing method is that high temperature causes the different alliin of part to go bad.
Summary of the invention
It is an object of the invention to provide a kind of nitrogen pressurization ethanol replacement and extract the method for different alliin in onion cells; the method at room temperature nitrogen protection preparation different alliin of sterling; do not destroy cellularity; there is not enzymolysis; nitrogen is protected, and completely cuts off air, and room temperature is extracted; avoid high temperature, prevent oxidation deterioration.
To achieve these goals; the technical scheme that the present invention takes is as follows: a kind of nitrogen pressurization ethanol replacement extracts the method for different alliin in onion cells, extraction step successively: different alliin is extracted in Bulbus Allii Cepae pretreatment, nitrogen pressurization ethanol replacement, nitrogen protection decompression distillation and concentration extracting solution, cation exchange resin separate different alliin and polysaccharide, are recrystallized to give different alliin；Concrete grammar is as follows:
(1) Bulbus Allii Cepae pretreatment
Bulbus Allii Cepae is removed crust, and crop truncates, and one slices into halves, and is washed with deionized water clean otch immediately, it is to avoid nickase remains, and is divided into lobe；It is the ratio of 1/3 according to Bulbus Allii Cepae mass parts/dehydrated alcohol parts by volume, after being weighed by Bulbus Allii Cepae, puts into rustless steel extraction pot.
(2) nitrogen pressurization ethanol replacement extracts different alliin
Add dehydrated alcohol to rustless steel extraction pot, seal, protect with nitrogen；Nitrogen pressure is regulated to 0.1～0.7MPa with the air relief valve of nitrogen cylinder; open extraction pot intake valve; the nitrogen that pressure is 0.1～0.7MPa is passed through in extraction pot; make extraction pot pressure reach to set pressure that is 0.1～0.7MPa, dwell time 0.5～2.5h, open extraction pot top vent valve; pressure release is to after 0.1MPa; extracting solution is all released from bottom sampling valve, is transferred quickly in Rotary Evaporators, protects with nitrogen.
(3) nitrogen protection decompression distillation and concentration extracting solution
With Rotary Evaporators decompression distillation and concentration extracting solution, vacuum 0.08MPa, temperature 50 C, Rotary Evaporators is passed through nitrogen isolation air, prevents different alliin high-temperature oxydation, after reclaiming about 95% to alcohol solvent, stop concentrating, obtain thick yellow liquid.
(4) nitrogen atmosphere cation exchange resin separates different alliin and polysaccharide
Thick yellow liquid 20g is added sucking filtration after 200mL ultra-pure water dissolves, removes the major part insoluble substances such as fiber and obtain faint yellow clear liquid；Measure the 732 resin dress posts that 200mL handles well, colourless to effluent with ultrapure water；Again by faint yellow clear liquid HCl regulation to pH be 2, under nitrogen atmosphere with the flow velocity loading of 0.5BV/h；Rinsing extremely ink formula reagent detection with the hydrochloric acid solution that pH is 3～4 with the flow velocity of 1BV/h afterwards to be negative, whether hydrochloric acid solution has aminoacid to reveal with ninhydrin solution detection during rinsing during affination；Whether clean water pillar to pH be 7, clean water during still with ninhydrin solution detection affination during have aminoacid reveal, effluent is polysaccharide solution if using instead again；Afterwards with 0.5mol/L ammonia spirit by different alliin eluting.
(5) it is recrystallized to give different alliin
Ammonia eluent is reduced pressure under nitrogen protection distillation, vacuum 0.08MPa, temperature 50 C, obtain concentrated solution, dissolve concentrated solution with ultra-pure water, again filter upper prop, use ammonia eluting, distillation of again reducing pressure, add dehydrated alcohol, be recrystallized to give the different alliin of sterling.
Step (1): during Bulbus Allii Cepae pretreatment, its substantive distinguishing features is not crush Bulbus Allii Cepae, does not heats, not enzyme denaturing；Step (2) its substantive distinguishing features is: under nitrogen pressure, ethanol enters in cell by cell wall, Cytoplasm is cemented out, different alliin is diffused in extracellular ethanol therewith, it is dissolved in ethanol water, due to the obstruction of fine and close tonoplast, ethanol does not enters in vacuole, and enzyme will not diffuse out；Step (3) is passed through in Rotary Evaporators nitrogen isolation air, prevents different alliin high-temperature oxydation；Step (4) its substantive distinguishing features is: be dissolved in by distillate in appropriate ultra-pure water, and with the 732 cationic exchange resin adsorption separating polyoses processed and different alliin after filtration, effluent is polysaccharide, and different alliin adsorbs on resin, collects with ammonia eluting.
Present invention nitrogen pressurization dehydrated alcohol replacement extraction method individually extracts different alliin, is the at room temperature nitrogen protection preparation different alliin of sterling, does not destroy cellularity, enzymolysis does not occur, and nitrogen is protected, and completely cuts off air, and room temperature is extracted, and prevents oxidation deterioration.Feature is not crush Bulbus Allii Cepae; not high temperature or microwave heating; do not inactivate the enzyme in Bulbus Allii Cepae; under nitrogen protection; ethanol is pressed in onion cells the different alliin displacing in Cytoplasm, but does not extract the enzyme in vacuole, the enzyme digestion reaction of different alliin does not occur; the different alliin of sterling rather than enzymatic hydrolysate allicin (onion essential oil) can be obtained.Different alliin extraction ratio can reach 0.426mg/g, is the 70% of theoretical extraction rate；Through being recrystallized to give the different alliin that purity is 99%.
Accompanying drawing explanation
Fig. 1 is different alliin standard substance working curve diagram；
Fig. 2 is different alliin standard substance high-efficient liquid phase chromatogram, and wherein peak 4 is alliin:
Fig. 3 is to extract pressure 0.6MPa, and the high-efficient liquid phase chromatogram of the extracting solution of extraction time 1.5, wherein peak 5 is different alliin；
Fig. 4 is under different pressures, the graph of relation of different alliin content C and extraction time T in extracting solution；
Fig. 5 is different alliin content C, pressure P and the change curve of extraction time T in extracting solution.
Detailed description of the invention
This part provides 35 comparative experimental example of the present invention, and main contrast's test data is extraction time and the extraction pressure of step 2, and wherein extraction time selects: 0.5h, 1h, 1.5h, 2h and five time values of 2.5h；Extraction pressure selects: 0.1MPa, 0.2MPa, 0.3MPa, 0.4MPa, 0.5MPa, 0.6MPa and seven force value of 0.7MPa；In 35 tests, each test uses a Bulbus Allii Cepae sample i.e. through the 200g Bulbus Allii Cepae of pretreatment；Under the pressure of 0.1MPa, make the test example of 0.5h, 1h, 1.5h, 2h and five time values of 2.5h respectively, under the pressure of 0.2MPa, make the test example of 0.5h, 1h, 1.5h, 2h and five time values of 2.5h the most respectively, by that analogy, 35 test examples are completed.
Test example 1；
1, Bulbus Allii Cepae pretreatment
Bulbus Allii Cepae is removed crust, and crop truncates, and one cuts open two halves, is divided into scattered lobe, first rinses 3 times with clear water, then is washed with deionized 3 times, cleans otch juice, it is to avoid otch residual enzyme and there is enzyme digestion reaction.
2, different alliin is extracted
Selecting dehydrated alcohol to make solvent, nitrogen pressurizes, and is put into by 200g Bulbus Allii Cepae lobe equipped with in the rustless steel extraction pot of 600mL ethanol, extracts with nitrogen protection, pressurization closed.Regulating the nitrogen pressure of nitrogen cylinder to 0.1MPa with air relief valve, open extraction pot intake valve, be passed through in extraction pot by the nitrogen that pressure is 0.1MPa, keeping extraction pot pressure is 0.1MPa, dwell time 0.5h；Then open extraction pot vent valve pressure release, then extracting solution is all released from extraction pot sampling valve port, be transferred quickly in Rotary Evaporators, protect with nitrogen.
3, the process of extracting solution
3.1 take out 2mL from extracting solution, and centrifugal 15min under 10000rad/min, liquid-10 DEG C preservation after being centrifuged, for efficient liquid phase chromatographic analysis；
3.2 by extracting solution nitrogen protect under conditions of, under the process conditions of temperature 50 C vacuum 0.08MPa, with Rotary Evaporators decompression distillation, remove 95% alcohol solvent, obtain yellow, viscous liquid.The nitrogen isolation air being passed through in Rotary Evaporators, prevents different alliin high-temperature oxydation.
4, nitrogen atmosphere cation exchange resin separates different alliin and polysaccharide
Rotation is steamed the yellow, viscous liquid 20g obtained by 4.1 adds sucking filtration after 200mL ultra-pure water dissolves, and removes the major part insoluble substances such as fiber and obtains faint yellow clear liquid.Measure the 732 resin dress posts that 200mL handles well, colourless to effluent with ultrapure water, again by above-mentioned faint yellow clear liquid HCl regulation to pH=2, under nitrogen atmosphere with the flow velocity loading of 0.5BV/h, rinse to ink formula reagent to detect be negative (whether period has aminoacid to reveal with ninhydrin solution detection during affination) with the hydrochloric acid solution of pH3.5 with the flow velocity of 1BV/h afterwards,, to pH=7, effluent is polysaccharide solution to use clean water pillar (period, whether aminoacid to be detected was revealed) instead.Afterwards with 0.5mol/L ammonia spirit by different alliin eluting, elution process detects with TLC, by identical flow point enrichment.
The detection (TLC) of 4.2 different alliins: dry up after point sample on chromatographic sheet with capillary glass tube, with n-butyl alcohol: glacial acetic acid: water=4:1:1 launches as developping solution, take out during about 1cm to forward position, dry up, dip ninhydrin solution, dry up 75 DEG C of drying 10min in rear baking oven, observe speckle and the colour developing situation of speckle.
The HPLC of 4.3 different alliin content measures: use reversed-phase high-performance liquid chromatography to detect the content of alliin different in extract.Chromatographic column is ODSC18 (250mm × 4.6mm, 5 μm), diode dot matrix detector, detects wavelength 210nm, and the volume ratio of flowing phase is methanol: aqueous systems=3:7, flow velocity is 0.8mL/min, and column temperature is 25 DEG C, sample size 20 μ L.
The impact on different alliin separating degree of this effects difference proportion of mobile phase, employing methanol: water=1:99～99:1(volume ratio) in the range of.Result represents methanol: during aqueous systems=3:7, separating effect is best.
5, it is recrystallized to give different alliin
Ammonia eluent is reduced pressure under nitrogen protection distillation, vacuum 0.08MPa, temperature 50 C, obtain concentrated solution, dissolve concentrated solution with ultra-pure water, again filter upper prop, use ammonia eluting, distillation of again reducing pressure, add dehydrated alcohol, be recrystallized to give the different alliin of sterling.
Test example 2；
1, Bulbus Allii Cepae pretreatment: with test example 1；
2, different alliin is extracted: dwell time 1.0h；Remaining with test example 1；
3, the process of extracting solution: with test example 1；
4, cation exchange resin separates different alliin and polysaccharide: with test example 1；
5, it is recrystallized to give different alliin；With test example 1.
Test example 3；
1, Bulbus Allii Cepae pretreatment: with test example 1；
2, different alliin is extracted: dwell time 1.5h；Remaining with test example 1；
3, the process of extracting solution: with test example 1；
4, cation exchange resin separates different alliin and polysaccharide: with test example 1；
5, it is recrystallized to give different alliin；With test example 1.
Test example 4；
1, Bulbus Allii Cepae pretreatment: with test example 1；
2, different alliin is extracted: dwell time 2.0h；Remaining with test example 1；
3, the process of extracting solution: with test example 1；
4, cation exchange resin separates different alliin and polysaccharide: with test example 1；
5, it is recrystallized to give different alliin；With test example 1.
Test example 5；
1, Bulbus Allii Cepae pretreatment: with test example 1；
2, different alliin is extracted: dwell time 2.5h；Remaining with test example 1；
3, the process of extracting solution: with test example 1；
4, cation exchange resin separates different alliin and polysaccharide: with test example 1；
5, it is recrystallized to give different alliin；With test example 1.
Test example 6；
1, Bulbus Allii Cepae pretreatment: with test example 1；
2, extracting different alliin: regulate the nitrogen pressure of nitrogen cylinder to 0.2MPa with air relief valve, open extraction pot intake valve, be passed through in extraction pot by the nitrogen that pressure is 0.2MPa, keeping extraction pot pressure is 0.2MPa, dwell time 0.5h；Remaining with test example 1；
3, the process of extracting solution: with test example 1；
4, cation exchange resin separates different alliin and polysaccharide: with test example 1；
5, it is recrystallized to give different alliin；With test example 1.
Test example 7；In step 2: dwell time 1.0h, remaining same test example 6.Other step is with test example 1.
Test example 8；In step 2, dwell time 1.5h, remaining same test example 6.Other step is with test example 1.
Test example 9；In step 2, dwell time 2.0h, remaining same test example 6.Other step is with test example 1.
Test example 10；In step 2, dwell time 2.5h, remaining same test example 6.Other step is with test example 1.
Test example 11；In step 2: regulate the nitrogen pressure of nitrogen cylinder to 0.3MPa with air relief valve, open extraction pot intake valve, be passed through in extraction pot by the nitrogen that pressure is 0.3MPa, keeping extraction pot pressure is 0.3MPa, dwell time 0.5h；Remaining with test example 1.Other step is with test example 1.
Test example 12；In step 2: dwell time 1.0h, remaining same test example 11.Other step is with test example 1.
Test example 13；In step 2: dwell time 1.5h, remaining same test example 11.Other step is with test example 1.
Test example 14；In step 2: dwell time 2.0h, remaining same test example 11.Other step is with test example 1.
Test example 15；In step 2: dwell time 2.5h, remaining same test example 11.Other step is with test example 1.
Test example 16；In step 2: regulate the nitrogen pressure of nitrogen cylinder to 0.4MPa with air relief valve, open extraction pot intake valve, be passed through in extraction pot by the nitrogen that pressure is 0.4MPa, keeping extraction pot pressure is 0.4MPa, dwell time 0.5h；Remaining with test example 1.Other step is with test example 1.
Test example 17；In step 2: dwell time 1.0h, remaining same test example 16.Other step is with test example 1.
Test example 18；In step 2: dwell time 1.5h, remaining same test example 16.Other step is with test example 1.
Test example 19；In step 2: dwell time 2.0h, remaining same test example 16.Other step is with test example 1.
Test example 20；In step 2: dwell time 1.5h, remaining same test example 16.Other step is with test example 1.
Test example 21；In step 2: regulate the nitrogen pressure of nitrogen cylinder to 0.5MPa with air relief valve, open extraction pot intake valve, be passed through in extraction pot by the nitrogen that pressure is 0.5MPa, keeping extraction pot pressure is 0.5MPa, dwell time 0.5h；Remaining with test example 1.Other step is with test example 1.
Test example 22；In step 2: dwell time 1.0h, remaining same test example 21.Other step is with test example 1.
Test example 23；In step 2: dwell time 1.5h, remaining same test example 21.Other step is with test example 1.
Test example 24；In step 2: dwell time 2.0h, remaining same test example 21.Other step is with test example 1.
Test example 25；In step 2: dwell time 2.5h, remaining same test example 21.Other step is with test example 1.
Test example 26；In step 2: regulate the nitrogen pressure of nitrogen cylinder to 0.6MPa with air relief valve, open extraction pot intake valve, be passed through in extraction pot by the nitrogen that pressure is 0.6MPa, keeping extraction pot pressure is 0.6MPa, dwell time 0.5h；Remaining with test example 1.Other step is with test example 1.
Test example 27；In step 2: dwell time 1.0h, remaining same test example 26.Other step is with test example 1.
Test example 28；In step 2: dwell time 1.5h, remaining same test example 26.Other step is with test example 1.
Test example 29；In step 2: dwell time 2.0h, remaining same test example 26.Other step is with test example 1.Test example 30；In step 2: dwell time 2.5h, remaining same test example 26.Other step is with test example 1.Test example 31；In step 2: regulate the nitrogen pressure of nitrogen cylinder to 0.7MPa with air relief valve, open extraction pot intake valve, be passed through in extraction pot by the nitrogen that pressure is 0.7MPa, keeping extraction pot pressure is 0.7MPa, dwell time 0.5h；Remaining with test example 1.Other step is with test example 1.
Test example 32；In step 2: dwell time 1.0h, remaining same test example 31.Other step is with test example 1.
Test example 33；In step 2: dwell time 1.5h, remaining same test example 31.Other step is with test example 1.Test example 34；In step 2: dwell time 2.0h, remaining same test example 31.Other step is with test example 1.
Test example 35；In step 2: dwell time 2.5h, remaining same test example 31.Other step is with test example 1.
Test result analysis is as follows:
1, the straight alcohol adding extraction pot in extracting is 600mL, and the extracting solution obtained is 640 ~ 650mL, has portion of water to leach into from onion cells in ethanol solution during illustrating to extract, and part ethanol enters in cell and displaced moisture.
2, different alliin standard working curve
It is 25 μ g/mL that different alliin standard substance are configured to concentration respectively, 37.5 μ g/mL, 50 μ g/mL, 62.5 μ g/mL, 75 μ g/mL, 100 μ g/mL, 125 μ g/mL, the aqueous solution of 150 μ g/mL, use high performance liquid chromatography to measure its integral area respectively, with concentration as abscissa, integral area is that vertical coordinate makes working curve as shown in Figure 1.
3, different alliin standard substance and extraction sample high-efficient liquid phase chromatogram
Different alliin standard substance high-efficient liquid phase chromatogram is as in figure 2 it is shown, peak 4 is different alliin；Being the extracting solution high-efficient liquid phase chromatogram extracting pressure 0.6MPa, extraction time 1.5h shown in Fig. 3, peak 5 is different alliin.
4, different alliin content and extract pressure and the relation of extraction time in extracting solution
Under different pressures in extracting solution different alliin content with extraction time variation relation as shown in Figure 4, extraction ratio no longer rises after 1.5h, different alliin concentration reaches balance in the ethanol solution inside and outside onion cells.Pressure on the impact of extraction ratio as it is shown in figure 5, during pressure 0.6MPa, extraction ratio is the highest, when pressure reaches 0.7MPa, extraction ratio declines on the contrary, and reason is that the vacuole in cell ruptures, out, by different for part alliin enzymolysis, extraction ratio declines substantially enzyme r e lease than 0.6MP.
Claims (1)
1. nitrogen pressurization ethanol replacement extracts the method for different alliin in onion cells, it is characterised in that extraction step successively: different alliin is extracted in Bulbus Allii Cepae pretreatment, nitrogen pressurization ethanol replacement, nitrogen protection decompression distillation and concentration extracting solution, nitrogen atmosphere cation exchange resin separate different alliin and polysaccharide, are recrystallized to give different alliin；Concrete grammar is as follows:
(1) Bulbus Allii Cepae pretreatment
Bulbus Allii Cepae is removed crust, and crop truncates, and one slices into halves, and is washed with deionized water clean otch immediately, it is to avoid nickase remains, and is divided into lobe；It is the ratio of 1/3 according to Bulbus Allii Cepae mass parts/dehydrated alcohol parts by volume, after being weighed by Bulbus Allii Cepae, puts into rustless steel extraction pot；
(2) nitrogen pressurization ethanol replacement extracts different alliin
Add dehydrated alcohol to rustless steel extraction pot, seal, protect with nitrogen；Nitrogen pressure is regulated to 0.1～0.7MPa with the air relief valve of nitrogen cylinder; open extraction pot intake valve; the nitrogen that pressure is 0.1～0.7MPa is passed through in extraction pot; make extraction pot pressure reach 0.1～0.7MPa, the dwell time 0.5～2.5h, open extraction pot top vent valve; pressure release is to after 0.1MPa; extracting solution is all released from bottom sampling valve, is transferred quickly in Rotary Evaporators, protects with nitrogen；
(3) nitrogen protection decompression distillation and concentration extracting solution
With Rotary Evaporators decompression distillation and concentration extracting solution, vacuum 0.08MPa, temperature 50 C, Rotary Evaporators is passed through nitrogen isolation air, after reclaiming about 95% to alcohol solvent, stops concentrating, obtain thick yellow liquid；
(4) nitrogen atmosphere cation exchange resin separates different alliin and polysaccharide
Thick yellow liquid 20g is added sucking filtration after 200mL ultra-pure water dissolves, removes the major part insoluble substances such as fiber and obtain faint yellow clear liquid；Measure the 732 resin dress posts that 200mL handles well, colourless to effluent with ultrapure water；Again by faint yellow clear liquid HCl regulation to pH be 2, under nitrogen atmosphere with the flow velocity loading of 0.5BV/h；Rinsing extremely ink formula reagent detection with the hydrochloric acid solution that pH is 3～4 with the flow velocity of 1BV/h afterwards to be negative, whether hydrochloric acid solution has aminoacid to reveal with ninhydrin solution detection during rinsing during affination；Whether use instead more still has different alliin to reveal with ninhydrin solution detection during clean water pillar is 7, clean water to pH during affination；Afterwards with 0.5mol/L ammonia spirit by different alliin eluting；
(5) it is recrystallized to give different alliin
Ammonia eluent is reduced pressure under nitrogen protection distillation, vacuum 0.08MPa, temperature 50 C, obtain concentrated solution, dissolve concentrated solution with ultra-pure water, again filter upper prop, use ammonia eluting, distillation of again reducing pressure, add dehydrated alcohol, be recrystallized to give the different alliin of sterling.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106307436A (en) * | 2016-08-17 | 2017-01-11 | 中国农业科学院农产品加工研究所 | Processing and preserving method of convenient marinated meat dishes |
| CN110615748A (en) * | 2019-10-15 | 2019-12-27 | 兰州大学 | Alliin extraction process optimization method |
| CN111587883A (en) * | 2020-06-04 | 2020-08-28 | 兰州大学 | Plant pesticide and preparation method and use method thereof |
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| JP2009254344A (en) * | 2008-03-28 | 2009-11-05 | House Foods Corp | Method for separating lfs and prencso contained in onion |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106307436A (en) * | 2016-08-17 | 2017-01-11 | 中国农业科学院农产品加工研究所 | Processing and preserving method of convenient marinated meat dishes |
| CN110615748A (en) * | 2019-10-15 | 2019-12-27 | 兰州大学 | Alliin extraction process optimization method |
| CN110615748B (en) * | 2019-10-15 | 2022-03-22 | 兰州大学 | Alliin extraction process optimization method |
| CN111587883A (en) * | 2020-06-04 | 2020-08-28 | 兰州大学 | Plant pesticide and preparation method and use method thereof |
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| CN105820097B (en) | 2017-10-13 |
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