CN109170796B - Method for extracting thaumatin from African arrowroot - Google Patents
Method for extracting thaumatin from African arrowroot Download PDFInfo
- Publication number
- CN109170796B CN109170796B CN201811141257.3A CN201811141257A CN109170796B CN 109170796 B CN109170796 B CN 109170796B CN 201811141257 A CN201811141257 A CN 201811141257A CN 109170796 B CN109170796 B CN 109170796B
- Authority
- CN
- China
- Prior art keywords
- thaumatin
- type
- aqueous solution
- solution
- sepharose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 235000010436 thaumatin Nutrition 0.000 title claims abstract description 109
- 239000000892 thaumatin Substances 0.000 title claims abstract description 109
- 238000000034 method Methods 0.000 title claims abstract description 34
- 244000292211 Canna coccinea Species 0.000 title claims abstract description 22
- 235000005273 Canna coccinea Nutrition 0.000 title claims abstract description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 48
- 238000001728 nano-filtration Methods 0.000 claims abstract description 30
- 239000011543 agarose gel Substances 0.000 claims abstract description 24
- 238000001816 cooling Methods 0.000 claims abstract description 21
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000003480 eluent Substances 0.000 claims abstract description 19
- 238000001914 filtration Methods 0.000 claims abstract description 18
- 239000003729 cation exchange resin Substances 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 14
- 239000005457 ice water Substances 0.000 claims abstract description 12
- 238000003756 stirring Methods 0.000 claims abstract description 12
- 239000012530 fluid Substances 0.000 claims abstract description 9
- 239000012528 membrane Substances 0.000 claims abstract description 9
- 238000002425 crystallisation Methods 0.000 claims abstract description 8
- 230000008025 crystallization Effects 0.000 claims abstract description 8
- 238000001035 drying Methods 0.000 claims abstract description 7
- 238000004537 pulping Methods 0.000 claims abstract description 7
- 238000004140 cleaning Methods 0.000 claims abstract description 6
- 239000013078 crystal Substances 0.000 claims abstract description 6
- 238000005406 washing Methods 0.000 claims abstract description 6
- 230000001376 precipitating effect Effects 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 36
- 239000007864 aqueous solution Substances 0.000 claims description 31
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 24
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 21
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical group [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 18
- 229920002684 Sepharose Polymers 0.000 claims description 16
- 238000001556 precipitation Methods 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 239000003513 alkali Substances 0.000 claims description 12
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 10
- 235000019270 ammonium chloride Nutrition 0.000 claims description 9
- 239000011552 falling film Substances 0.000 claims description 9
- 238000000926 separation method Methods 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 8
- 239000012564 Q sepharose fast flow resin Substances 0.000 claims description 7
- 235000013399 edible fruits Nutrition 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000001103 potassium chloride Substances 0.000 claims description 5
- 235000011164 potassium chloride Nutrition 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 229920002271 DEAE-Sepharose Polymers 0.000 claims description 4
- 230000001476 alcoholic effect Effects 0.000 claims 1
- 239000000047 product Substances 0.000 abstract description 24
- 238000011068 loading method Methods 0.000 abstract description 7
- 238000005227 gel permeation chromatography Methods 0.000 abstract description 6
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 3
- 230000002860 competitive effect Effects 0.000 abstract description 2
- 239000012465 retentate Substances 0.000 abstract 1
- 101710135233 Thaumatin I Proteins 0.000 description 9
- 101710135323 Thaumatin II Proteins 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 239000000463 material Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 238000010812 external standard method Methods 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 239000003456 ion exchange resin Substances 0.000 description 4
- 229920003303 ion-exchange polymer Polymers 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000007863 gel particle Substances 0.000 description 3
- 239000000049 pigment Substances 0.000 description 3
- 238000007670 refining Methods 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 244000299461 Theobroma cacao Species 0.000 description 2
- 235000019658 bitter taste Nutrition 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 235000013599 spices Nutrition 0.000 description 2
- 229960004793 sucrose Drugs 0.000 description 2
- 244000205574 Acorus calamus Species 0.000 description 1
- 235000011996 Calamus deerratus Nutrition 0.000 description 1
- 241001061906 Caragana Species 0.000 description 1
- 206010013911 Dysgeusia Diseases 0.000 description 1
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 description 1
- 241000862513 Kandelia Species 0.000 description 1
- 235000009470 Theobroma cacao Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 235000019606 astringent taste Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 229960001948 caffeine Drugs 0.000 description 1
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 description 1
- 229940023913 cation exchange resins Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000019219 chocolate Nutrition 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 235000016213 coffee Nutrition 0.000 description 1
- 235000013353 coffee beverage Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 235000013615 non-nutritive sweetener Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
- 239000001648 tannin Substances 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/10—Natural spices, flavouring agents or condiments; Extracts thereof
Landscapes
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Peptides Or Proteins (AREA)
Abstract
A method for extracting thaumatin from African arrowroot comprises the following steps: (1) cleaning fresh African arrowroot, removing core, adding water, pulping, filtering, centrifuging to obtain a centrifugal clear liquid; (2) after the centrifugal clear liquid is put on a cation exchange resin column, eluting with an eluant, performing nanofiltration on the eluent by using a nanofiltration membrane, and collecting nanofiltration trapped fluid; (3) loading the nanofiltration retentate to an agarose gel chromatography column, eluting with an eluent, concentrating the eluent under reduced pressure, cooling to room temperature, precipitating with ethanol, centrifuging, and filtering to obtain a crude thaumatin product; (4) dissolving the crude thaumatin product with warm water, cooling to room temperature, adjusting the pH value to be alkaline, cooling, stirring for crystallization, centrifuging, filtering, washing crystals with ice water, and drying to obtain the refined thaumatin product. The quality content of the thaumatin in the thaumatin competitive product obtained by the method is up to 99.2 percent, and the total yield of the thaumatin is up to 95.4 percent; the method has the advantages of simple equipment, simple and convenient operation and environmental protection, and is suitable for industrial production.
Description
Technical Field
The invention relates to a method for extracting thaumatin, in particular to a method for extracting thaumatin from African arrowroot.
Background
Thaumatin (A)Thaumatin) Alias: the thaumatin is prepared from West African arrowroot (West African arrowroot)Thaurnatocuccus daniell) The sweetness of the super-sweet substance extracted from the extract is 2000-2500 times that of cane sugar, is one of the sweet substances discovered so far, belongs to natural protein, and has delayed sweetness, longer aftertaste, slow coming and slow disappearance. The thaumatin has 2 kinds of components, thaumatin I is a compound with 207 amino acids combined in straight chain, the molecular weight is 22204, thaumatin II is a protein formed by combining 198 amino acids, and the molecular weight is 21000 +/-500. Thaumatin is a natural non-caloric sweetener and flavoring agent, and is preferably used in combination with sugar such as sucrose in coffee, chocolate, beverage, and dessertBaked goods, and the like. In addition, the thaumatin has an enhancing effect on the fragrance, and has obvious synergy when being used together with spices and the like. The thaumatin can also improve the bitterness of certain amino acids, the astringent taste of tannins and the bitterness of caffeine, enhance the flavors of dairy products and cocoa, and has an enhancing effect when used for spices and the like.
Thaumatin is generally prepared by the following steps: the method comprises the steps of freeze-drying aril of the African arrowroot fruit, separating seeds, extracting with water (the pH value is 2.5-4.0), removing low molecular substances through ultrafiltration, refining and drying.
At present, few reports exist on the separation method of high-quality thaumatin.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art and provides the method for extracting the thaumatin from the African arrowroot, which has high quality content and yield of the obtained thaumatin, simple process and environmental friendliness and is suitable for industrial production.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for extracting thaumatin from African arrowroot comprises the following steps:
(1) cleaning fresh African arrowroot, removing core, adding water, pulping, filtering, centrifuging to obtain a centrifugal clear liquid;
(2) after the centrifugal clear liquid obtained in the step (1) is put on a cation exchange resin column, eluting with an eluant, performing nanofiltration on the eluent by using a nanofiltration membrane, and collecting nanofiltration trapped fluid;
(3) putting the nanofiltration trapped fluid obtained in the step (2) on an agarose gel chromatographic column, eluting by using an eluant, concentrating the eluant under reduced pressure, cooling to room temperature, precipitating with ethanol, centrifuging, and filtering to obtain a crude thaumatin product;
(4) and (4) dissolving the crude thaumatin product obtained in the step (3) by using warm water, cooling to room temperature, adjusting the pH value to be alkaline, cooling, stirring for crystallization, centrifuging, filtering, washing crystals by using ice water, and drying to obtain the refined thaumatin product.
Preferably, in the step (1), the thaumatin content in the fresh African arrowroot fruits is 2-5% by mass.
Preferably, in the step (1), the amount of the added water is 5-10 times of the mass of the fresh Katemon emarginata fruits.
Preferably, in the step (1), the slurry is carried out until the particle size is less than or equal to 100 mu m.
Preferably, in the step (1), the centrifugal separation factor is 5000-30000 (more preferably 8000-20000), and the rotation speed is 6000-10000 r/min. The purpose of centrifugation is to remove suspended substances from the extract and to clarify the material.
Preferably, in step (1), the centrifugation is butterfly centrifugation.
Preferably, in the step (2), the flow rate of the upper column is 0.5-1.0 BV/h.
Preferably, in the step (2), the volume-to-mass ratio (L/kg) of the cation exchange resin to the fresh African arrowroot is 0.05-0.30: 1 (more preferably 0.1-0.2: 1). The purpose of using cation exchange resin is to adsorb the total protein in the centrifuged supernatant in order to extract the thaumatin belonging to the native protein to the maximum extent.
Preferably, in the step (2), the height-diameter ratio of the cation exchange resin column is 4-10: 1.
If the flow rate of the upper column is too fast, the dosage of the ion exchange resin is too small or the height-diameter ratio is too small, the adsorption of the total protein is insufficient, and the yield of the total protein is low; if the flow rate of the upper column is too slow, the dosage of the ion exchange resin is too much or the height-diameter ratio is too large, the waste of materials and energy sources is caused.
Preferably, in the step (2), the type of the cation exchange resin is one or more of 001 × 7 type, 001 × 8 type, 001 × 16 type, LSD-001 type, and the like.
Preferably, in the step (2), the flow rate of the elution is 1.0-2.0 BV/h.
Preferably, in the step (2), the amount of the eluent is 2-4 BV. The purpose of the elution is to desorb the total protein adsorbed on the ion exchange resin.
Preferably, in the step (2), the mass concentration of the sodium chloride aqueous solution or the potassium chloride aqueous solution is 1-5%.
Preferably, in step (2), the eluent is an aqueous solution of sodium chloride or potassium chloride.
Preferably, in the step (2), the molecular weight cut-off of the nanofiltration membrane is 500-1000 Da. The purpose of nanofiltration is to remove salts and small organic molecules from the eluate of the ion exchange resin column.
Preferably, in the step (2), the pressure of the nanofiltration is 0.5-1.5 MPa.
Preferably, in the step (3), the flow rate of the upper column is 0.1-0.5 BV/h. The separation principle of the agarose gel chromatography is similar to that of a molecular sieve, total protein to be separated passes through the agarose gel chromatography column, and different proteins move in the agarose gel chromatography column at different speeds due to different molecular weights and different blocking effects on the agarose gel chromatography column; substances with molecular weight larger than the range of the allowed gel meshes are completely excluded by the gel, cannot enter the interior of gel particles, have small blocking effect, and flow out of the chromatographic column firstly along with the flowing of the solution among the gel particles, so the flow path is short; the substances with small molecular weight can completely enter the meshes of the gel particles, so that the retardation effect is large, the flow is prolonged, and the substances finally flow out of the chromatographic column; by selecting the appropriate type and model of agarose gel, the molecular weight of the target product thaumatin is between the molecular weight of the complete exclusion and the molecular weight of the complete entry into the mesh material, and the two products flow out of the column, thereby achieving the purpose of separation.
Preferably, in the step (3), the volume-to-mass ratio (L/kg) of the agarose gel to the fresh African arrowroot fruits is 0.1-0.2: 1.
Preferably, in the step (3), the ratio of the height to the diameter of the agarose gel chromatography column is 8-15: 1.
If the flow rate of the upper column is too high, the dosage of the agarose gel is too small or the height-diameter ratio is too small, the thaumatin is insufficiently adsorbed, and the yield of the thaumatin is low; if the flow rate of the upper column is too low, the dosage of the agarose gel is too much or the height-diameter ratio is too large, the waste of materials and energy sources is caused.
Preferably, in step (3), the type of the Sepharose is one or more of Q Sepharose FF type, DEAE Sepharose FF type, SP Sepharose FF type, CM Sepharose FF type, or the like.
Preferably, in the step (3), the amount of the eluent is 2-4 BV. The purpose of elution is to desorb crude thaumatin adsorbed on the sepharose chromatography column.
Preferably, in step (3), the eluent is an aqueous ammonium chloride solution.
Preferably, in the step (3), the mass concentration of the ammonium chloride aqueous solution is 1 to 5% (more preferably 1.5 to 3.5%).
Preferably, in the step (3), the flow rate of the elution is 1.0-2.0 BV/h.
Preferably, in the step (3), the temperature of the reduced pressure concentration is 55-65 ℃, the vacuum degree is-0.08-0.09 MPa, and the concentration is carried out until the solid content is 30-40%.
Preferably, in the step (3), the reduced-pressure concentration is a falling-film type reduced-pressure concentration. The falling film type reduced pressure concentration can complete the concentration process of the materials in a short time at a relatively low temperature, and the damage to the molecular structure of the thaumatin caused by a long-time high-temperature environment is prevented.
Preferably, in the step (3), the volume ratio of the alcohol solution used for alcohol precipitation to the concentrated solution is 1-2: 1. The purpose of alcohol precipitation is to precipitate the crude thaumatin in the concentrated solution by means of alcohol precipitation so as to remove ammonium salt and a small amount of water-soluble pigment in the concentrated solution.
Preferably, in the step (3), the volume fraction of the alcohol solution used for alcohol precipitation is 85-100%.
Preferably, in the step (3), the alcohol solution used for alcohol precipitation is an ethanol solution.
If the dosage of the alcohol solution is too small or the volume fraction is too small, the purification purpose is difficult to achieve, so that the yield of the thaumatin is low; if the dosage of the alcohol solution is too much or the volume fraction is too large, the waste of materials and energy sources is caused, and a large amount of impurities are possibly separated out, so that the content of the crude thaumatin product is low.
Preferably, in the step (4), the amount of the warm water is 2-5 times of the wet weight of the crude thaumatin product.
Preferably, in the step (4), the temperature of the warm water is 50-60 ℃.
Preferably, in the step (4), the pH value is adjusted to 11-13. The purpose of adjusting the pH is to bring the pH of the solution within the isoelectric point range of thaumatin, which is the minimum solubility of thaumatin when the pH is within the isoelectric point range, so that it can be precipitated to the maximum extent.
The method comprises the following steps of (1) carrying out alcohol precipitation in the step (3), then adjusting the pH value to be alkaline in the step (4), and finally cooling and refining: the thaumatin is difficult to crystallize and separate out in the concentrated solution containing a large amount of ammonium chloride and water-soluble pigment, and the impurities can be fully separated out and separated by alcohol precipitation; the crude thaumatin product is dissolved by hot water in the refining process, the aim is to re-dissolve the crude thaumatin product and a small amount of impurities (ammonium chloride and water-soluble pigments) embedded in the crude product, the pH value is adjusted to the isoelectric point of thaumatin, and then the temperature is reduced, so that the aim of preparing the refined thaumatin product is fulfilled by utilizing the principle that thaumatin has the minimum solubility and is easy to separate out under the alkaline pH value, and the impurities cannot be separated out.
Preferably, in the step (4), dilute alkali liquor with the mass concentration of 0.5-2.0% is used for adjusting the pH value.
Preferably, in the step (4), the dilute alkali solution is an aqueous solution of sodium hydroxide or an aqueous solution of potassium hydroxide.
Preferably, in the step (4), the temperature is reduced to 5-10 ℃. The purpose of cooling is to reduce the solubility of thaumatin, thereby causing thaumatin to be precipitated to the maximum extent.
Preferably, in the step (4), the rotation speed of stirring and crystallizing is 20-60 r/min, and the time is 12-24 h.
Preferably, in the step (4), the temperature of the ice water is 0-10 ℃.
The method has the following beneficial effects:
(1) the quality content of thaumatin (the total amount of thaumatin I and thaumatin II) in the thaumatin competitive product obtained by the method is up to 99.2 percent, and the total yield of thaumatin (the total amount of thaumatin I and thaumatin II) is up to 95.4 percent;
(2) the method has the advantages of simple equipment, simple and convenient operation and environmental protection, and is suitable for industrial production.
Detailed Description
The present invention will be further described with reference to the following examples.
The fresh Kandelia calamus fruits used in the embodiment of the invention are purchased from Caragana, wherein the mass content of thaumatin is 3.72%; the 001X 16 type, 001X 8 type, LSD-001 type cation exchange resins, SP Sepharose FF type, Q Sepharose FF type, CM Sepharose FF type agarose gels used in the examples of the present invention are commercially available from New scientific and technical materials, Inc. of Xian blue; the nanofiltration membranes of 800Da, 900Da and 500Da used in the embodiment of the invention are all purchased from Hangzhou Seffy membrane separation technology, Inc.; the chemical reagents used in the examples of the present invention, unless otherwise specified, are commercially available in a conventional manner.
In the embodiment of the invention, the content of thaumatin is detected by adopting a high performance liquid chromatography external standard method.
Example 1
(1) Cleaning 10kg of fresh African arrowroot, removing core, adding 80kg of water, pulping until the granularity is less than or equal to 200 mu m, filtering, and performing butterfly centrifugation under the conditions that the separation factor is 10000 and the rotation speed is 8000 r/min to obtain a centrifugal clear liquid;
(2) loading the centrifugal clear liquid obtained in the step (1) on a 001X 16 type cation exchange resin column (the using amount of the 001X 16 type cation exchange resin is 2L, and the height-diameter ratio is 8: 1) at the flow rate of 0.8BV/h, eluting with a sodium chloride aqueous solution with the mass concentration of 3% and the flow rate of 1.5BV/h, performing nanofiltration on the eluent with a nanofiltration membrane with the molecular weight cutoff of 900Da at the pressure of 1.2MPa, and collecting nanofiltration trapped fluid;
(3) loading the nanofiltration trapped fluid obtained in the step (2) onto an SP Sepharose FF agarose gel chromatographic column (the dosage of the SP Sepharose FF agarose gel is 2L, and the height-diameter ratio is 8: 1) at the flow rate of 0.3BV/h, eluting with 3BV of ammonium chloride aqueous solution with the mass concentration of 2.5% at the flow rate of 1.5BV/h, carrying out falling film type reduced pressure concentration on eluent at 60 ℃ and 0.08MPa until the solid content is 40% to obtain 2.4L of concentrated solution, cooling to room temperature, adding 3.6L of ethanol solution with the volume fraction of 87%, carrying out alcohol precipitation, centrifuging and filtering to obtain 0.87kg (wet weight) of crude thaumatin;
(4) and (3) dissolving 0.87kg (wet weight) of crude thaumatin product obtained in the step (3) by using 1.75L of warm water at 60 ℃, cooling to room temperature, adjusting the pH value to 12.5 by using a potassium hydroxide aqueous solution with the mass concentration of 2%, cooling to 5 ℃, stirring and crystallizing for 12 hours at the speed of 30 r/min, centrifuging, filtering, washing crystals by using ice water at the temperature of 5 ℃, and drying to obtain 0.35kg of refined thaumatin product.
Through detection of a high performance liquid chromatography external standard method, the mass content of thaumatin (the total amount of thaumatin I and thaumatin II) in the thaumatin refined product obtained in the embodiment of the invention is 99.2%, and the total yield of thaumatin (the total amount of thaumatin I and thaumatin II) is 93.3%.
Example 2
(1) Cleaning 20kg of fresh African arrowroot, removing core, adding 100kg of water, pulping until the granularity is less than or equal to 150 mu m, filtering, and performing butterfly centrifugation at a separation factor of 8000 and a rotation speed of 6000 r/min to obtain a centrifugal clear liquid;
(2) loading the centrifugal clear liquid obtained in the step (1) on a 001X 8 type cation exchange resin column (the using amount of the 001X 8 type cation exchange resin is 3L, and the height-diameter ratio is 5: 1) at the flow rate of 0.5BV/h, eluting with a sodium chloride aqueous solution with the mass concentration of 1.5% and the flow rate of 3BV/h, performing nanofiltration on the eluent with a nanofiltration membrane with the molecular weight cutoff of 800Da at the pressure of 1.0MPa, and collecting nanofiltration trapped liquid;
(3) loading the nanofiltration trapped fluid obtained in the step (2) onto a Q Sepharose FF agarose gel chromatographic column at the flow rate of 0.2BV/h (the dosage of Q Sepharose FF agarose gel is 2L, and the height-diameter ratio is 10: 1), eluting with 2.5BV of ammonium chloride aqueous solution with the mass concentration of 2% at the flow rate of 1.0BV/h, carrying out falling film type reduced pressure concentration on eluent at 55 ℃ and-0.09 MPa until the solid content is 30% to obtain 6L of concentrated solution, cooling to room temperature, adding 12L of ethanol solution with the volume fraction of 90%, carrying out alcohol precipitation, centrifuging and filtering to obtain 1.3kg (wet weight) of crude thaumatin;
(4) and (3) dissolving 1.3kg (wet weight) of crude thaumatin product obtained in the step (3) by using 3.3L of warm water at 50 ℃, cooling to room temperature, adjusting the pH value to 11.5 by using a sodium hydroxide aqueous solution with the mass concentration of 1%, cooling to 10 ℃, stirring and crystallizing for 24 hours at the speed of 20 r/min, centrifuging, filtering, washing the crystal by using ice water at the temperature of 0 ℃, and drying to obtain 0.72kg of refined thaumatin product.
Through detection of a high performance liquid chromatography external standard method, the mass content of thaumatin (the total amount of thaumatin I and thaumatin II) in the thaumatin refined product obtained in the embodiment of the invention is 98.6%, and the total yield of thaumatin (the total amount of thaumatin I and thaumatin II) is 95.4%.
Example 3
(1) Cleaning 30kg of fresh African arrowroot, removing core, adding 180kg of water, pulping until the granularity is less than or equal to 100 mu m, filtering, and performing butterfly centrifugation under the condition that the separation factor is 15000 and the rotation speed is 10000 r/min to obtain a centrifugal clear liquid;
(2) loading the centrifugal clear liquid obtained in the step (1) on an LSD-001 type cation exchange resin column (the using amount of the LSD-001 type cation exchange resin is 3L, and the height-diameter ratio is 6: 1) at the flow rate of 1.0BV/h, eluting with 3.5BV of potassium chloride aqueous solution with the mass concentration of 2%, subjecting the eluent to nanofiltration with the molecular weight cutoff of 500Da at the pressure of 1.5MPa, and collecting nanofiltration cutoff liquid;
(3) loading the nanofiltration trapped fluid obtained in the step (2) onto a CM Sepharose FF agarose gel chromatographic column (the use amount of the CM Sepharose FF agarose gel is 4.5L, and the height-diameter ratio is 12: 1) at the flow rate of 0.4BV/h, eluting with 4BV of ammonium chloride aqueous solution with the mass concentration of 1.8% at the flow rate of 2.0BV/h, carrying out falling film type reduced pressure concentration on eluent at 63 ℃ and-0.08 MPa until the solid content is 35% to obtain 8.5L of concentrated solution, cooling to room temperature, adding 10L of ethanol solution with the volume fraction of 85%, carrying out alcohol precipitation, centrifuging and filtering to obtain 2.5kg (wet weight) of crude thaumatin;
(4) and (3) dissolving 2.5kg (wet weight) of the crude thaumatin product obtained in the step (3) by using 7.5L warm water at 55 ℃, cooling to room temperature, adjusting the pH value to 12 by using a sodium hydroxide aqueous solution with the mass concentration of 1.5%, cooling to 7 ℃, stirring and crystallizing for 18h at 40r/min, centrifuging, filtering, washing the crystal by using ice water at 8 ℃, and drying to obtain 1.07kg of the refined thaumatin product.
Through detection of a high performance liquid chromatography external standard method, the mass content of thaumatin (the total amount of thaumatin I and thaumatin II) in the thaumatin refined product obtained in the embodiment of the invention is 97.8%, and the total yield of thaumatin (the total amount of thaumatin I and thaumatin II) is 93.8%.
Claims (17)
1. A method for extracting thaumatin from African arrowroot is characterized by comprising the following steps:
(1) cleaning fresh African arrowroot, removing core, adding water, pulping, filtering, centrifuging to obtain a centrifugal clear liquid;
(2) after the centrifugal clear liquid obtained in the step (1) is put on a cation exchange resin column, eluting with an eluant, performing nanofiltration on the eluent by using a nanofiltration membrane, and collecting nanofiltration trapped fluid; the flow rate of the upper column is 0.5-1.0 BV/h; the volume mass ratio of the cation exchange resin to the fresh African arrowroot is 0.05-0.30: 1; the height-diameter ratio of the cation exchange resin column is 4-10: 1; the flow rate of elution is 1.0-2.0 BV/h; the dosage of the eluent is 2-4 BV; the eluent is sodium chloride aqueous solution or potassium chloride aqueous solution; the mass concentration of the sodium chloride aqueous solution or the potassium chloride aqueous solution is 1-5%; the molecular weight cut-off of the nanofiltration membrane is 500-1000 Da;
(3) putting the nanofiltration trapped fluid obtained in the step (2) on an agarose gel chromatographic column, eluting by using an eluant, concentrating the eluant under reduced pressure, cooling to room temperature, precipitating with ethanol, centrifuging, and filtering to obtain a crude thaumatin product; the flow rate of the upper column is 0.1-0.5 BV/h; the volume-mass ratio of the agarose gel to the fresh African arrowroot is 0.1-0.2: 1; the height-diameter ratio of the agarose gel chromatographic column is 8-15: 1; the eluent is ammonium chloride aqueous solution; the mass concentration of the ammonium chloride aqueous solution is 1-5%; the flow rate of elution is 1.0-2.0 BV/h, and the dosage of the eluent is 2-4 BV; the temperature of the reduced pressure concentration is 55-65 ℃, the vacuum degree is-0.08-0.09 MPa, and the concentration is carried out until the solid content is 30-40%; the volume ratio of the alcohol solution used for alcohol precipitation to the concentrated solution is 1-2: 1; the volume fraction of the alcoholic solution used for alcohol precipitation is 85-100%;
(4) dissolving the crude thaumatin product obtained in the step (3) by using warm water, cooling to room temperature, adjusting the pH value to be alkaline, cooling, stirring for crystallization, centrifuging, filtering, washing crystals by using ice water, and drying to obtain a refined thaumatin product; the amount of the warm water is 2-5 times of the wet weight of the crude thaumatin product; the temperature of the warm water is 50-60 ℃; adjusting the pH value to 11-13; cooling to 5-10 ℃.
2. The method of claim 1 for extracting thaumatin from thaumatin, wherein the thaumatin is obtained by the steps of: in the step (1), the amount of the added water is 5-10 times of the mass of the fresh African arrowroot fruits; pulping until the granularity is less than or equal to 100 mu m; the centrifugal separation factor is 5000-30000, and the rotating speed is 6000-10000 r/min; the centrifugation is butterfly centrifugation.
3. The method for extracting thaumatin from thaumatin according to claim 1 or 2, characterized in that: in the step (2), the type of the cation exchange resin is one or more of 001 × 7 type, 001 × 8 type, 001 × 16 type or LSD-001 type.
4. The method for extracting thaumatin from thaumatin according to claim 1 or 2, characterized in that: in the step (2), the nanofiltration pressure is 0.5-1.5 MPa.
5. The method of claim 3, wherein the thaumatin is extracted from thaumatin: in the step (2), the nanofiltration pressure is 0.5-1.5 MPa.
6. The method for extracting thaumatin from thaumatin according to claim 1 or 2, characterized in that: in the step (3), the type of the agarose gel is one or more of Q Sepharose FF type, DEAE Sepharose FF type, SP Sepharose FF type or CM Sepharose FF type.
7. The method of claim 3, wherein the thaumatin is extracted from thaumatin: in the step (3), the type of the agarose gel is one or more of Q Sepharose FF type, DEAE Sepharose FF type, SP Sepharose FF type or CM Sepharose FF type.
8. The method of claim 4, wherein the thaumatin is extracted from thaumatin: in the step (3), the type of the agarose gel is one or more of Q Sepharose FF type, DEAE Sepharose FF type, SP Sepharose FF type or CM Sepharose FF type.
9. The method for extracting thaumatin from thaumatin according to claim 1 or 2, characterized in that: in the step (3); the reduced pressure concentration is a falling film type reduced pressure concentration; the alcohol solution used for alcohol precipitation is an ethanol solution.
10. The method of claim 3, wherein the thaumatin is extracted from thaumatin: in the step (3); the reduced pressure concentration is a falling film type reduced pressure concentration; the alcohol solution used for alcohol precipitation is an ethanol solution.
11. The method of claim 4, wherein the thaumatin is extracted from thaumatin: in the step (3); the reduced pressure concentration is a falling film type reduced pressure concentration; the alcohol solution used for alcohol precipitation is an ethanol solution.
12. The method of claim 6, wherein the thaumatin is extracted from thaumatin: in the step (3); the reduced pressure concentration is a falling film type reduced pressure concentration; the alcohol solution used for alcohol precipitation is an ethanol solution.
13. The method for extracting thaumatin from thaumatin according to claim 1 or 2, characterized in that: in the step (4), dilute alkali liquor with the mass concentration of 0.5-2.0% is used for adjusting the pH value; the dilute alkali solution is sodium hydroxide aqueous solution or potassium hydroxide aqueous solution; the rotating speed of stirring crystallization is 20-60 r/min, and the time is 12-24 h; the temperature of the ice water is 0-10 ℃.
14. The method of claim 3, wherein the thaumatin is extracted from thaumatin: in the step (4), dilute alkali liquor with the mass concentration of 0.5-2.0% is used for adjusting the pH value; the dilute alkali solution is sodium hydroxide aqueous solution or potassium hydroxide aqueous solution; the rotating speed of stirring crystallization is 20-60 r/min, and the time is 12-24 h; the temperature of the ice water is 0-10 ℃.
15. The method of claim 4, wherein the thaumatin is extracted from thaumatin: in the step (4), dilute alkali liquor with the mass concentration of 0.5-2.0% is used for adjusting the pH value; the dilute alkali solution is sodium hydroxide aqueous solution or potassium hydroxide aqueous solution; the rotating speed of stirring crystallization is 20-60 r/min, and the time is 12-24 h; the temperature of the ice water is 0-10 ℃.
16. The method of claim 6, wherein the thaumatin is extracted from thaumatin: in the step (4), dilute alkali liquor with the mass concentration of 0.5-2.0% is used for adjusting the pH value; the dilute alkali solution is sodium hydroxide aqueous solution or potassium hydroxide aqueous solution; the rotating speed of stirring crystallization is 20-60 r/min, and the time is 12-24 h; the temperature of the ice water is 0-10 ℃.
17. The method of claim 9 for extracting thaumatin from thaumatin, wherein the thaumatin is obtained by: in the step (4), dilute alkali liquor with the mass concentration of 0.5-2.0% is used for adjusting the pH value; the dilute alkali solution is sodium hydroxide aqueous solution or potassium hydroxide aqueous solution; the rotating speed of stirring crystallization is 20-60 r/min, and the time is 12-24 h; the temperature of the ice water is 0-10 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811141257.3A CN109170796B (en) | 2018-09-28 | 2018-09-28 | Method for extracting thaumatin from African arrowroot |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811141257.3A CN109170796B (en) | 2018-09-28 | 2018-09-28 | Method for extracting thaumatin from African arrowroot |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109170796A CN109170796A (en) | 2019-01-11 |
| CN109170796B true CN109170796B (en) | 2021-07-20 |
Family
ID=64907654
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811141257.3A Active CN109170796B (en) | 2018-09-28 | 2018-09-28 | Method for extracting thaumatin from African arrowroot |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109170796B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111574607B (en) * | 2020-05-12 | 2023-09-05 | 武汉市华甜生物科技有限公司 | Method for extracting thaumatin from African arrowroot based on microbial fermentation |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101153311A (en) * | 2007-09-27 | 2008-04-02 | 华中农业大学 | Preparation method and product of rapeseed oligopeptide |
| CN102276515A (en) * | 2011-09-13 | 2011-12-14 | 西南大学 | Method for extracting deoxynojirimycin |
| CN108396049A (en) * | 2018-03-08 | 2018-08-14 | 浙江汇能生物股份有限公司 | The preparation method of silkworm pupa protein polypeptide |
-
2018
- 2018-09-28 CN CN201811141257.3A patent/CN109170796B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101153311A (en) * | 2007-09-27 | 2008-04-02 | 华中农业大学 | Preparation method and product of rapeseed oligopeptide |
| CN102276515A (en) * | 2011-09-13 | 2011-12-14 | 西南大学 | Method for extracting deoxynojirimycin |
| CN108396049A (en) * | 2018-03-08 | 2018-08-14 | 浙江汇能生物股份有限公司 | The preparation method of silkworm pupa protein polypeptide |
Non-Patent Citations (5)
| Title |
|---|
| "New technologies for taste modifying proteins";Michael Witty;《Trends in Food Science & Technology》;19981231;275-280 * |
| "Solubility of Thaumatin";Neer Asherie et al;《Crystal Growth & Design》;20080630;第8卷(第6期);4712-4713 * |
| "天然甜味蛋白索马甜的研究进展";张泽生等;《中国食品添加剂》;20180531(第05期);186-189 * |
| "甜味植物研究进展";张桂玲等;《安徽农业科学》;20060930(第18期);4712-4713 * |
| "甜味蛋白Thaumatin研究进展及在食品工业中的应用";杨阳等;《中国食品添加剂》;20121031(第5期);171-176 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109170796A (en) | 2019-01-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108516997B (en) | Method for extracting rubusoside from sweet tea leaves | |
| US4861870A (en) | Process for purifying anthracyclinone glycosides by selective adsorption on resins | |
| CN105061526B (en) | A kind of extracting method of high-purity Rubusoside | |
| CN111793099B (en) | Method for separating hesperidin, neohesperidin, naringin and synephrine from immature bitter orange | |
| CN108752231B (en) | Method for extracting theanine from sweet tea and simultaneously extracting rubusoside and tea polyphenol | |
| WO2018028144A1 (en) | Separation and purification method for high-purity mogroside v | |
| CN107936079B (en) | Method for preparing high-purity mogroside V | |
| EP3528646B1 (en) | Extracts from fruits of the cucurbitaceae family, and methods of preparing thereof | |
| CZ2002948A3 (en) | Process for simultaneous purification and separation of pectin and pectin-containing sugars or oligomers from sugar beet pulp | |
| CN106967142A (en) | It is a kind of at the same extract momordica glycoside V, VI and 11 O base glycosides V method | |
| CN109170796B (en) | Method for extracting thaumatin from African arrowroot | |
| US12193458B2 (en) | Sweetening composition and preparation method and use thereof | |
| CN110981921A (en) | Continuous method for synchronously extracting multiple effective components from figs | |
| CN104892717B (en) | A kind of technical grade preparative liquid chromatography separation method of momordica glycoside V | |
| CN107573255B (en) | A kind of method for separating and purifying capsaicin and dihydrocapsaicin from pepper fruit | |
| US10968470B2 (en) | Method for preparing rubusoside | |
| CN105418703A (en) | Method for extracting and purifying stevioside from stevia rebaudiana | |
| CN108997359B (en) | A kind of method for extracting chlorophyll from stevioside production waste residue | |
| CN102648750B (en) | A kind of refined jujube sucrose and jujube fructose syrup and preparation method thereof | |
| CN110526892B (en) | Method for extracting anthocyanin from blueberry | |
| CN103408614A (en) | Novel preparation technique of steviosin and Rebaudioside-A | |
| CN116284173B (en) | A method for simultaneously preparing rubusoside and tea polyphenols from sweet tea | |
| CN112300231A (en) | Method for extracting high-purity stevioside | |
| CN111848705A (en) | A kind of preparation and separation method of glycoside-bound aroma precursor substances in tea | |
| JPH04214767A (en) | Process for preventing precipitation of yellow pigment of safflower |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |