CN102462701A - Enzyme method for refining Chinese medicine extract - Google Patents
Enzyme method for refining Chinese medicine extract Download PDFInfo
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Abstract
The invention relates to an enzyme method for refining a Chinese medicine extract. The method comprises the following steps of: (1) acquiring a water or alcohol extract of a Chinese medicinal material, and concentrating to obtain a concentrated Chinese medicine extract; (2) adding the concentrated Chinese medicine extract obtained in the step (1) to a reactor, adding 0.01-5 parts by weight of one or more of cellulase, pectinase, xylanase, neutral proteinase, papain and beta-glucanase to the reactor in terms of 100 parts by weight of the concentrated Chinese medicine extract to form a mixture, and stirring the obtained mixture in the reactor to form a uniform mixture; and (3) reacting the uniform mixture obtained in the step (2) at 40-60 DEG C with pH being 3-5 for 2-3 hours to obtain a clear Chinese medicine extract liquid. The method can be carried out multiple times according to needs.
Description
Technical field
The present invention relates to a kind of process for purification of Chinese medicine extract, promptly relate to a kind of method that adopts the refining Chinese medicine extract of enzyme process.
Background technology
Enzyme engineering is very extensive in Application in Food Processing in recent years; Used enzyme class is more and more; Quantity is also increasing; If the human multiple enzyme glue enzyme that is applied in fruit and vegerable, medicated beer, the beverage, pectinesterase, cellulase, rhamnosidase, neutral protease, galactomannan enzyme, the liquefaction glucosidase etc. developed, wherein using maximum is pectase and cellulase.
Use enzyme abroad the thirties in 20th century industrialization.Domestic research then starts from the sixties in last century, just begins suitability for industrialized production the end of the eighties.In the natural product production process, pectase can remove pectin fast up hill and dale, reduces the fruit juice viscosity, and it is stable to be beneficial to fruit juice filtration clear filtrate and clarity.
Pectase (pectolytic enzyme or pectinase) is meant the general name that is prevalent in antibacterial, fungus and the plant plurality of enzymes that is decompose pectin class material.In garden stuff processing, feedstuff, textile and paper industry, use very extensive.
In fruit and vegetable juice was produced, pectase was one of most important enzyme preparation, be widely used in fruit and vegetable juice extraction and clarification, improve the filterability of fruit and vegetable juice and the dipping of plant tissue and extraction.In the fruit and vegetable juice processing industry, extensively adopt pectase degraded fruit and vegerable cell wall at present to improve pressability, reduce viscosity, to increase crushing juice rate and progressive nutritional labeling.Use pectase, cellulase and hemicellulase can promote cell separation, the cell wall deliquescing, this is particularly suitable for fruits and vegetables [1].Pectase acts on the glycosidic bond between the D-galacturonic acid residue in the pectic substance, makes high molecular polygalacturonic acid reduce to small-molecule substance.Can improve the greatly crushing juice rate of fruit and vegetable varieties improves its rate of filtration and guarantees product bin stability [2].
Pectase also can produce flocculation except that reducing viscosity, make the fruit and vegetable juice clarification.The essence of clarification mechanism comprises the enzymatic hydrolysis of pectin and electrostatic bunching two parts of non-enzyme.It is the principal element that causes fruit juice muddy that influences such as a lot of materials such as cellulose, protein, starch, pectin substance clarification and pectin substance are arranged in the fruit juice.In the course of processing of cherry juice, add pectase, make hydrolyzed pectin, thereby the cherry juice viscosity is reduced, filtration resistance reduces, and the rate of filtration is accelerated; Because the suspension fruit grain in the cherry juice loses the protection of macromolecule pectin, be easy to take place sedimentation, and the consumption of gelatin clarifier in the limpid clarifying process afterwards of upper strata juice just can be significantly reduced, even exempt to add clarifier [3] simultaneously.
In the Chinese crude drug, especially usually contain many stickums in the root class medical material, generally speaking, these stickums generally include protein, starch, tannin, pectin, phlegmatic temperament etc.The existence of these materials can influence the viscosity and the clarity of Chinese medicine extraction liquid, thereby can influence being further purified of effective ingredient in Chinese.For this reason, the extract of this type of medical material usually needs clarification steps after extracting, according to the type of impurity, and the defecation method commonly used such as the water extraction alcohol sedimentation method, alcohol extraction water precipitation method etc.Although said method has certain effect, do not reach requirement far away for the production of high-purity Chinese medicine, especially contain the more medical material of pectase, not only cause productive rate to descend, also consume more energy and processing cost is increased.
At present, the industrial applications of enzyme aspect Chinese medicine extraction also got into the elementary development phase.The cell wall of part Chinese crude drug is made up of cellulose, and the effective ingredient of plant often is wrapped in the cell wall.Cellulose be β-D-glucose with 1,4-β glucoside chain connects, and can destroy β-D-glucose key with cellulose, and then help the extraction of effective ingredient.According to this principle, the domestic people of having uses it for Cortex Phellodendri and extracts the pretreatment in the berberine, and compares experiment with not enzyme-added extraction, and yield improves.Someone is used for Fructus Psoraleae with papain and fries in shallow oil pretreatment in advance, and Fructus Psoraleae is the seed of legumes psoraleae, contains rich in protein, and protein is met thermocoagulation in decoction process, influence the fried of other effective ingredient.Therefore can carry out pretreatment with papain to the Fructus Psoraleae powder frying in shallow oil in advance, proteolysis is become peptide and amino acids, meet not resolidification of heat, thereby promote the increase of the fried amount of various effective ingredient.
Domestic have some enterprises and scientific research institution to research and develop technology and product that a collection of cellulase is applied to herbal pharmaceutical, and obtained domestic patent, but few in number.In these patents, except only adopt cellulase, adopt cellulase and another kind of or several kinds of enzymes to form compound enzyme class enzymolysis and extraction Chinese medicines in addition, but be not used in clarification and reduce viscosity.
This shows that present above-mentioned enzyme process for purification is used very extensive in food processing, and only limits in the Chinese medicine extraction in the Chinese medicine sector application, purpose is not used in extract refining is handled in order to increase extraction ratio.Therefore need a kind of technology simple, easy to operate, quick, reduce the technology that Chinese crude drug extract refining that water, electricity, clarifier (perhaps ethanol) consumes is handled greatly, the processing of the product that is used for clarity is had relatively high expectations.
Summary of the invention
For this reason, the invention provides the method (to call " method of the present invention " in the following text) of the refining Chinese medicine extract of a kind of enzyme process, this method may further comprise the steps:
1) obtain the water or the alcohol extract of Chinese crude drug, concentrating becomes the Chinese medicine concentrated extracting solution;
2) be added in the reaction vessel in the Chinese medicine concentrated extracting solution with the step 1) acquisition; The amount that in reaction vessel, adds the 0.01-5 weight portion by the said Chinese medicine concentrated extracting solution of per 100 weight portions then adds one or more and is selected from following enzyme: cellulase, pectase, xylanase, neutral protease, xylanase, neutral protease, papain and 1,4 beta-glucanase form mixture and the mixture that obtains are stirred in reaction vessel to form uniform mixture;
3) with step 2) the middle uniform mixture that obtains is under 40～60 ℃, and reaction is 2-3 hour under the condition of pH 3-5, obtains the Chinese medicine extract clear liquor.
In an embodiment of method of the present invention, wherein said Chinese crude drug is the medical material that contains stickum.
In another embodiment of method of the present invention, wherein the consumption of enzyme described in the step 1 is for adding the 0.01-5 weight portion by the said Chinese medicine concentrated extracting solution of per 100 weight portions, preferred 0.05-1 weight portion.
In an embodiment of method of the present invention, step 2 wherein) enzyme that uses in is cellulase or pectase.
In an embodiment of method of the present invention, step 2 wherein) enzyme that uses in is the compound enzyme that two kinds of enzymes are formed, the combination of preferred cellulose enzyme and pectase, and the proportioning of said two kinds of enzymes is 1: 1 by weight.
In an embodiment of method of the present invention; This method also comprises: the viscosity that detects the Chinese medicine extract clear liquor that obtains in the step 3); If the viscosity of the Chinese medicine concentrated extract that obtains in this viscosity and the step 1) is compared and reduced approximately below 5%, then adopt the combination repeating step 1 that is different from last employed enzyme or enzyme)-3).
In the method for the invention, the viscosity of the Chinese medicine concentrated extracting solution that obtains in the viscosity of the Chinese medicine extract clear liquor that obtains in the step 3) and the step 1) is compared and is reduced 5-95%, more preferably reduces 10-95% and most preferably reduces 20-95%.
In an embodiment of method of the present invention, this method also comprises:
4) the Chinese medicine extract clear liquor that step 3) is obtained is through absorption with macroporous adsorbent resin, and collects eluent.
In a preferred embodiment of method of the present invention, wherein the macroporous adsorbent resin in the step 4) is selected from nonpolar to the low pole macroporous resin any.
The present invention is through the pectin in adding pectase, cellulase, the mixed enzyme decomposition Chinese medicine extract, cellulose etc.; Reduced intrinsic viscosity; Replace alcohol deposition method and removed pectin, cellulose, polysaccharide etc.; Reduced the lot of consumption of precipitant such as water, electricity, ethanol, help further separation, purification effective ingredient in Chinese composition (
Annotate: the present invention receives state natural sciences fund 30772755,30830118; The great special 2009ZX09103-317 of national science and technology subsidizes).
The specific embodiment
Method of the present invention is the method for the refining Chinese medicine extract of a kind of enzyme process, and this method may further comprise the steps:
1) obtain the water or the alcohol extract of Chinese crude drug, concentrating becomes the Chinese medicine concentrated extracting solution;
2) be added in the reaction vessel in the Chinese medicine concentrated extracting solution with the step 1) acquisition; The amount that in reaction vessel, adds the 0.01-5 weight portion by the said Chinese medicine concentrated extracting solution of per 100 weight portions then adds one or more and is selected from following enzyme: cellulase, pectase, xylanase, neutral protease, xylanase, neutral protease, papain and 1,4 beta-glucanase form mixture and the mixture that obtains are stirred in reaction vessel to form uniform mixture;
3) with step 2) the middle uniform mixture that obtains is under 40～60 ℃, and reaction is 2-3 hour under the condition of pH 3-5, obtains the Chinese medicine extract clear liquor.
In an embodiment of method of the present invention, wherein said Chinese crude drug is the medical material that contains stickum, comprises the animal and plant medical material.For vegetable drug, the position of containing stickum comprises different agents areas, for example root class, tubers, skin class, flower, fruit, seed etc.The example of such medical material comprises; But be not limited to Radix Rehmanniae Preparata, Fructus Lycii, Arillus Longan, Fructus Corni, Rhizoma Polygonati, Rhizoma Polygonati Odorati, Pollen Typhae, Radix Asparagi, Radix Ophiopogonis, the Rhizoma Atractylodis Macrocephalae, Radix Vladimiriae, Semen Plantaginis, Semen Sterculiae Lychnophorae, Radix Et Rhizoma Rhei, the Rhizoma Anemarrhenae, Herba Dendrobii, Flos Farfarae, Poria, Os Draconis, Semen Lepidii (Semen Descurainiae) etc.
In the method for the invention, the extraction of Chinese crude drug can make water or organic solvent for example alcohol extract, said alcohol can be that concentration is methanol, ethanol of 30-100% etc., preferably uses 50～95% ethanol.The Chinese medicine extraction method can be with reference to the Chinese medicine extraction method of routine, and general step is: with pulverizing medicinal materials, (for example add suitable quantity of water or organic solvent at every turn; 6～10 times of medical material weight), heated and boiled keep 1～2 hour/inferior, extract 2-5 time; Preferred 2-3 time, the gained extracting solution is merged.
In the embodiment in the method for the invention, carry out enzyme refining before, obtain in the step 1) Chinese medicine extraction liquid can be concentrated to relative density 1.05～1.30 (60 ℃ of heat are surveyed) to reduce enzyme dosage as far as possible.
In the method for the invention, before being added to reaction vessel, the Chinese medicine concentrated extracting solution that obtains in the step 1) can be cooled to room temperature in advance, and then is added in the reaction vessel.
In another embodiment of method of the present invention, wherein the consumption of enzyme described in the step 1) is for adding the 0.01-5 weight portion by the said Chinese medicine concentrated extracting solution of per 100 weight portions, preferred 0.05-1 weight portion.Said enzyme can directly be added to after taking by weighing suitable weight in the Chinese medicine concentrated extracting solution, also can be with a spot of distilled water or the organic solvent that is used to extract Chinese medicine dissolve and dilute the back a little and add.For example,, add a small amount of distilled water or Chinese medicine extraction liquid (soak into be as the criterion, approximately 2g/ml) back furnishing pasty state after can weighing, be added to then in the Chinese medicine concentrated extracting solution so that disperse like the dissolubility of enzyme in water hour.Those skilled in the art can wait the comprehensive consumption of confirming enzyme according to the combination of the viscosity of used medical material, concentrated traditional Chinese medicine extract, used enzyme or enzyme.
In an embodiment of method of the present invention, step 2 wherein) the enzyme preferred cellulose enzyme or the pectase that use in, because Chinese crude drug is plant mostly, wherein cellulose and pectin content are than horn of plenty.
In an embodiment of method of the present invention, step 2 wherein) enzyme that uses in is the compound enzyme that two kinds of enzymes are formed, the combination of preferred cellulose enzyme and pectase, and the proportioning of said two kinds of enzymes is 1: 1 by weight.If adopt the combination of other enzymes, also can use according to 1: 1 weight proportion.
Employed in the method for the invention enzyme is unrestricted, can be commercially available, also can be according to conventional method preparation known in the field.
In the method for the invention, the selection of reaction vessel is unrestricted, as long as can carry out the extraction of Chinese crude drug, can use container agitated reactor, evaporator etc. according to the extraction scale.In the method for the invention, the method for mixed enzyme and Chinese medicine concentrated extracting solution is unrestricted in reaction vessel, can adopt method as known in the art according to the scale and the type of reaction vessel, like stirring rod, agitator, blender etc.When mixing, preferably control the temperature of reactant mixture, make the interior temperature of reaction vessel be no more than the denaturation temperature or the reaction temperature of employed enzyme, for example, be no more than 60 ℃ usually.
Through said method, the Chinese medicine extract clear liquor that obtains is according to the method for the invention compared with the viscosity (like intrinsic viscosity) of handling Chinese medicine concentrated extracting solution before with enzyme can be reduced by at least about 5%, preferably at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, more preferably at least 85% and most preferably at least 90%; For example, can reduce 5-95%, 10-90%, 20-90%, 30-90%, 10-80%, 20-80%, 30-80%, 10-70%, 20-70%, 30-70%, 10-60%, 20-60%, 30-60%, more preferably 20-95% and most preferably 30-95%.
If the Chinese medicine that is adopted can't be confirmed possibly originating of its viscosity in advance; And after the combined treatment that adopts a kind of enzyme or enzyme; The degree that the viscosity of Chinese medicine concentrated extracting solution reduces is less than about 5%; Then can take other kinds or combination in the enzyme recited above to come the method step of repetition the invention described above, be reduced by at least more than 5% until viscosity.Such repeating step is also included within the scope of the present invention; Promptly in another embodiment of method of the present invention; This method also comprises: the viscosity that detects the Chinese medicine extract clear liquor that obtains in the step 3); Reduce (to be the about more than 95% of the latter) 5% below approximately if the viscosity of the Chinese medicine concentrated extract that obtains in this viscosity and the step 1) is compared, then employing is different from the combination repeating step 1 of last employed enzyme or enzyme)-3).As required, this repeating step can carry out 2-4 time.
In an embodiment of method of the present invention, this method also comprises:
4) the Chinese medicine extract clear liquor that step 3) is obtained is through absorption with macroporous adsorbent resin, and collects eluent.
In a preferred embodiment of method of the present invention, wherein the macroporous adsorbent resin in the step 4) is selected from nonpolar to the low pole macroporous resin any.This resinoid can use commercially available resin, comprises AB-8, the D101 type, and the ADS-9 type, HP-20 type etc., the aperture of above-mentioned resin is generally 100-1000nm.Resin absorption step of the present invention can adopt the method for well known to a person skilled in the art and step; Generally carry out at ambient temperature; For example under room temperature (about 25 ℃), carry out; Adsorption rate be generally 1～2 bed volume/hour, and with suitable solvent, for example water or 10%～70% ethanol gradient elution.
The eluent that obtains according to the method for the invention can further extract and purification as required.
To describe the present invention through concrete embodiment below,, repeat no more with top description repetition part among the embodiment for for simplicity.In addition, be noted that following embodiment only represents some preferred implementation of the present invention, although some embodiment is not obtained significant especially beneficial effect, this does not also mean that restriction scope of the present invention or its enforcement.On the contrary; According to instruction of the present invention; Those skilled in the art can be according to principle of the present invention, selects suitable enzyme to carry out the clarifying treatment of Chinese medicine extract according to factors such as the viscosity of the kind of employed Chinese crude drug, Chinese medicine extract, reaction temperature, reaction vessels.
Embodiment
Embodiment 1
Getting Fructus Gardeniae processes the water extract and is concentrated into relative density 1.036 (25 ℃); (Beijing ease pavilion Bioisystech Co., Ltd provides to add the cellulase of the 0.5-1 weight % of entry extract respectively; 4000IU/g tires), (Shanghai pattern ocean Bioisystech Co., Ltd provides pectase; Tire＞500U/mg), (Beijing ease pavilion Bioisystech Co., Ltd provides xylanase, tires 1,000,000/g), (Beijing rope comes precious Bioisystech Co., Ltd to provide to neutral protease, tires 1,000,000/g), (Beijing ease pavilion Bioisystech Co., Ltd provides papain; Tire 1,000,000/g), (Beijing ease pavilion Bioisystech Co., Ltd provides 1,4 beta-glucanase; Tired 1: 3000), compound enzyme (pectase: cellulase=1: 1), at pH value=3-5, under the condition that temperature is 50 ℃ enzymolysis 2-3 hour.Use black formula viscosimeter to measure the intrinsic viscosity of water extract behind the enzymolysis, the result is presented in the table 1.
Add the result behind the different enzymes in the table 1 Fructus Gardeniae aqueous extract
From table 1, it is thus clear that after utilizing enzymatic treatment, can significantly reduce the viscosity of Chinese medicine extraction liquid, wherein the effect of cellulase, pectase and compound enzyme is best.
Embodiment 2
Get Radix Asparagi, the Cortex Eucommiae, Flos Farfarae, Fructus Lycii, 5 kinds of medical materials of Rhizoma Polygonati respectively; Process the water extract and be concentrated into more than the relative density 1.026 (25 ℃); 1 weight % with the water extract adds compound enzyme (pectase: cellulase=1: 1) in various water extracts respectively; In PH=3～5, enzymolysis is 2～3 hours under the condition that temperature is 50 ℃.Use black formula viscosimeter to measure the viscosity of water extract clear liquor behind the enzymolysis.
Viscosity changes before and after the enzyme-added processing of the aqueous extract of the different medical materials of table 2
It is thus clear that after utilizing enzymatic treatment, compound enzyme can significantly reduce the viscosity of plurality of Chinese extracting solution.
List of references
[1] Yang Jun, Zhao Xuehui. pectase is to the research [J] of fruit and vegerable system juice effect. food science and technology .1998 (3): 27～29.
[2] Zhang Yingjiu, Jin Chengri, Wang Hongmei, etc. the immobilization research [J] of pectase. biotechnology .1996,6 (2): 26～29.
[3] Liu Songtao. several kinds of application [J] of defecation method in fruit-vegetable juice beverage is produced. Guangxi light industry 1999 (2): 37～38.
Claims (9)
1. the method for the refining Chinese medicine extract of an enzyme process, this method may further comprise the steps:
1) obtain the water or the alcohol extract of Chinese crude drug, concentrating becomes the Chinese medicine concentrated extracting solution;
2) the Chinese medicine concentrated extracting solution that step 1) is obtained is added in the reaction vessel; The amount that in reaction vessel, adds the 0.01-5 weight portion by the said Chinese medicine concentrated extracting solution of per 100 weight portions then adds one or more and is selected from following enzyme: cellulase, pectase, neutral protease, xylanase, neutral protease, papain and 1,4 beta-glucanase form mixture and the mixture that obtains are stirred in reaction vessel to form uniform mixture;
3) with step 2) the middle uniform mixture that obtains is under 40～60 ℃, and reaction is 2-3 hour under the condition of pH 3-5, obtains the Chinese medicine extract clear liquor.
2. method according to claim 1 is characterized in that described Chinese crude drug is the medical material that contains stickum.
3. method according to claim 1 and 2, the consumption that it is characterized in that enzyme described in the step 1) is for adding the 0.01-5 weight portion by the said Chinese medicine concentrated extracting solution of per 100 weight portions, preferred 0.05-1 weight portion.
4. according to each described method among the claim 1-3, it is characterized in that step 2) in the enzyme that uses be cellulase or pectase.
5. according to each described method among the claim 1-4, it is characterized in that step 2) in the enzyme that uses be the compound enzyme that two kinds of enzymes are formed, the combination of preferred cellulose enzyme and pectase, the proportioning of said two kinds of enzymes is 1: 1 by weight.
6. according to each described method among the claim 1-5; This method also comprises: the viscosity that detects the Chinese medicine extract clear liquor that obtains in the step 3); If the viscosity of the Chinese medicine concentrated extract that obtains in this viscosity and the step 1) is compared and reduced approximately below 5%, then adopt the combination repeating step 1 that is different from last employed enzyme or enzyme)-3).
7. according to each described method among the claim 1-5; The viscosity of the Chinese medicine concentrated extracting solution that obtains in the viscosity of the Chinese medicine extract clear liquor that it is characterized in that obtaining in the step 3) and the step 1) is compared and is reduced 5-95%, more preferably reduces 10-95% and most preferably reduces 20-95%.
8. according to each described method among the claim 1-6, this method also comprises: the Chinese medicine extract clear liquor that 4) step 3) is obtained is through absorption with macroporous adsorbent resin, and collects eluent.
9. method according to claim 8 is characterized in that the macroporous adsorbent resin that uses in the step 4) is selected from nonpolar to the low pole macroporous resin any.
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| CN106333912A (en) * | 2016-11-11 | 2017-01-18 | 福建师范大学 | Preparation method of dendrobium officinale essence |
| CN107691956A (en) * | 2017-09-27 | 2018-02-16 | 广西宜州市宜源桑杆菌业有限公司 | A kind of mulberry leaf beverage particle and preparation method thereof |
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| CN113398186A (en) * | 2021-08-20 | 2021-09-17 | 山东中医药大学附属医院 | Viola yedoensis makino extract and preparation method and application thereof |
| CN113827522A (en) * | 2021-10-18 | 2021-12-24 | 山东花物堂生物科技有限公司 | A cortex Phellodendri extract for cosmetic and its preparation method |
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| CN102964461B (en) * | 2012-11-20 | 2015-02-04 | 六安同济生生物科技有限公司 | Auxiliary extraction method of biological enzyme for improving dissolution rate of dendrobe bioactive polysaccharide |
| CN103070970A (en) * | 2013-01-07 | 2013-05-01 | 河南中医学院 | Preparation method of fresh rehmannia root granule |
| CN103070970B (en) * | 2013-01-07 | 2014-05-21 | 河南中医学院 | Preparation method of fresh rehmannia root granule |
| CN106333912A (en) * | 2016-11-11 | 2017-01-18 | 福建师范大学 | Preparation method of dendrobium officinale essence |
| CN107691956A (en) * | 2017-09-27 | 2018-02-16 | 广西宜州市宜源桑杆菌业有限公司 | A kind of mulberry leaf beverage particle and preparation method thereof |
| CN108434381A (en) * | 2018-05-14 | 2018-08-24 | 雷山县三元科技发展有限责任公司 | A kind of Chinese medicine and preparation method thereof for treating Low Back Pain kidney deficiency |
| CN113398186A (en) * | 2021-08-20 | 2021-09-17 | 山东中医药大学附属医院 | Viola yedoensis makino extract and preparation method and application thereof |
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| CN113827522A (en) * | 2021-10-18 | 2021-12-24 | 山东花物堂生物科技有限公司 | A cortex Phellodendri extract for cosmetic and its preparation method |
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