JP2021048794A - Ceramide aglycone-containing composition, and production method - Google Patents

Abstract

To provide a composition containing ceramide aglycone (free ceramide) obtained from sphingoglycolipid, and a production method for the same.SOLUTION: The ceramide aglycone-containing composition is obtained by making enzyme derived from black Aspergillus act on a sphingoglycolipid-containing composition; in particular, the sphingoglycolipid is β-glucosylceramide; the enzyme derived from black Aspergillus is enzyme having a β-glucosidase activity derived from Aspergillus, and by making the enzyme act, the composition containing ceramide aglycone can be produced at an arbitrary ratio.SELECTED DRAWING: Figure 1

Description

The present invention relates to a ceramide aglycone-containing composition obtained by allowing an enzyme to act on glycosphingolipid, and a method for producing the same. More specifically, the present invention relates to a ceramide aglycone-containing composition obtained by reacting β-glucosylceramide with an enzyme having β-glucosidase activity derived from Jiuqu, and a method for producing the same.

It has been reported that free ceramide is widely distributed in living organisms and has various physiological activities such as cell proliferation inhibitory action such as cell differentiation, apoptosis, and cell cycle arrest in cells. Besides that, the major role of free ceramide is that it exists as the main component of lipids between skin keratinocytes, and it has the function of protecting the skin from external stimuli as a barrier film between the living body and the outside world, which is inherent in the skin, and water It is responsible for the moisturizing function of retaining and the adhesive function of keratinocytes.

It is known that when the amount of free ceramide in keratinocytes decreases due to aging or skin disease, its barrier function and water retention function decrease, the skin becomes dry easily, and skin aging is promoted. In fact, skin diseases such as dry skin, rough skin, atopic dermatitis, and xerosis (dry skin) may be caused by a decrease in moisturizing function and barrier function due to a decrease in free ceramide in the stratum corneum. It has been reported. It is known that external supplementation of free ceramide is effective for reducing the barrier function and water retention function of the stratum corneum.

In recent years, cosmetics and supplements containing ceramides (free ceramide and / or glycosphingolipid, etc.) have been attracting attention. In the past, the source of ceramides was limited to horse spinal cord and bovine brain extract, but due to price, purity, BSE (bovine spongiform encephalopathy) problems, etc., use a large amount of safe high-purity products. Was difficult. In order to solve this, studies have been carried out to extract ceramides from various plants. As a result, rice, soybeans, wheat, corn, konjac, maitake mushrooms, peaches, pineapples, etc. have become known as effective sources of ceramides.

These plants have the potential to supply large quantities of ceramides at low cost. However, ceramides in plants are glycosphingolipids in which sugar is bound to ceramide, and most of them are β-glucosylceramide in which glucose is β-bonded to ceramide.
After β-glucosylceramide is taken up in the living body, especially in the skin, glucose is excised by enzymes such as glucocerebrosidase existing in the living body to produce ceramide aglycone and exert various useful physiological activities. However, the abundance of glucocerebrosidase decreases with aging, or the presence of β-glucosyl group of glycosphingolipid reduces lipophilicity, so that it is difficult to be absorbed transdermally. It is desirable to administer as ceramide).

In addition, ceramides have been attracting attention as a functional food material in recent years, and in addition to their skin function improving effect, they exhibit physiological activities useful for maintaining human health, such as anti-inflammatory action on the intestinal tract and suppression of colon cancer. Has been reported.
When glucosylceramide is taken into the body, it is thought that it is absorbed from the intestinal wall as ceramide aglycone after the sugar portion is cut by the action of degrading enzymes present in the intestine and intestinal bacteria. That is, even when used orally, ceramide aglycone (free ceramide) from which the sugar portion has been removed in advance is desirable.

β-Glucocerebroside is a type of glycoside. Generally, in order to obtain aglycone from glycosides, there is classically an acid hydrolysis method, and recently, a method such as exposure to high temperature and high pressure conditions such as subcriticality has been proposed. No efficient method for producing ceramide aglycone using such a chemical or physical decomposition reaction has been reported.
Further, it is disclosed that a method for selectively removing terminal sugars from glycosides using β-glucosidase is used for the production of various aglycones (Patent Document 1 and Non-Patent Document 1). There is no example of application to β-glucosylceramide.

Furthermore, there have been some reports on the hydrolysis reaction under mild conditions using enzymes, and endoglycoceramidese (EGCase), which hydrolyzes the sugar-ceramide bond of glycosphingolipid (β-glucosylceramide), is used. A method of releasing ceramide from glycosphingolipid using it is known (Patent Documents 2 and 3).

It has been reported that the EGCase used in this method is produced by Rhodococcus microorganisms, Corynebacterium microorganisms, leeches, earthworms, freshwater clams, jellyfish, hydra and the like. Further, artificial variants of EGCase with improved activity and substrate specificity have been developed (Patent Documents 4 and 5).
However, until now, a means for obtaining a large amount of EGCase at a low cost, for example, a mass production method using a microorganism incorporating an enzyme gene, has not been known, and therefore, an enzymatic reaction of ceramide aglycone from glycosphingolipid industrially. There was no production method to liberate in.

On the other hand, it is known that fermented lees, which is a by-product of the production of fermented products using koji such as sake, shochu, beer, or soy sauce, contains glycosphingolipid and a trace amount of free ceramide. Has been done. This is because by fermenting a plant as a raw material that originally contains glycosphingolipid, the sugar portion of glycosphingolipid is cut out by the action of enzymes produced by aspergillus and yeast in the production process.
A method for producing a ceramide aglycone-containing substance for extracting the fermented meal thus obtained with alcohol or the like has been reported (Patent Documents 6 and 7).

However, such fermented lees contain a wide variety of substances other than the target ceramide aglycone, and the purity of the ceramide aglycone in the alcohol extract is relatively low. In order to obtain a composition containing a high concentration of ceramide aglycone, it is necessary to additionally perform various complicated purification operations such as chromatograph fee for the alcohol extract. It's hard to say.

Japanese Unexamined Patent Publication No. 2001-204486 Special Fair 07-089914 JP-A-2017-195855 Japanese Unexamined Patent Publication No. 06-007161 Special Table 2008-502329 Gazette JP-A-2007-82427 Japanese Unexamined Patent Publication No. 2000-80394

Wakako Egashira: Research Report of Toyo College of Food Research, Vol. 30, 101-103 (2014)

An object of the present invention is to prepare a composition containing ceramide aglycone (free ceramide) from glycosphingolipid in an arbitrary ratio by using an inexpensive and mass-available food processing enzyme instead of an expensive enzyme such as EGCase. It is an object of the present invention to provide a method for efficiently producing a composition containing high-purity ceramide aglycone (free ceramide) without using a multi-step purification method.

The present inventors have made diligent studies to solve the above problems, and when a food processing enzyme derived from black aspergillus is allowed to act on glycosphingolipid, an efficient hydrolysis reaction occurs and ceramide aglycone can be easily produced. I found.
The present invention has been completed by repeated diligent studies based on such findings.

That is, the present invention provides a composition containing ceramide aglycone, and a method for producing the same. Furthermore, it is used orally because it contains a large amount of free ceramide, which is effective as a component of cosmetics or external skin preparations useful for healing atopic dermatitis as a functional food or moisturizer, or free ceramide, which is extremely rare in nature. The present invention has been completed by finding that it is possible to provide a ceramide aglycon-containing composition capable of providing the same composition.

A more specific invention has the following configuration.
(1) A ceramide aglycone-containing composition obtained by allowing an enzyme derived from Jiuqu to act on a glycosphingolipid-containing composition;
(2) The ceramide aglycone-containing composition according to (1) above, wherein the glycosphingolipid is β-glucosylceramide;
(3) The ceramide aglycone-containing composition according to (1) above, wherein the enzyme derived from Aspergillus is an enzyme derived from Aspergillus and having β-glucosidase activity;
(4) A method for producing a ceramide aglycone-containing composition, which comprises allowing an enzyme derived from Jiuqu to act on a glycosphingolipid-containing composition;
(5) The method for producing a ceramide aglycone-containing composition according to (4) above, wherein the glycosphingolipid is β-glucosylceramide;
(6) The method for producing a ceramide aglycone-containing composition according to (4) above, wherein the enzyme of Aspergillus japonicus is an enzyme derived from Aspergillus and having β-glucosidase activity.

The ceramide aglycone-containing composition produced by the present invention is characterized by containing free ceramide.
Therefore, the ceramide-containing composition produced by the present invention can be used as a raw material for cosmetics and functional foods. In particular, the ceramide aglycon-containing composition of the present invention can be obtained by using only substances used in food production in the production method thereof, and therefore, cosmetic raw materials and functionality in which safety is particularly important. It is extremely useful as a food raw material.
In addition, since an inexpensive food processing enzyme is used and the operation is simple, it is possible to industrially obtain a large amount of a composition containing an arbitrary ratio of human-type-like free ceramide, which is extremely rare in nature. It is.

FIG. 1 is a chart showing LC / MS of enzymatic decomposition of rice ceramide by Morcin F, and shows a comparison between before and after the enzymatic reaction.

Hereinafter, the present invention will be described in detail.
First, the glycosphingolipid-containing composition which is the raw material of the present invention is not particularly limited whether it is derived from a plant or an animal, but for the purpose of the present invention, it is derived from a plant having a high content ratio of glycolipid in which a sugar residue is present. Is desirable. Of these, glycosphingolipids are preferably glucocerebrosides (β-glucosylceramides) because the enzymatic reaction is likely to proceed. That is, specific examples of the raw material include β-glucosylceramide derived from rice, soybean, peach, corn, wheat, konjac, pineapple and the like.

The enzyme to be acted on next is not particularly limited as long as it is commercially available, is inexpensively distributed, can be used in large quantities, and has β-glucosidase activity. However, from the viewpoint of the efficiency of the hydrolysis reaction, the one derived from black aspergillus is superior.
Among the black aspergillus, Aspergillus is preferable. More specifically, the food processing enzyme "Morcin F" derived from Aspergillus saitoi (Kikkoman Biochemifa Co., Ltd.), the food processing enzyme "Sumiteam BGA" derived from Aspergillus niger (Shin Nihon Kagaku Kogyo Co., Ltd.), Aspergillus Examples thereof include niger-derived food processing enzyme "cellulase A" (Amano Enzyme Co., Ltd.), but "Morcin F" is preferable because of its particularly high reactivity.

The solvent used for the enzymatic reaction is particularly limited as long as it is commercially available, can be distributed inexpensively, can be used in large quantities, and can dissolve the raw material glucosylceramides and / or the enzyme for food processing. Not done.
Examples of solvents that can dissolve both are DMF (dimethylformamide) and DMSO (dimethylsulfoxide), and examples of solvents that mainly dissolve glucosylceramides are n-hexane, toluene, and acetone. Examples thereof include ethyl methyl ketone, tetrahydrofuran, 1,2-dioxane, n-butanol, 1,2-dichloroethane and the like.

An example of a solvent primarily intended to dissolve food processing enzymes is water. These may be used alone or in combination. Further, it can be used in the form of a two-phase reaction in which both n-hexane and water do not mix.

The concentration when used in the reaction is also not particularly limited, but is, for example, 1 to 50% (w / v), preferably 5 to 20% (w / v) for glucosylceramide, and 1 to 50% for food processing enzymes, for example. (W / v), preferably 3 to 15% (w / v) is dissolved and used.

Other than the solvent can be added into the reaction system as long as it does not interfere with the progress of the reaction. For example, during a two-phase reaction, salt can be added to improve the separation of the two phases. The liquid property (pH) of the reaction is usually close to neutral, but the liquid property may be adjusted for the purpose of adjusting to the optimum pH of the enzyme for food processing. In order to adjust the liquid property, not only hydrochloric acid and sodium hydroxide, which are inorganic acids and alkalis, but also organic acids and organic bases such as acetic acid and triethylamine can be used.

As another condition for carrying out the enzymatic reaction, the reaction temperature is not particularly limited as long as the solvent does not evaporate or freeze, but is usually carried out at room temperature to 60 ° C.
As for the reaction time, the reaction can be continued until the raw material glucosylceramide disappears, but it is not always necessary to eliminate all of them. An example of the reaction time is 1 to 30 days.

In some cases, the raw material glucosylceramide and / or the enzyme for food processing may be added during the reaction. The reaction solution may be allowed to stand or stirred, but it is desirable to stir the reaction solution in order to make the temperature, concentration and the like uniform. In particular, when carrying out a two-phase reaction, it is desirable to stir vigorously so that contact between the two phases can easily occur.

As a treatment method after the reaction is completed, first, if necessary, filtration is performed to remove insoluble matter. The filter medium is not particularly limited, but one in which the ceramide-containing composition is not irreversibly adsorbed is preferable. Examples include glass filters, cotton plugs, filter papers, filter cloths, membrane filters, cellulose powders, cellites, activated carbon and the like.
A crude product can be obtained by removing the solvent of the obtained filtrate by a method such as distilling off at normal pressure or reduced pressure, freeze-drying, and spray-drying. The crude product may be used as it is as a composition containing ceramic aglycon, but if necessary, chromatography using a carrier such as silica gel, ODS, or alumina, deadsorption using an ion exchange resin, a synthetic adsorbent, or the like. , Precipitation treatment using a difference in solubility, purification treatment using a method such as recrystallization may be performed.

The content of ceramide aglycone in the ceramide aglycone-containing composition produced according to the present invention can be arbitrarily adjusted by adjusting the reaction conditions, the ratio of the amount of the substrate to the enzyme, and other conditions of the enzyme reaction. It is also possible to isolate only ceramide aglycone if necessary.

The ceramide aglycone-containing composition produced according to the present invention is characterized by containing free ceramide.
In particular, since the ceramide aglycon-containing composition of the present invention can be obtained by using only substances used in food production in its production method, cosmetic raw materials and functions in which safety is particularly important. It is extremely useful as a raw material for sex foods.

Hereinafter, the method for producing the ceramide aglycone-containing composition of the present invention will be described in more detail based on specific examples and the like. However, the present invention is not limited thereto.

Example 1: Production of rice-derived ceramide aglycone (1) 3.0 g of rice-derived glycosphingolipid (manufactured by Okayas Co., Ltd., glucosylceramide content 77.2%) is dissolved in 15 mL of n-hexane and placed in a glass container. Stirred.
(2) Add 60 mL of 0.1 M acetate buffer (pH 4.5) to this, and add 0.45 g of a food processing enzyme (manufactured by Kikkoman Biochemifa Co., Ltd., trade name "Morcin F") at 40 ° C. The mixture was allowed to react for 2 days while stirring well. Then the stirring was stopped and the aqueous layer was removed using a separatory funnel.
(3) After repeating the operation of (2) above 5 times, the hexane layer was filtered with a cotton stopper to remove insoluble substances, and then concentrated to dryness under reduced pressure with a rotary evaporator. 2.8 g of the substance was obtained.
(4) The obtained crude product was subjected to silica gel column chromatography (silica gel 60, 140 g manufactured by Merck Co., Ltd.) and eluted with chloroform: methanol: water (65: 3: 0.3) to obtain a ceramide aglycon fraction 1. 0.04 g was obtained (yield 57%). Further, a raw material glucosylceramide fraction of 0.60 g was obtained (recovery rate 26%).

(5) The results of liquid chromatography-mass spectrometry (LC-MS analysis: Kazusa DNA Research Institute, LipidSearch, positive ion mode) of the ceramide aglycone fraction obtained in (4) above are shown in FIGS. 1 and 1. It was.

(6) For reference, the results of LC-MS analysis (Kazusa DNA Laboratory, LipidSearch, positive ion mode) of the raw material rice-derived glycosphingolipid (manufactured by Okayas Co., Ltd.) are also shown in FIG. And are shown in Table 1.
In Table 1, what is labeled as "before reaction (1HexCer)" is the result of the raw material glycosphingolipid derived from rice, and what is labeled as "after reaction (Cer)" is the obtained ceramide aglycone fraction. The result.

Table 1: Analysis results of LC-MS peaks before and after the enzymatic reaction

1HexCer: β-glucosylceramide
Cer: Ceramide aglycone
SG: Sphingosine
FA: 2-Hydroxy fatty acid
M + H: Molecular weight + H

As can be seen from the results shown in FIG. 1 and Table 1, in the obtained ceramide aglycone fraction, the main peak (RT = 40.59 min) was m / z (M + H); 608.5619, Lipid Class; It was identified as Cer, Fatty Acid; d18: 2_20: 0 + O), Formula; C38H74O4N1.
In addition, it was confirmed that five types of rice-derived sphingolipids were produced.

On the other hand, in the rice-derived glycosphingolipid (manufactured by Okayas Co., Ltd.), which was examined for reference, the main peak (RT = 37.43 min) was m / z (M + H) 770.6146, Lipid Class; Hex1Cer. , Fatty Acid; d18: 2_20: 0 + O, Formula; C44H84O9N1.
In addition, five types of glycosphingolipids, which are raw materials for ceramide aglycone, were also identified.

(7) In order to precisely purify the obtained ceramide aglycone fraction, 200 mg thereof was subjected to preparative ODS column chromatography (Universal column ODS premium 30 μL manufactured by Yamazen Corporation) and eluted with methanol. A fraction of 40 mg was obtained.
Similarly, 200 mg of rice-derived glycosphingolipid (manufactured by Okayas Co., Ltd.), which is a raw material, was precisely purified by preparative ODS column chromatography to obtain a main fraction of 30 mg.

The NMR of these precision purified fractions was measured, and N-2'-hydroxyicosanoyel-4E, 8Z-sphingadiene (corresponding to Cer1 in Table 1), and N-2'-hydroxyicosanoyl-O-β-D-glucopyayanoyyl-, respectively. It was confirmed that it was 4E, 8Z-sphingagene (corresponding to HexCer1 in Table 1).

From the above, it can be seen that ceramide aglycone can be easily obtained by allowing a specific food processing enzyme having β-glucosidase activity to act on rice-derived glucosylceramide. The purification operation (chromatography) performed here is for the purpose of calculating the yield and analyzing the chemical structure, and is not necessarily an essential operation when producing the ceramide aglycone-containing composition.

Example 2: Production of peach-derived ceramide aglycone (1) 1.0 g of peach-derived glycosphingolipid (manufactured by Okayas Co., Ltd., glucosylceramide content 37%) was dissolved in 20 mL of toluene, placed in a glass container, and stirred.
(2) Add 20 mL of 0.1 M acetate buffer (pH 4.5), and add 0.15 g of a food processing enzyme (manufactured by Kikkoman Biochemifa Co., Ltd., trade name "Morcin F") at 50 ° C. The mixture was allowed to react for 24 hours while stirring well. Then the stirring was stopped and the aqueous layer was removed using a separatory funnel.
(3) After repeating the above operation (2) three times, the toluene layer was filtered with a cotton stopper to remove insoluble substances, and then concentrated to dryness under reduced pressure with a rotary evaporator. 0.9 g of the substance in the form was obtained.
(4) The obtained crude product was subjected to silica gel column chromatography (silica gel 60, 50 g manufactured by Merck Co., Ltd.) and eluted with chloroform: methanol: water (90:10: 1) to obtain a ceramide aglycone fraction of 110 mg. (Yield 38%). Moreover, 75 mg of the raw material glucosylceramide fraction was obtained (recovery rate 20% with a molecular weight of 714).

Example 3: Production of soybean-derived ceramic aglycon (1) 100 mg of glycosphingolipid derived from soybean (manufactured by Nagara Science Co., Ltd., glucosylceramide content 100%) is placed in a glass container, and 10 mL of distilled water is added to suspend it. 50 mg of sodium stearate and 40 mg of a surfactant were added thereto, and the mixture was stirred and dissolved.
(2) 100 mg of a food processing enzyme (manufactured by Amano Enzyme Co., Ltd., trade name "cellulase A") was added thereto, and the mixture was reacted at 55 ° C. for 3 days while stirring well.
When the stirring was stopped and the mixture was concentrated to dryness under reduced pressure with a rotary evaporator, 95 mg of an oily substance was obtained as a crude reaction product.
(3) The obtained crude product was subjected to silica gel chromatography (silica gel 60, 10 g manufactured by Merck Co., Ltd.) and eluted with chloroform: methanol: water (90:10: 1) to obtain a ceramide aglycone fraction of 11 mg. (Yield 14%). Although the glucosylceramide fraction of the raw material remained, it was not separated.

Reference Example 1: Production of free ceramide using endoglycoceramide (EGCase) (1) 100 mg of rice-derived glycosphingolipid (manufactured by Okayas Co., Ltd., glucosylceramide content 77.2%) dissolved in 0.5 mL of n-hexane. Then, it was placed in a glass container and stirred.
(2) To this, add 2 mL of 0.02 M tris-phosphate buffer (pH 7.5), add 10 mg of endoglycoceramidase I (manufactured by EGCase I, BioLabs Inc.), and react at 40 ° C. for 2 days while stirring well. It was. The stirring was then stopped and the aqueous layer was removed using a Komagome pipette.
(3) After repeating the above operation (2) three times, the hexane layer was filtered with a cotton stopper to remove insoluble substances, and then concentrated to dryness under reduced pressure with a rotary evaporator. 90 mg of the substance was obtained.
(4) The obtained crude product was subjected to silica gel column chromatography (silica gel 60, 10 g manufactured by Merck Co., Ltd.) and eluted with chloroform: methanol: water (65: 3: 0.3) to obtain a free ceramide fraction of 23 mg. Was obtained (yield 38%). Although the glucosylceramide fraction of the raw material remained, it was not separated.
(5) Silica gel thin-layer chromatographic analysis of the free ceramide fraction obtained in (4) above and the ceramide aglycone fraction obtained in (4) of Example 1 (TLCSilicagel60 manufactured by Merck, Glassplate, developing solvent). Chloroform: methanol: water (65:10:0.3)). Sulfuric acid spray-heating method was used for color development. As a result, the Rf values of both were completely the same. That is, it was found that the same ceramide aglycone was produced by the reaction with rice-derived glucosylceramide with Morsin F or endoglycoceramidese I.

Reference Example 2: Reaction of rice-derived glycosphingolipid with various hydrolases (1) 100 mg of rice-derived glycosphingolipid (manufactured by Okayas Co., Ltd., glucosylceramide content 77.2%) was dissolved in 0.5 mL of n-hexane. , Placed in a glass container and stirred.
(2) 2 mL of 0.1 M acetate buffer was added thereto, and 100 mg of each of the various hydrolases shown in Table 2 below was further added, and the mixture was reacted for 3 days with good stirring. For the liquid property and temperature, the optimum conditions for the enzymes used were applied.
(3) After filtering the hexane layer of the obtained reaction solution with a cotton plug, 0.5 mL of n-hexane was added and mixed to prepare a test solution for analysis. 1 μL of this analytical test solution was subjected to silica gel thin layer chromatography analysis (TLCSilicagel60 manufactured by Merck & Co., Glassplate, developing solvent; chloroform: methanol: water (65:10: 0.3)). Sulfuric acid spray-heating method was used for color development. Ceramide aglycone was detected as an orange-brown spot near Rf = 0.57.
The results of silica gel thin layer chromalography analysis are shown in Table 2 below.
As is clear from Table 2, it was shown that no ceramide aglycone was produced from hydrolases other than endoglycoceramidelase I, morcin F derived from Aspergillus, Sumiteam BGA, and cellulase A.

Table 2: Results of comparative experiments using various enzymes

View results:
〇: Reaction progresses △: Reaction progresses slightly ×: Almost ~ no reaction at all

As described above, the ceramide aglycone-containing composition produced by the method of the present invention is characterized by containing free ceramide, and can be used as a raw material for cosmetics and functional foods.
In particular, since the ceramide aglycon-containing composition of the present invention can be obtained by using only substances used in food production in its production method, cosmetic raw materials and functions in which safety is particularly important. It is extremely useful as a raw material for sex foods.
In addition, since it uses a safe and inexpensive enzyme for food processing and is easy to operate, it is possible to industrially obtain a large amount of a composition containing an arbitrary ratio of human-type-like free ceramide, which is extremely rare in nature. In that it can be used, its industrial applicability is enormous.

Claims (6)

A ceramide aglycone-containing composition obtained by allowing an enzyme derived from Jiuqu to act on a glycosphingolipid-containing composition. The ceramide aglycone-containing composition according to claim 1, wherein the glycosphingolipid is β-glucosylceramide. The ceramide aglycone-containing composition according to claim 1, wherein the enzyme derived from Aspergillus is an enzyme having β-glucosidase activity derived from Aspergillus. A method for producing a ceramide aglycone-containing composition, which comprises allowing an enzyme derived from Jiuqu to act on a glycosphingolipid-containing composition. The method for producing a ceramide aglycone-containing composition according to claim 4, wherein the glycosphingolipid is β-glucosylceramide. The method for producing a ceramide aglycone-containing composition according to claim 4, wherein the enzyme of Aspergillus japonicus is an enzyme having β-glucosidase activity derived from Aspergillus.

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