JP2926412B2 - α-Glycosyl-L-ascorbic acid, its production method and use - Google Patents

Description

The present invention relates to an α-glycosyl-L which does not exhibit direct reducing properties.
-With respect to ascorbic acid and its production method and use, more specifically, a novel substance which does not exhibit direct reducibility α-
Glycosyl-L-ascorbic acid and its biochemical production method, beverages and foods containing α-glycosyl-L-ascorbic acid, foods and drinks such as preference foods, preventive and therapeutic agents for susceptible diseases, ie, anti-sensitive diseases The present invention relates to the use of the composition in various compositions such as cosmetics such as skin care agents and skin lightening agents.

[Prior art] L-ascorbic acid has the formula [I]: And cannot be synthesized in vivo for humans, monkeys and guinea pigs, and is an essential nutrient vitamin C.

L-ascorbic acid participates in the living body, for example, in the hydroxylation reaction of proline and lysine necessary for the synthesis of collagen, which is a main component of biological connective tissue, and also reduces, for example, Fe ⁺⁺⁺ of cytochrome C. It is known to be involved in an oxidation-reduction reaction such as conversion to Fe ⁺⁺ , and furthermore to be involved in an immunopotentiating effect due to an increase in leukocytes, and plays an important role in maintaining and promoting the health of a living body. Scurvy is L-
It has long been known as an ascorbic acid deficiency and presents with weakened skin, petechiae, ecchymosis, gingival and submedullary hemorrhage. In order to prevent this and maintain health, the recommended daily requirement (RDA) of L-ascorbic acid is determined, and according to it, it is 60 mg for an adult male and 50 mg for an adult female in Japan.

The use of L-ascorbic acid is not limited to vitamin C fortification as an essential nutrient, but various chemicals such as acidulants, reducing agents, antioxidants, bleaching agents, stabilizers, etc. In reaction bases, foods and drinks, and also as prophylactic and therapeutic agents for susceptible diseases such as viral diseases, bacterial diseases, and malignant tumors, ie, anti-susceptible disease agents, as well as reducing agents, ultraviolet absorbers, and melanin production inhibitors To cosmetics as skin-beautifying agents, skin-whitening agents, etc.
The range is extremely wide.

The biggest drawback of L-ascorbic acid is that it is extremely unstable, susceptible to oxidative degradation, because it exhibits direct reducing properties,
Easily lose its biological activity.

As a method for stabilizing L-ascorbic acid,
Sugar derivatives of ascorbic acid have been proposed. For example,
Previously, the present inventors, "vitamins" Volume 43, 205-209
(1971), "Vitamin", Vol. 47, pp. 259 to 267 (1973) and JP-B-48-38158, disclose a method for synthesizing L-ascorbic acid glucoside by a biochemical method. .

However, all of these ascorbic acid glucosides are prepared in the same manner, and the ascorbic acid obtained is described in, for example, column 2, lines 14 to 16 of the publication, "The obtained derivative is No. 6 of ascorbic acid. Carbon first
It is described that the alcohol group forms a glucoside by an ether bond, '' and that the production is a sugar transfer reaction of an α-glucosyl group from maltose. This chemical structure has the formula [II]: The degree of stabilization is also superior to L-ascorbic acid, as is clear from the results in the table of Example 1 of the publication, but is still unstable. It has not yet been put to practical use.

Also, Ishido et al. In Japanese Patent Publication No. 58-5920 discloses a method for synthesizing an L-ascorbic acid sugar derivative by an organic chemical technique.

However, this ascorbic acid sugar derivative is described in column 7, line 6 to column 8, line 11 of the publication as 2,3-di-O-.
As apparent from the description of 21 types of β-D-glucopyranosyl-type L-ascorbic acid sugar derivatives such as (β-D-glucopyranosyl) -L-ascorbic acid, all D-glucose is β-linked L-ascorbic acid sugar derivative.

Also, Masamoto et al. Disclosed a method for synthesizing an L-ascorbic acid sugar derivative by an organic chemical method in Japanese Patent Application Laid-Open No. 58-198498, but this method is also a β-glucosyl-type L-ascorbic acid sugar derivative. is there.

In addition, the present inventors have examined these β-D-glucopyranosyl-type L-ascorbic acid sugar derivatives, and found that it is difficult to sufficiently exert a biological activity in a living body, particularly in a human. Furthermore, the synthesis method by the organic chemistry method has a complicated reaction and a low yield, and therefore is inferior in economic efficiency. In addition, in securing non-toxicity and safety of the derivative, considerable difficulty is required. It was also found that there were accompanying drawbacks.

As described above, all of the conventionally known L-ascorbic acid sugar derivatives are insufficient in stability, safety, physiological activity, economy and the like, and their realization has not yet been seen.

[Problems to be Solved by the Invention] To solve the drawbacks of the conventional L-ascorbic acid sugar derivative,
There is a strong demand for an L-ascorbic acid sugar derivative that has excellent stability, sufficiently exhibits the physiological activity of L-ascorbic acid in vivo, and is nontoxic and can be used with confidence.

[Means for Solving the Problems] The present invention has been made to solve the drawbacks of the conventional sugar derivatives of L-ascorbic acid. We have been working diligently toward L-ascorbic acid sugar derivatives.

As a result, a novel L-ascorbic acid sugar derivative which does not show direct reducing properties, is excellent in stability, is easily hydrolyzed in a living body, and is satisfactory in terms of bioactivity has been found.
Furthermore, the present invention was completed by establishing a method for producing the same, and establishing uses of the novel L-ascorbic acid sugar derivative in various compositions such as foods and drinks, anti-sensitive diseases and cosmetics. .

Further, it has been found that the α-glycosyl-L-ascorbic acid of the present invention is a substance that is produced and metabolized in vivo by ingesting L-ascorbic acid and an α-glucosyl sugar compound orally. By the α- of the present invention
Glycosyl-L-ascorbic acid is a substance in the body by nature, and can be said to be an ideal novel sugar derivative of L-ascorbic acid from the viewpoint of safety.

By the way, β-D synthesized by organic chemistry
-Glucosyl-type L-ascorbic acid sugar derivatives are not produced in vivo, and are therefore considered to be foreign substances for living organisms.

The α-glycosyl-L-ascorbic acid of the present invention may be produced by a biochemical method or an organic chemical method, regardless of the production method.

However, in general, L-
It is desirable to produce the solution containing ascorbic acid and the α-glucosyl sugar compound by a biochemical method in which a glycosyltransferase is allowed to act on the solution.

The term "not exhibiting direct reducibility" as used herein means, unlike L-ascorbic acid, that 2,6-dichlorophenolindophenol is not reductively decolorized.

As used herein, L-ascorbic acid means not only free L-ascorbic acid but also L-ascorbic acid salts such as an alkali metal salt or an alkaline earth metal salt of L-ascorbic acid, unless otherwise disadvantageous. Or
Means mixtures thereof. Therefore, as L-ascorbic acid used in the glycosyltransfer reaction of the present invention, not only free L-ascorbic acid, but also, if necessary, sodium L-ascorbate, calcium L-ascorbate and the like are appropriately used. .

Similarly, α-glycosyl-L-ascorbic acid, 2-O-α-D-glucosyl-L-ascorbic acid and the like referred to in the present specification are not limited to free acids, unless otherwise disadvantageous. It also means those salts.

The α-glucosyl sugar compound used in the present invention may be any compound capable of producing α-glycosyl-L-ascorbic acid that does not directly show a reducibility from L-ascorbic acid by simultaneously using a glycosyltransferase, such as maltose and maltose. Lamtooligosaccharides such as triose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and maltooctaose, dextrin,
Starch partial hydrolysates such as cyclodextrin and amylose, as well as liquefied starch, gelatinized starch, and soluble starch can be appropriately selected.

Therefore, in order to facilitate the production of α-glycosyl-L-ascorbic acid, an α-glucosyl sugar compound suitable for a glycosyltransferase is selected.

For example, as a glycosyltransferase, α-glucosidase (EC
3.2.1.20) when using maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, malto oligosaccharides such as maltooctaose, or a dextrin having a DE of about 5 to 60, A starch hydrolyzate or the like is preferable. When cyclomaltodextrin glucanotransferase (EC2.4.1.19) is used, the starch portion from cyclodextrin or a gelatinized product of less than DE1 to a dextrin of DE about 60 is used. Hydrolysates and the like are preferred, and when α-amylase (EC 3.2.1.1) is used, starch partially hydrolyzed from gelatinized starch with a DE less than 1 to dextrin with a DE of about 30 is preferred.

The concentration of L-ascorbic acid during the reaction is usually 1 W / V%
As described above, the content of α-glucosyl sugar compound is preferably about 0.5 to 30 times the concentration of L-ascorbic acid.

The glycosyltransferase used in the present invention, when acted on a solution containing L-ascorbic acid and an α-glucosyl sugar compound suitable for this enzyme, does not decompose L-ascorbic acid and at least L-ascorbic acid. One to several α-glucosyl groups are transferred to the alcohol group at the 2-position carbon to form α-glucosyl group.
Any substance that produces glycosyl-L-ascorbic acid may be used.

For example, α-glycosidase is an enzyme derived from animals such as kidneys of mice, intestinal mucosa of rats, small intestines of dogs and pigs, enzymes derived from plants such as rice seeds and corn seeds, furthermore, genus Mucor and Penicilliu (Penicilliu).
m) Mold belonging to the genus or Saccharomyces (Sacch
An enzyme derived from a culture obtained by culturing a microorganism such as yeast belonging to the genus aromyces or the like in a nutrient medium is a cyclomaltodextrin / glucanotransferase, and a bacterial culture belonging to the genus Bacillus or Klebsiella or the like. As the α-amylase, an enzyme derived from a bacterial culture belonging to the genus Bacillus or the like can be appropriately selected.

These glycosyltransferases do not necessarily need to be purified and used as long as the above conditions are satisfied. Usually, the purpose of the present invention can be achieved with a crude enzyme. If necessary, it may be used after being purified by various known methods. Also,
Commercially available glycosyltransferases can also be used. There is a close relationship between the amount of enzyme used and the reaction time, and usually the amount of enzyme is selected such that the reaction is completed in about 3 to 80 hours from the viewpoint of economy.

In addition, the immobilized glycosyltransferase can be advantageously used in a batch-wise or continuous manner for the reaction.

In the reaction method of the present invention, a glycosyltransferase is usually added to a solution containing L-ascorbic acid and an α-glucosyl sugar compound, and the enzyme is allowed to act sufficiently, usually at a pH of about 3
To 9 and a temperature of about 30 to 80 ° C. In addition, since L-ascorbic acid is susceptible to oxidative decomposition during the reaction, it is desirable to keep the anaerobic or reduced state as much as possible under light shielding, and if necessary, the reaction may be carried out in the coexistence of thiourea, sulfite and the like. It can be implemented advantageously.

If necessary, L-ascorbic acid and an α-glucosyl sugar compound can coexist during the growth of a microorganism having a transglycosylation reaction ability to produce the target substance.

The α-glycosyl-L- of the present invention which does not exhibit direct reducibility
Describing ascorbic acid, an α-D-glucosyl group is bonded to an alcohol group of at least the 2-position carbon of L-ascorbic acid, and the number of bonds is about 1 to 7 glucosyl groups having α-1,4 bonds. The individual substances are, for example, 2-O-α-D-glucosyl-L-ascorbic acid, 2-O-α-D-maltosyl-L-ascorbic acid, 2-O-α-D --- Malttriosyl-L
-Ascorbic acid, 2-O-α-D-maltotetraosyl-L-ascorbic acid, 2-O-α-D-maltopentaosyl-L-ascorbic acid, 2-O-α-D-maltohexa Osyl-L-ascorbic acid, 2-O-α-D
-Maltoheptaosyl-L-ascorbic acid. When produced by α-glucosidase,
Usually, only 2-O-α-D-glucosyl-L-ascorbic acid can be produced.
O-α-D-glucosyl-L-ascorbic acid has 2-
O-α-D-maltosyl-L-ascorbic acid, 2-O
-Α-D-maltotriosyl-L-ascorbic acid or the like can be mixed and produced.

When it is produced by cyclomaltodextrin glucanotransferase or α-amylase, it is generally possible to produce a mixture having a larger number of α-D-glucosyl groups than α-glucosidase. -It varies depending on the glucosyl sugar compound, but in general, in the case of cyclomaltodextrin glucanotransferase, the number of α-D-glucoxy groups is distributed to about 1 to 7, and in the case of α-amylase, Has a slightly narrower distribution than this. The product which is such a mixture is optionally subjected to α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.
2), it can be partially hydrolyzed by glucoamylase (EC 3.2.1.3) or the like to reduce the number of α-D-glucosyl groups. For example, when glucoamylase is allowed to act, 2-O-α-D-maltosyl-L-ascorbic acid or higher is hydrolyzed to give 2-O-α-
D-glucosyl-L-ascorbic acid can be accumulated and produced, and when β-amylase is acted on, it is mainly used in the production of 2-O-α-D-maltotriosyl-L-ascorbic acid or higher. Hydrolyzing the molecular substance, 2-O-α
-D-glucosyl-L-ascorbic acid and 2-O-
A mixture of α-D-maltosyl-L-ascorbic acid can accumulate and form.

As described above, the α-glycosyl-L-ascorbic acid-containing solution which does not show direct reducibility and is produced by various methods is generally not only α-glycosyl-L-ascorbic acid but also unreacted L-ascorbic acid. Although it contains an acid, an α-glucosyl sugar compound, etc., it can be directly used as an α-glycosyl-L-ascorbic acid-containing product. Usually, the enzyme is inactivated by heating the reaction solution or the like, and then filtered and concentrated to obtain a syrup-shaped product, and further to a powdery product containing α-glycosyl-L-ascorbic acid.

Further, when producing a purified α-glycosyl-L-ascorbic acid product, it comprises α-glycosyl-L-ascorbic acid and unreacted L-ascorbic acid, D-glucose, α-glucosyl sugar compound and the like. Separation means utilizing differences in molecular weight and affinity with contaminants, such as membrane separation, column chromatography, high-performance liquid chromatography, gel filtration chromatography, ion-exchange chromatography, etc.
Obtaining high-purity products can also be easily achieved. At this time, L-ascorbic acid, α-glucosyl sugar compound and the like to be separated can be advantageously used again as a raw material for the transglycosylation reaction. Also, after completion of the sugar transfer reaction, before the separation reaction such as chromatography, if necessary, for example, the insolubles generated by heating the reaction solution are removed by filtration or treated with activated carbon or the like. Protein substances and coloring substances in the reaction solution are adsorbed and removed, demineralized with a cation exchange resin (H type), adsorbed on an anion exchange resin (OH type), and washed with water. It is also possible to optionally use a combination of purification methods such as removal of glucose, α-glucose sugar compound, etc., followed by desorption treatment with an anion or salt solution.

The α-glycosyl-L-ascorbic acid thus obtained has the following features.

(1) It is extremely stable without direct reduction. L-
Unlike ascorbic acid, it is hard to cause Maillard reaction. Therefore, even if it coexists with amino acids, peptides, proteins, lipids, carbohydrates, physiologically active substances, etc., it does not cause useless reactions, but rather stabilizes these substances.

(2) undergoing hydrolysis to produce L-ascorbic acid,
It exhibits the same reducing and antioxidant effects as L-ascorbic acid.

(3) It is easily hydrolyzed into L-ascorbic acid and D-glucose by enzymes in the body, and exhibits the natural physiological activity of L-ascorbic acid.

(4) Since L-ascorbic acid and α-glucosyl sugar compound are substances produced and metabolized in vivo by oral ingestion, their safety is extremely high.

(5) In the case of a product containing a saccharide such as an α-glucosyl sugar compound, not only the effect of α-glycosyl-L-ascorbic acid is exerted, but also the saccharide is shaped, increased in volume,
In the case of a purified product from which saccharides have been removed, a sweetening effect can be exerted, and in the case of a purified product from which saccharides have been removed, although the shaping and weight-increasing effects are low, α-glycosyl-L-ascorbic acid can exert its original effect with a small amount. .

Due to these features, α-glycosyl-L-ascorbic acid, which does not show direct reducibility, is not only a highly stable and safe natural vitamin C enhancer, but also a taste improver, an acidulant, and a stabilizer. , As a quality improving agent, antioxidant, ultraviolet absorber, etc., foods and drinks, taste foods, also preventive agents for susceptibility diseases such as viral diseases, bacterial diseases, malignant tumors,
It can be advantageously used in various compositions, such as a therapeutic agent, further, a cosmetic agent such as a skin beautifying agent and a fair skin agent, and more preferably 0.001 W / W% or more.

Further, α-glycosyl-L-ascorbic acid is well harmonized with various substances having tastes such as sourness, salty taste, astringency, umami, and bitterness, and has high acid resistance and heat resistance.
Ordinary general foods and drinks, such as soy sauce, powdered soy sauce,
Miso, powdered miso, moromi, hishio, frikake, mayonnaise, dressing, vinegar, three tablespoons vinegar, powdered sushi vinegar, Chinese noodles, tentsuyu, noodle soup, sauce, ketchup, grilled meat sauce, curry roux, stew noodles, soup noodles, Various seasonings such as Dashi-no-Matsu, complex seasonings, mirin, new mirin, table sugar, coffee sugar, rice crackers, hail, rice, kalinto, fertilizer, rice cake, manju,
Various Japanese sweets such as uiro, bean jam, yokan, mizuyokan, nishikidama, jelly, castella, candy, bread, biscuit, cracker, cookies, pie, pudding, cream puff, waffle, sponge cake, donut, chocolate, chewing gum, caramel , Candy and other Western sweets, ice cream, sherbet and other ice confections, ice honey and other syrups, butter cream, custard cream, flower paste, peanut paste, fruit paste and other spreads, pastes, jams, marmalades, syrup pickles, Fruits such as sugar fruits, processed foods of vegetables, breads, noodles, cooked rice, cereal processed foods such as artificial meat, salad oils, oils and fats foods such as margarine, pickles such as Fukujinzuke, Betarazuke, Senmaizuke, Rakkyozuke 、 Takuan pickles, Motorui of pickles, such as elements of Na漬, ham, meat products such as sausage, fish meat ham, fish meat sausage, boiled fish paste, chikuwa, fish meat products such as halves,
Various delicacies such as sea urchin, salted squid, vinegar kelp, Sakisame, Fugu mirin dried, seaweed, wild vegetables, sardines, small fish,
Boiled tsukuda, boiled beans, potato salad, etc.
Vegetables such as kelp, tempura, kinshi eggs, milk drinks,
Eggs such as butter and cheese, dairy products, fish meat, meat, fruits,
Bottled vegetables, canned foods, synthetic liquor, brewed liquor, fruit liquor,
Alcoholic beverages such as Western liquor, coffee, cocoa, juice, tea, black tea, oolong tea, carbonated drinks, soft drinks such as lactic acid drinks, lactic acid drinks, pudding mix, hot cake mix, instant juice, instant coffee, instant shirako, instant soup, etc. It can be advantageously used in instant foods and drinks for purposes such as vitamin C enhancers, taste improvers, sour agents, quality improvers, stabilizers, and antioxidants. Livestock, poultry,
It is also convenient to use it in feeds and feeds for breeding animals such as bees, silkworms, fish, etc., for the purpose of enhancing vitamin C, taste improver, antioxidant, and improving palatability.

In addition, tobacco, troches, liver oil drops, multivitamin preparations, mouthwash, mouthwash tablets, mouthwash, tube feeding, oral medicine, injection, toothpaste, lipstick, eyeshadow, milky lotion, lotion, cream, foundation , Sunscreen, face-wash soap, shampoo, rinse, etc. various solid, paste, liquid taste, susceptibility disease prevention agent,
The therapeutic agent, ie, an anti-sensitivity disease agent, a skin beautifying agent, can also be advantageously used in combination with cosmetics such as a whitening agent,
Furthermore, it can be advantageously used by blending it into a plastic product or the like as an ultraviolet absorber or a deterioration inhibitor, or using it as a substrate for measuring α-glycoside hydrolase.

The susceptibility disease referred to in the present invention is a disease which is prevented or treated by α-glycosyl-L-ascorbic acid, and is, for example, a viral disease, a bacterial disease, a traumatic disease, an immune disease, an allergy. It may be a disease, diabetes, cataract, malignant tumor or the like. The shape of the prophylactic or therapeutic agent for α-glycosyl-L-ascorbic acid-sensitive disease can be freely selected depending on the purpose. Examples thereof include liquid preparations such as sprays, eye drops, nasal drops, gargles, and injections, pastes such as ointments, plaques, creams, and solid preparations such as powders, granules, capsules, and tablets. In the formulation, if necessary, other components,
For example, it may be used in combination with one or more of a therapeutic agent, a physiologically active substance, an antibiotic, an auxiliary agent, a bulking agent, a stabilizer, a coloring agent, a flavoring agent and the like.

The dose can be appropriately adjusted depending on the content, administration route, administration frequency and the like. Usually, about 0.00 per adult day
A range of 1 to 100 grams is preferred.

Also, in the case of cosmetics, it can be used according to the above-mentioned prophylactic and therapeutic agents.

As a method for incorporating α-glycosyl-L-ascorbic acid, in the steps until the products of the various compositions are completed, for example, known methods such as kneading, kneading, dissolving, dipping, spraying, coating, spraying, and pouring are used. Is appropriately selected.

When the α-glycosyl-L-ascorbic acid or 2-O-α-D-glucosyl-L-ascorbic acid of the present invention is a free acid, if necessary, it is converted to a metal hydroxide or a carbonate. Reaction with an aqueous solution of metal or the like
O-α-D-glucopyranosyl-L-ascorbic acid salts, for example, sodium salts, calcium salts, magnesium salts, iron salts, copper salts, zinc salts, etc., have pH adjustment and have both vitamin C action and minerals It can also be advantageous to prepare substances and utilize them as nutrient enhancers, chemicals and the like.

Hereinafter, typical examples of the α-glycosyl-L-ascorbic acid that does not exhibit direct reducing properties of the present invention will be described in detail using experiments.

Experiment 1 α-glucosidase preparation Rat small intestine was immersed in a 0.1 M phosphate buffer (pH 7.0) at 20 W / W.
%, Homogenized with a homogenizer, centrifuged (4,000 × g, 10 minutes), and added trypsin (manufactured by Merck) to the supernatant to a final concentration of 0.1 g / L. Maintain for 4 hours, then add 2 volumes of cold ethanol, centrifuge, dissolve the precipitate in 0.01 M phosphate buffer (pH 7.0), place in semi-permeable membrane, Dialysis was performed for 15 hours.

Then, according to a conventional method, DEAE-cellulose column chromatography, subjected to hydroxylapatite chromatography, to collect α- glucosidase active fraction,
This was freeze-dried to obtain an α-glucosidase standard.

This product has a specific activity of 60.6 (unit / mg protein) and a purification factor of 460
The activity yield was about 47%.

As used herein, one unit of activity is 0.1 M containing 1.35 mM EDTA.
750 μL acetate buffer (pH 6.0) and 4 W / V% maltose 250
After adding 100 μL of an appropriately diluted enzyme solution to the mixture with μL and reacting at 37 ° C. for 30 minutes, the reaction was stopped by keeping the mixture in boiling water for 3 minutes, centrifuged, and 20 μL of the supernatant was taken. Then, add 1 mL of a coloring reagent (glucose oxidase method, manufactured by Wako Pure Chemical Industries, trade name: glucose B test), and add
The amount of the enzyme that releases 1 μmole of glucose per minute at 37 ° C. under the condition that color development is maintained at 7 ° C. for 20 minutes and then absorbance at 505 nm is measured.

Experiment 2 α-D-glucosyl-L-ascorbic acid (1) Glycosyl transfer reaction 7.04 parts by weight of L-ascorbic acid, 12.8 parts by weight of maltose and 0.2 parts by weight of thiourea were added to a 0.2 M acetate buffer (pH 5.3).
Dissolved in 100 parts by weight, and the partially purified α-glucosidase preparation prepared by the method of Experiment 1 was used per maltose gram.
0.5 units were added and reacted at 50 ° C. for 5 hours under light shielding. Next, 4 volumes of a 1.06 W / V% metaphosphoric acid solution was added to inactivate the enzyme, and the reaction was stopped.

When this product was measured by high performance liquid chromatography, about 30% of L-ascorbic acid used in the reaction was converted to a sugar derivative.

(2) Purification The reaction stop solution of (1) was subjected to gel filtration chromatography using a gel filtration agent (trade name: Bio-Gel P-2) manufactured by Bio-Rad with water as an eluent to obtain α- A fraction containing a high content of D-glucosyl-L-ascorbic acid was collected, and then subjected to high performance liquid chromatography using a column manufactured by Shimadzu (Shim-pack ODS) with 0.3 W / W% acetic acid as an eluent. The fraction containing α-D-glucosyl-L-ascorbic acid was collected, concentrated under reduced pressure, freeze-dried and powdered to obtain a high-purity α-D-glucosyl-
An L-ascorbic acid sample was obtained at a yield of about 80 W / W% based on α-D-glucosyl-L-ascorbic acid in the reaction solution.

(3) Physicochemical properties As a typical example of the α-glycosyl-L-ascorbic acid of the present invention, the following high-purity α-D-glucosyl-L-ascorbic acid sample prepared in (2) was used. Properties were investigated.

Incidentally, as an example of those having a larger number of α-D-glucosyl groups than α-D-glucosyl-L-ascorbic acid,
Using α-D-glycosyl-L-ascorbic acid obtained by the method of Example A-1 to be described later, it was examined as much as possible and the properties thereof are shown in parentheses [].

Elemental analysis: C ₁₂ H ₁₈ O ₁₁ Theoretical value C = 42.6% H = 5.36% Actual value C = 42.3% H = 5.38% N <0.01% Molecular weight: Mass spectrometer (M-80B, manufactured by Hitachi, Ltd.)
As a result of measuring FD mass spectrum by using (M + H)
339 was observed as ⁺ (C ₁₂ H ₁₈ O ₁₁ MW = 338).

UV absorption spectrum: 260 nm at pH 7.0, 238 n at pH 2.0
m shows the absorption maximum.

[Shows properties substantially the same as this property. Infrared absorption spectrum: measured by the KBr tablet method.

 The results are shown in the figure.

[Shows properties substantially the same as this property. NMR: Measured using an NMR measurement device (JNM-GX400, manufactured by JEOL Ltd.).

The measurement solvent used was heavy water, and the pH of the solution at the time of measurement was 2.8.

TSP (sodium 3-trimethyl-silylprop) as internal standard
ionate-2,2,3,3-d ₄₎ was used. _{^{1 H-NMR δppm (D 2}} O) 3.50 (1H, dd, J = 9.5, J = 9.7Hz) 3.56 (1H, dd, J = 3.4, J = 9.5Hz) 3.75 (2H, d, J = 6.4Hz 3.78 (2H, d, J = 3.0Hz) 3.86 (1H, dd, J = 9.5, J = 9.5Hz) 4.02 (1H, dt, J = 9.7, J = 3.0Hz) 4.08 (1H, td, J = 6.4, J = 1.5 Hz) 4.91 (1H, d, J = 1.5 Hz) 5.52 (1H, d, J = 3.4 Hz)-Dissociation constant: pKa = 3.0 This value can be calculated by using a method such as J. Jernow. Tetrahedron, 35, 1483-1486 (197
9 years) Table 1 and Pao-Wen L
u) etc., Journal of Agricultural Food Chemistr
y) Referring to the dissociation constants (pKa) of various ascorbic acid derivatives shown in Table 2 of Vol. 32, pp. 21 to 28 (1984), in the case of this substance, the second position of the ascorbic acid portion is shown. Is involved in the α-D-glucosyl bond, and it is determined that the alcohol group at position 3 remains free.

-Methylation analysis L-ascorbic acid described in the above-mentioned Pao-Wen Lu et al. Document is methylated with diazomethane to mainly produce 3-O-methyl-L-ascorbic acid. The substance was methylated by the method of methylation reaction, and the obtained methylated product was hydrolyzed and analyzed. As a result, mainly, 3-O-methyl-L-ascorbic acid and D-glucose were produced.

From these NMR, dissociation constant, and methylation analysis data,
It is determined that the alcohol group at the 2-position carbon of L-ascorbic acid forms an α-glucoside bond with D-glucose through an ether bond.

-Solubility in solvent: water, 0.1N-caustic soda, 0.1N
-Easily soluble in acetic acid, slightly soluble in methanol and ethanol, insoluble in ether, benzene, chloroform and ethyl acetate.

[Shows properties substantially the same as this property. -Color reaction: does not show direct reducibility and does not decolorize 2,6-dichlorophenol indophenol. Does not show 2,4-dinitrophenylhydrazine reaction. A green color is produced by the anthrone sulfate reaction.

[Shows properties substantially the same as this property. Stability: (a) α-glucosidase action or 1N-hydrochloric acid, 100
Hydrolysis at 5 ° C. for 5 minutes produces L-ascorbic acid and D-glucose at a molar ratio of 1: 1.

[Hydrolyzed by glucoamylase, 2-O-α-
Produces D-glucosyl-L-ascorbic acid and D-glucose. (B) Not hydrolyzed by β-glucosidase.

[Shows properties substantially the same as this property. (C) 2-O-α-D-glucosyl-L-ascorbic acid and 6-O disclosed in JP-B-48-38158.
-Α-D-glucosyl-L-ascorbic acid and L-
The stability in aqueous solution with ascorbic acid was compared. That is, each sample was prepared at a concentration of 70 μM, pH 7.0 or pH
Adjusted to 2.0 and taken in the cell for the spectrophotometer, maintain this at 20 ° C, and with the spectrophotometer over time, pH 7.0, 260 nm
In the case of pH 2.0, the absorbance was measured at 245 nm, and the residual ratio (%) was measured and compared. The results are shown in the following table.

As is clear from the results in the table, 2-O-α-D-glucosyl-L-ascorbic acid is different from both 6-O-α-D-glucosyl-L-ascorbic acid and L-ascorbic acid. Very stable in aqueous solution.

[Shows properties substantially the same as those of 2-O-α-D-glucosyl-L-ascorbic acid. Physiological activity (a) Cytochrome C reducing activity Using 2-O-α-D-glucosyl-L-ascorbic acid, 6-O-α-D-glucosyl-L-ascorbic acid and L-ascorbic acid , Cytochrome C
Were compared.

That is, 0.1 M potassium phosphate buffer (pH 7.8) -0.2
0.5 mL of mM EDTA, 0.1 mL of 0.1 mM cytochrome C, and a fixed amount of water to make a final solution volume of 1 mL, and to this, 10 mM of each sample
Was added, and the change in absorbance at 550 nm at room temperature was measured with a spectrophotometer and compared. The reduction activity was determined by determining the absorbance difference (ΔA) / min / 10 μL from the initial reaction rate.

As a result, 2-O-α-D-glucosyl-L-ascorbic acid does not show a reducing activity unlike any of 6-O-α-D-glucosyl-L-ascorbic acid and L-ascorbic acid. There was found.

In addition, it was found that 2-O-α-D-glucosyl-L-ascorbic acid exhibited this reducing activity when hydrolyzed by the α-glucosidase preparation prepared by the method of Experiment 1.

(B) Collagen synthesis activity Using 2-O-α-D-glucosyl-L-ascorbic acid, 6-O-α-D-glucosyl-L-ascorbic acid and L-ascorbic acid, the collagen synthesis activity was measured. Examined.

That is, human fibroblasts (7 × 10 ⁴ cells / plate) were cultured in Eagle's MEM (containing 10 FBS) medium for 1 week, and ³ H-proline was added thereto at 4 μCi / mL and β-aminopropionitrile was added at mL. 20 μg per sample and each sample
The mixture was added to a concentration of 0.25 mM, and further cultured for 24 hours. 10 W / V% trichloroacetic acid was added thereto, and the collagen component in the culture was recovered and freeze-dried. Dissolve this preparation and adjust pH
After the treatment, collagenase (type III) treatment was performed at 37 ° C. for 90 minutes, centrifuged, and the radioactivity contained in the supernatant was measured to determine the collagen synthesis activity.

As a result, it was found that 2-O-α-D-glucosyl-L-ascorbic acid has a collagen synthesis activity equivalent to that of ascorbic acid.

In addition, the collagen synthesis activity of 6-O-α-D-glucosyl-L-ascorbic acid tended to be slightly inferior to these.

Based on the above physicochemical properties, α-glycosyl-L-ascorbic acid which does not show direct reducibility prepared in this experiment has the formula [III]: Has a chemical structure represented by

Also, 2-O-α-D-glucosyl-L-ascorbic acid as a representative example thereof has the formula [IV]: Has a chemical structure represented by

Experiment 3 Production in vivo In rats, L-ascorbic acid 1g and maltose 500mg
(10W / V% solution, 5mL) and orally, blood is collected over time,
Centrifugation was performed, and the supernatant (plasma) was subjected to high-performance liquid chromatography to confirm the peaks corresponding to α-D-glycosyl-L-ascorbic acid and a small amount of α-D-maltosyl-L-ascorbic acid. After oral administration
It was observed around 30 minutes, peaked at 180 minutes, then decreased rapidly, and disappeared from the blood at 360 minutes.

Among these peaks, a substance showing α-D-glycosyl-L-ascorbic acid which occupies most of the peaks was isolated and examined in detail. As a result, the aforementioned 2-O-α-D-glucosyl-L-
It turned out to be identical to the physicochemical properties of ascorbic acid.

Therefore, since α-glycosyl-L-ascorbic acid is a biological substance that is produced, metabolized, and eliminated in the living body, it can be said that its safety is extremely high.

Experiment 4 Acute toxicity test Using 7-week-old dd mice, orally administered the high-purity α-D-glucosyl-L-ascorbic acid sample prepared by the method of Experiment 2 (2), and acute toxicity test As a result, no deaths were observed up to 5 g per kg of body weight.

 Therefore, the toxicity of this substance is extremely low.

In addition, α- manufactured by the method of Example A-1 described later.
When this test was performed using glycosyl-L-ascorbic acid, similar results were obtained, and it was found that the toxicity was extremely low.

Hereinafter, as an example of the present invention, a production example of α-glycosyl-L-ascorbic acid that does not show direct reducibility is shown in Example A, and examples of use of α-glycosyl-L-ascorbic acid in various compositions are shown below. This will be described in Example B.

Example A-1 α-Glycosyl-L-ascorbic acid 9 parts by weight of α-cyclodextrin was dissolved by heating in 20 parts by weight of water, kept under reduction, and 3 parts by weight of L-ascorbic acid was added thereto. While maintaining the temperature at 60 ° C., cyclomaltodextrin glucanotransferase (available from Hayashibara Biochemical Laboratory Co., Ltd.) was added to the mixture at 150 units per α-cyclodextrin lamella, and reacted for 40 hours. The reaction solution was subjected to high performance liquid chromatography (manufactured by Shimadzu Corporation, LC-6;
Column, YMC AQ303 ODS; Eluent, 0.1M KH ₂ PO ₄ −H ₃ PO ₄ (pH
2.0); Flow rate, 0.5mL / min, detection, manufactured by JASCO Corporation, MULT-
340), L-ascorbic acid appeared at the position of 9.5 minutes, whereas newly generated α-D-glycosyl-L-ascorbic acid was 11.2 minutes, and α-D-maltosyl-L-ascorbic acid was found. Acid for 15.7 minutes, α-D-maltotriosyl-L-ascorbic acid for 20.6 minutes, α-D-maltotetraosyl-L-ascorbic acid for 24.9 minutes, α-D-
Maltopentaosyl-L-ascorbic acid was 28.1 minutes, α
-D-maltohexaosyl-L-ascorbic acid is 32.1
Min and α-D-maltoheptaosyl-L-ascorbic acid appeared at 38.6 minutes. About L-ascorbic acid
50% had converted to these α-glycosyl-L-ascorbic acids. The reaction solution is heated to inactivate the enzyme, filtered, and the filtrate is subjected to purification and isolation of each component of α-glycosyl-L-ascorbic acid according to the method of Experiment 2 (2). The α-glycosyl-L-ascorbic acid contained was collected, concentrated under reduced pressure, and powdered to obtain a powdered product of α-glycosyl-L-ascorbic acid of about 90 W relative to the raw material L-ascorbic acid per solid. / W% yield.

This product does not show direct reduction, has sufficient stability and physiological activity, and is not only a vitamin C enhancer, but also a stabilizer,
It can be freely used as a quality improving agent, an antioxidant, a physiologically active agent, an ultraviolet absorber and the like for various compositions such as foods and drinks, anti-sensitivity agents and cosmetics.

Example A-2 40 parts by weight of α-glycosyl-L-ascorbic acid dextrin (about 6 DE) was dissolved in 50 parts by weight of water under heating, kept under reduction, and 13 parts by weight of L-ascorbic acid was added. 6. While maintaining the temperature at 65 ° C., 270 units of cyclomaltodextrin glucanotransferase was added thereto, and the mixture was reacted for 40 hours. The reaction solution was analyzed by high performance liquid chromatography in the same manner as in Example A-1.
65% was α-D-glycosyl- as in Example A-1.
L-ascorbic acid, α-D-maltosyl-L-ascorbic acid, α-D-maltotriosyl-L-ascorbic acid, α-D-maltotetraosyl-L-ascorbic acid, α-D-maltopenta It was converted to α-glycosyl-L-ascorbic acid such as osyl-L-ascorbic acid and α-D-maltohexaosyl-L-ascorbic acid. The reaction solution was heated to inactivate the enzyme, filtered, and the filtrate was decolorized and purified with activated carbon by a conventional method, concentrated and concentrated to obtain a syrup-like α-glycosyl-containing α-glucosyl sugar compound.
An L-ascorbic acid product was obtained at about 90% w / w based on the weight of the raw material per solid.

In this product, α-glycosyl-L-ascorbic acid contained in the product does not show direct reducing properties, has sufficient stability and physiological activity, and is used not only as a vitamin C enhancer but also as a seasoning,
Humectants, stabilizers, quality improvers, antioxidants, bioactive agents,
As an ultraviolet absorber, it can be advantageously used for various compositions such as foods and drinks, anti-sensitivity agents, and cosmetics.

Example A-3 2-O-α-D-glucosyl-L-ascorbic acid A syrup-like α prepared according to the method of Example A-2
Α-Glycosyl-L- containing a glucosyl sugar compound
One part by weight of an ascorbic acid product is dissolved in 4 parts by weight of water, and glucoamylase (EC 3.2.1.3, sold by Toyobo Co., Ltd.) is added thereto at 100 units per gram of the solid product.
The reaction was performed for 5 hours. When the reaction solution was analyzed by high performance liquid chromatography, α-glycosyl-L-ascorbic acid containing each component was converted to 2-O-α-D-glucosyl-L-ascorbic acid.

The reaction solution is heated to inactivate the enzyme, filtered, and the filtrate is purified according to the method of Experiment 2 (2) to collect a 2-O-α-D-glucosyl-L-ascorbic acid-rich fraction. , Concentrated under reduced pressure, powdered, and high purity 2-O with a purity of 99.0% or more.
-Α-D-glucosyl-L-ascorbic acid as raw material L
-Obtained in a yield of about 80 W / W% relative to ascorbic acid.

When the physicochemical properties of this product were examined, it was substantially the same as 2-O-α-D-glucosyl-L-ascorbic acid shown in Experiment 2 (3).

The present 2-O-α-D-glucosyl-L-ascorbic acid is a vitamin C enhancer that does not exhibit direct reductivity and has sufficient stability and physiological activity, as a stabilizer, quality improver, antioxidant, Activators, UV absorbers, chemicals, pharmaceutical raw materials, etc., food and drink, anti-sensitivity agents, cosmetics, reagents, etc.
It can be advantageously used for applications to various compositions.

Example A-4 20 parts by weight of α-glycosyl-L-ascorbic acid dextrin (DE18) was dissolved by heating in 70 parts by weight of water, kept under reduction, and added with 10 parts by weight of L-ascorbic acid and the method of Experiment 1. 4 units of the partially purified α-glucosidase prepared in the above was added per dextrin gram,
The reaction was carried out at 5.0 and 50 ° C. for 8 hours. Next, the product was purified according to the method of Example A-2, concentrated, and then powdered to obtain a powdery product at a yield of about 90 W / W%.

This product contained about 10 W / W% of α-glycosyl-L-ascorbic acid.

This product has α-glycosyl-L-ascorbic acid contained in it, which does not show direct reducing properties, has sufficient stability, antioxidant, and physiological activity, and is not only a vitamin C enhancer, but also a seasoning and humectant. , Stabilizers, quality improvers, antioxidants,
It can be advantageously used as a physiologically active agent or an ultraviolet absorber in various compositions such as foods and drinks, anti-sensitivity agents, and cosmetics.

Example A-5 α-Glycosyl-L-ascorbic acid Maltose (10 parts by weight) was dissolved by heating in water (80 parts by weight) and kept under reduction, and L-ascorbic acid (10 parts by weight) and Sigma rice seeds were added thereto. Of α-glucosidase was added at 4 units per gram of maltose, and the mixture was reacted at pH 6.0 and 45 ° C. for 6 hours under light shielding. Next, the product was purified and concentrated according to the method of Example A-2, and then powdered to obtain a powdery product with a yield of about 90%.
Obtained in W / W%. This product contains about 15% α-glycosyl-L-
It contained ascorbic acid.

In this product, α-glycosyl-L-ascorbic acid contained in it does not show direct reducing properties, has sufficient stability and physiological activity, and is not only a vitamin C enhancer, but also a sweetener, a seasoning, a humectant, and a stable As an agent, a quality improving agent, an antioxidant, a bioactive agent, an ultraviolet absorber, etc., it can be advantageously used for various compositions such as foods and drinks, anti-sensitivity agents, cosmetics and the like.

Example A-6 α-Glycosyl-L-ascorbic acid (1) Preparation of α-glucosidase preparation Maltose 4 W / V%, potassium monophosphate 0.1 W / V%, ammonium nitrate 0.1 W / V%, magnesium sulfate 0.05 W / V%,
Mucor javanicus (Mucor javanicus) in a liquid medium consisting of 0.05W / V% potassium chloride, 0.2W / V% polypeptone, 1W / V% calcium carbonate (separately after dry heat sterilization and aseptically added) and water IFO 4570 was cultured with shaking at a temperature of 30 ° C. for 44 hours. After completion of the culture, the mycelium was collected and immobilized α
-A glucosidase standard was used.

(2) Production of α-glycosyl-L-ascorbic acid Maltose (Hayashibara Co., Ltd., registered trademark: Sanmalto) 40
The resulting solution was heated and dissolved in 70 parts by weight of water, kept under reduction, and 10 parts by weight of L-ascorbic acid and 10 units of the immobilized α-glucosidase standard prepared by the method of (1) were added thereto in an amount of 10 units per maltose gram. The reaction was performed at pH 5.5 and 50 ° C. for 3 hours under light shielding.

This was filtered, and an immobilized α-glucosidase preparation was recovered and reused. The resulting filtrate was heated and treated according to Example A
After purifying and concentrating according to the method of -2, powdering,
A powdery product was obtained with a yield of about 95 W / W%.

This product contained about 7 W / W% of α-glycosyl-L-ascorbic acid.

In this product, the α-glycosyl-L-ascorbic acid contained therein does not show direct reducing properties, has sufficient stability and physiological activity, and is not only a vitamin C enhancer, but also a sweetener,
It can be advantageously used as a seasoning, humectant, stabilizer, quality improving agent, antioxidant, physiologically active agent, ultraviolet absorber, etc. for various compositions such as food and drink, anti-sensitivity agents, cosmetics and the like.

Example B-1 Chewing gum 25 parts by weight of gum base and α obtained by the method of Example A-6
-Glycosyl-L-ascorbic acid-containing powder (20 parts by weight) is kneaded with a mixer at 60 ° C, and then anhydrous crystalline maltitol (Mayabit, sold by Hayashibara Shoji Co., Ltd.)
50 parts by weight, 1.5 parts by weight of calcium phosphate and 0.1 part by weight of a 1-menthol β-cyclodextrin inclusion compound were mixed, and finally a small amount of a seasoning was mixed and kneaded well, and roll processing and cutting were performed to obtain a chewing gum. . This product is a chewing gum enriched with vitamin C, and has low caries and low calories.

Example B-2 Composting 1.2 parts by weight of water was mixed with 1 part by weight of waxy rice starch, and 1.5 parts by weight of sugar and 0.7 parts by weight of crystalline β-maltose (manufactured by Hayashibara Co., Ltd., San Marto) while gelatinizing by heating. Part and α-glycosyl-L- obtained by the method of Example A-2.
0.5 part by weight of syrup containing ascorbic acid was mixed, and then molded and packaged according to a conventional method to produce a fertilizer.

This product is a kibi-dango-style Japanese confection that is fertilized with vitamin C fortification, has a reduced aging, is durable, has a good mouthfeel and a good taste.

Example B-3 100 parts by weight of a mixed sweetener honey, 50 parts by weight of isomerized sugar, 2 parts by weight of brown sugar, and high-purity 2-O-α- obtained by the method of Example A-3
1 part by weight of D-glucosyl-L-ascorbic acid powder was mixed to obtain a mixed sweetener.

This product is a sweetener enriched with vitamin C and is suitable as a health food.

Example B-4 40 parts by weight of chocolate cacao paste, 10 parts by weight of cocoa butter, 50 parts by weight of anhydrous crystalline maltitol and 1 part by weight of the α-glycosyl-L-ascorbic acid-containing powder obtained by the method of Example A-1 After mixing and passing through a refiner to reduce the particle size,
Put in a conche and knead it at 50 ° C for two days. During this time, 0.5 part by weight of lecithin was added and sufficiently dispersed. Then, the temperature was adjusted to 31 ° C. with a temperature controller, poured into a mold immediately before the butter hardened, and the mill was beaten with a vibrator, and then passed through a 10 ° C. cooling tunnel for 20 minutes to be solidified. This was die-cut and packaged to obtain a product.

This product has no hygroscopicity, good color and gloss, good internal structure, melts smoothly in the mouth, and has an elegant sweetness and a mellow flavor. The product is a vitamin C-enriched chocolate having low calories and low caries.

Example B-5 Sand cream, crystalline α-maltose (manufactured by Hayashibara Co., Ltd., fine tose registered trademark) 1,200 parts by weight, shortening 1,000 parts by weight, α-glycosyl-L- obtained by the method of Example A-4
10 parts by weight of ascorbic acid-containing powder, 1 part by weight of lecithin,
1 part by weight of lemon oil and 1 part by weight of vanilla oil were mixed by a conventional method to produce a sand cream.

This product is a sand cream fortified with vitamin C and has good mouthfeel, melting condition and flavor.

Example B-6 Tablet 20 parts by weight of the high-purity 2-O-α-D-glucosyl-L-ascorbic acid powder obtained by the method of Example A-3 and crystalline β
-13 parts by weight of maltose, 4 parts by weight of corn starch, 1 part by weight of rutin and 0.5 part by weight of riboflavin were uniformly mixed, and the mixture was tabletted according to a conventional method to produce a tablet of 150 mg per tablet.

The product, vitamin C, vitamin P, with vitamin complex of vitamin B _2, good stability, a easy tablet to drink.

Example B-7 Capsule Calcium acetate monohydrate 10 parts by weight, L-magnesium lactate trihydrate 50 parts by weight, maltose 57 parts by weight, Example A
-20 parts by weight of powder containing α-glycosyl-L-ascorbic acid and γ containing 20% of eicosapentaenoic acid obtained by the method of Example 3.
-After uniformly mixing 12 parts by weight of cyclodextrin clathrate compound and granulating with a granulator, according to a conventional method,
The capsule was enclosed in a gelatin capsule to prepare a capsule containing 150 mg of one capsule.

The product can be advantageously used as a blood cholesterol-lowering agent, an immunostimulant, a skin-beautifying agent, a preventive agent for susceptibility diseases, a therapeutic agent, a food for promoting health, and the like.

Example B-8 Ointment 1 part by weight of sodium acetate trihydrate and 4 parts by weight of DL-calcium lactate were uniformly mixed with 10 parts by weight of glycerin, and the mixed part was mixed with 50 parts by weight of petrolatum, 10 parts by weight of wood wax, To a mixture of 10 parts by weight of lanolin, 14.5 parts by weight of sesame oil, 1 part by weight of the α-glycosyl-L-ascorbic acid-containing powder obtained by the method of Example A-4, and 0.5 part by weight of peppermint oil, and further uniformly mixed. To produce an ointment.

The product can be advantageously used as a sunscreen, a skin beautifying agent, a fairing agent, etc., and also as an agent for promoting healing of wounds and burns.

Example B-9 Injection High-purity α-glycosyl-L obtained by the method of Experiment 2 (2)
-Dissolve and neutralize the ascorbic acid powder in water, purify and filter according to a conventional method to be pyrogen-free.
α-Glycosyl-L-ascorbic acid 50 in a mL ampoule
The solution was dispensed so as to be 0 mg, and this was sealed to produce an injection.

The injection can be administered alone or mixed with other vitamins, minerals, etc., intramuscularly or intravenously. The product is a stable preparation without the need for low-temperature storage.

Compared with L-ascorbic acid, the product has a longer residence time in the body of about 2 to 10 times, is gradually hydrolyzed, releases L-ascorbic acid, and has the natural physiological properties of L-ascorbic acid. It is effective.

In addition, this product is not only for supplementation of vitamin C,
It is hydrolyzed and acts as an antioxidant to remove active oxygen, suppress the production of lipid peroxide, etc., and is effective in viral diseases, bacterial diseases, traumatic diseases, immune diseases, allergic diseases, diabetes, cataracts, cardiovascular diseases , And can be advantageously used as preventive and therapeutic agents for various diseases such as malignant tumors.

Example B-10 Injection 6 parts by weight of sodium chloride, 0.3 part by weight of potassium chloride,
0.2 parts by weight of calcium chloride, 3.1 parts by weight of sodium lactate,
48 parts by weight of maltose and 2 parts by weight of high-purity 2-O-α-D-glucosyl-L-ascorbic acid powder obtained by the method of Example A-3 were dissolved in 1,000 parts by weight of water,
The solution was purified and filtered to be pyrogen-free, and this solution was filled into sterilized plastic containers in 250 mL portions to produce injections.

This product is used not only as a supplement for vitamin C, but also as a calorie supplement and mineral supplement. Furthermore, it is hydrolyzed, has the effect of removing active oxygen as an antioxidant, and suppressing the production of lipid peroxide. Demonstrates, promotes treatment during and after disease, promotes recovery, and furthermore, viral diseases, bacterial diseases,
It can be advantageously used as a preventive or therapeutic agent for various diseases such as traumatic disease, immunological disease, allergic disease, diabetes, cataract, cardiovascular disease, malignant tumor and the like.

Example B-11 Tube feeding nutrient 20 parts by weight of crystalline α-maltose, 1.1 parts by weight of glycine, 0.18 parts by weight of sodium glutamate, 1.2 parts by weight of sodium chloride, 1 part by weight of citric acid, 0.4 parts by weight of calcium lactate, 0.1 parts by weight of magnesium carbonate A mixture comprising 0.1 part by weight of the α-glycosyl-L-ascorbic acid-containing powder obtained by the method of Example A-5, 0.01 part by weight of thiamine and 0.01 part by weight of riboflavin is prepared.

Each 24 g of the blend was filled into a laminated aluminum pouch and heat sealed to prepare a tube nutritional supplement.

The tube-feeding nutrient is obtained by dissolving one bag in about 300 to 500 mL of water, and can be advantageously used as an oral or parenteral nutritional supplement for the nasal cavity, stomach, intestine, etc. by a tube-feeding method.

Example B-12 Bath agent DL-sodium lactate 21 parts by weight, sodium pyruvate 8 parts by weight, α-glycosyl- obtained by the method of Example A-1
5 parts by weight of L-ascorbic acid-containing powder and ethanol 40
Parts by weight were mixed with 26 parts by weight of purified water and an appropriate amount of a coloring agent and a flavor to prepare a bath agent.

The product is suitable as a skin-beautifying agent and a skin-whitening agent, and may be used after being diluted 100 to 10,000 times with hot water for bathing. As in the case of hot water for bathing, this product can be advantageously used by diluting it in face wash water, lotion and the like.

Example B-13 Emulsion 0.5 parts by weight of polyoxyethylene behenyl ether, 1 part by weight of polyoxyethylene sorbitol tetraoleate, 1 part by weight of lipophilic glyceryl monostearate, 0.5 part by weight of pyruvate, 0.5 part by weight of behenyl alcohol, avocado oil 1 part by weight, high purity 2- obtained by the method of Example A-3
1 part by weight of O-α-D-glucosyl-L-ascorbic acid powder, an appropriate amount of vitamin E and a preservative are dissolved by heating according to a conventional method, and 1 part by weight of sodium L-lactate and 1,3-
5 parts by weight of butylene glycol, 0.1 part by weight of carboxyvinyl polymer and 85.3 parts by weight of purified water were added, emulsified with a homogenizer, and an appropriate amount of flavor was further added, followed by stirring and mixing to prepare an emulsion.

This product can be advantageously used as a sunscreen, skin beautifier, fair skin agent and the like.

Example B-14 Cream 2 parts by weight of polyoxyethylene glycol monostearate, 5 parts by weight of self-emulsifying glyceryl monostearate, high-purity 2-O-α-D- obtained by the method of Example A-3
Glucosyl-L-ascorbic acid powder (2 parts by weight), liquid paraffin (1 part by weight), glyceryl trioctanoate (10 parts by weight) and an appropriate amount of a preservative were dissolved by heating according to a conventional method, and L-lactic acid (2 parts by weight) and 1,3- 5 parts by weight of butylene glycol and 66 parts by weight of purified water were added, and the mixture was emulsified with a homogenizer, followed by adding an appropriate amount of a flavor and mixing with stirring to produce a cream.

This product can be advantageously used as a sunscreen, skin beautifier, fair skin agent and the like.

[Effects of the Invention] As described in the text, the novel substance α-glycosyl-L-ascorbic acid of the present invention does not show direct reducing properties, is excellent in stability, and is easily hydrolyzed in vivo, and has a low L-ascorbic acid content. −
Demonstrates the natural antioxidant and physiological activities of ascorbic acid.
In addition, α-glycosyl-L-ascorbic acid is a substance whose safety is extremely high, as it has been found that it is also a substance produced and metabolized in vivo.

Also, since α-glycosyl-L-ascorbic acid can be easily produced by a biochemical method of allowing a glycosyltransferase to act on a solution containing L-ascorbic acid and an α-glucosyl sugar compound, it is economically excellent, Industrial implementation is also easy.

Further, α-glycosyl-L which does not exhibit direct reduction
-Ascorbic acid has sufficient stability and bioactivity, not only as a vitamin C enhancer, but also as a stabilizer, a quality improving agent, an antioxidant, a bioactive agent, an ultraviolet absorber, etc.
It can be advantageously used by being incorporated into various compositions such as beverages, processed foods, foods and drinks such as favorite foods, preventives and remedies for susceptible diseases, and cosmetics such as skin-skins and fair-skins. Therefore, the α-glycosyl-L-ascorbic acid of the present invention has a wide range of uses, and the industrial significance given to these industries is extremely large.

[Brief description of the drawings]

The figure shows an infrared absorption spectrum of α-D-glucosyl-L-ascorbic acid of the present invention.

Continuation of the front page (51) Int.Cl. ⁶ Identification code FI A61K 7/00 A61K 31/00 631 31/00 631 31/71 31/71 C12P 19/60 C12P 19/60 A23L 2/00 F (58) Surveyed field (Int.Cl. ⁶ , DB name) C07H 15/26 C12P 19/60

Claims (12)

(57) [Claims] (1) α-Glycosyl-L which does not show direct reducibility
-Ascorbic acid. 2. A solution containing L-ascorbic acid and an α-glucosyl sugar compound is allowed to act with a glycosyltransferase to produce α-glycosyl-L-ascorbic acid which does not exhibit direct reducing properties, and is collected. A process for producing α-glycosyl-L-ascorbic acid which does not exhibit direct reducibility. 3. The method of claim 1, wherein the glycosyltransferase is cyclomaltodextrin.
Glucanotransferase (EC 2.4.1.19) or α
3. The method for producing α-glycosyl-L-ascorbic acid according to claim 2, which is glucosidase (EC 3.2.1.20). 4. α-Glycosyl-L which does not show direct reducibility
-A food or drink, an antisensitizing agent or a cosmetic containing at least 0.001 w / w% of ascorbic acid. 5. An α-glycosyl-L which does not exhibit direct reducing properties.
-Ascorbic acid is a vitamin C enhancer, taste improver,
Stabilizers, quality improvers, antioxidants, humectants, bioactive agents,
The food / drink, anti-sensitivity agent or cosmetic according to claim 4, which is an ultraviolet absorber, a skin-smoothing agent or a skin-whitening agent. 6. A 2-O-α-D-glucosyl-L-ascorbic acid. 7. 2-O-α-D-glucosyl-L-ascorbic acid is a vitamin C enhancer, a taste improver, a stabilizer,
7. 2-O-.alpha.-D- according to claim 6, which is a quality improving agent, an antioxidant, a physiologically active agent, an ultraviolet absorber, a beautifying agent or a fairing agent.
Glucosyl-L-ascorbic acid. 8. A solution containing L-ascorbic acid and an α-glucosyl sugar compound is reacted with a glycosyltransferase or a glycosyltransferase and glucoamylase (EC 3.2.1.3),
2-O-α-D characterized by producing O-α-D-glucosyl-L-ascorbic acid and collecting it.
-A method for producing glucosyl-L-ascorbic acid. 9. The method according to claim 9, wherein the glycosyltransferase is cyclomaltodextrin.
Glucanotransferase (EC 2.4.1.19) or α
-O-α-D-glucosyl-L- according to claim 8, which is -glucosidase (EC 3.2.1.20).
A method for producing ascorbic acid. 10. A 2-O-α- obtained by reacting a solution containing L-ascorbic acid and an α-glucosyl sugar compound with a glycosyltransferase or a glycosyltransferase and glucoamylase (EC 3.2.1.3). The solution containing D-glucosyl-L-ascorbic acid, L-ascorbic acid and D-glucose is subjected to chromatography selected from high performance liquid chromatography, gel filtration chromatography and ion exchange chromatography.
A method for producing 2-O-α-D-glucosyl-L-ascorbic acid, comprising collecting a -O-α-D-glucosyl-L-ascorbic acid-rich fraction. (11) A food, drink, anti-sensitive disease agent or cosmetic product containing 0.001 w / w% or more of 2-O-α-D-glucosyl-L-ascorbic acid. 12. 2-O-α-D-glucosyl-L-ascorbic acid is a vitamin C enhancer, a taste improver, a stabilizer, a quality improver, an antioxidant, a humectant, a bioactive agent, an ultraviolet ray. 12. The food / drink, anti-sensitivity agent or cosmetic according to claim 11, which is an absorbent, a skin-brightening agent or a skin-whitening agent.