JPH06263790A - 2-o-beta-d-galactopyranosyl-l-ascorbic acid or its salt, its production and use thereof - Google Patents

Abstract

PURPOSE:To obtain the subject new compound used for medicines, cosmetics, etc., having good stability by subjecting beta-galactosidase to react with a solution containing 5,6-isopropylidene-L-ascorbic acid and a compound having a beta- galactosyl group. CONSTITUTION:5,6-isopropylidene-L-ascorbic acid and lactose are dissolved in distilled water and adjusted to pH 4.5, to which is added beta-galactosidase derived from Aspellgirus oryzae, and subjected to react at 40 deg.C for 60 minutes, then boiled for 5 minutes to dineactivate the enzyme and eliminate isopropylidene group. The obtained reactional product is subjected to high performance liquid chromatography and active fractions are treated by an active carbon column to eliminate L-ascorbic acid and isolate the reactional product to obtain the objective compound expressed by the formula, excellent in stability, exhibiting enough vitamin C activity in a living body and useful for foods and drinks, sensitive disease treating agents, cosmetics, etc., as a stabilizer, a quality improving agent, a biologically active agent, a UV adsorbent, etc.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a highly stable L-ascorbic acid sugar derivative, that is, 2-O-β-D-galactopyranosyl-L-ascorbic acid or a salt thereof, a process for producing the same and uses thereof. It is about.

[0002]

2. Description of the Related Art L-ascorbic acid (vitamin C) is
Since humans, monkeys, and guinea pigs lack the synthase, it must be taken in as an essential vitamin from the outside. In vivo, L-ascorbic acid is involved in the hydroxylation of proline and lysine, which are necessary for the synthesis of collagen, which is the main component of biological connective tissue, and reduces Fe ³⁺ of cytochrome C to Fe ²⁺ . It is known that it is involved in redox reactions such as syrup and the like, and further, it has an immune enhancing effect by leukocytosis, an anti-atherogenic effect by suppressing cholesterol production, an anti-allergic effect by an antihistamine effect, etc. It plays an important role.

As described above, L-ascorbic acid is used not only as a vitamin C enhancer as an essential nutrient supplier but also as a sour agent, a reducing agent and an antioxidant because of its chemical structure and physiological activity. , A bleaching agent, various chemical reaction bases such as stabilizers, foods and drinks, and also a preventive agent for therapeutic diseases such as viral diseases, bacterial diseases, malignant tumors, diabetes and anti-susceptive disease agents, Further, it has an extremely wide range of applications including cosmetics such as an ultraviolet absorber, a skin beautifying agent and a skin whitening agent due to the action of suppressing melanin production.

However, it is known that L-ascorbic acid is a very unstable substance and is easily decomposed or modified by heat, light, oxygen, metal ions or the like. There have been many attempts to stabilize such unstable L-ascorbic acid, and several derivatives have been developed. For example, ascorbic acid phosphoric acid ester (JP-A-57-140789), glucose-linked glycosyl ascorbic acid (JP-A-58-5920), ascorbic acid fatty acid ester (JP-A-62-84072) , L-ascorbic acid derivatives and a method for producing the same (Japanese Patent Laid-Open No. 2-311490) and the like can be mentioned as specific examples.

[0005]

As mentioned above, various ascorbic acid derivatives have been developed. Among them, the sugar derivatives are excellent in stability and are expected to be put to practical use. The yield at the time of reaction was low and there was a problem to be solved. INDUSTRIAL APPLICABILITY The present invention produces a L-ascorbic acid sugar derivative, which is safe, has excellent storage stability, and can sufficiently exhibit the physiological activity of L-ascorbic acid in vivo, and further produces the L-ascorbic acid sugar. The object of the present invention is to provide food and drink, an anti-susceptive disease agent and cosmetics containing a derivative as an active ingredient.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that 5,6-isopropylidene-L-ascorbic acid or a salt thereof and β
-By allowing β-galactosidase to act on a mixture with a compound containing a galactosyl group under mild conditions and utilizing the glycosyl transfer reaction of β-galactosidase, the desired 2-O-β-D-galactopyra can be obtained. It was found that nosyl-L-ascorbic acid can be synthesized, and the present invention has been completed. Further, its L-ascorbic acid derivative, 2-O-β-D-galactopyranosyl-
It was found that L-ascorbic acid or a salt thereof has excellent stability, is non-toxic because it is easily hydrolyzed in vivo, and exhibits sufficient vitamin C activity in vivo, and its food, drink and anti-susceptive disease. The present invention has been achieved by establishing applications for agents, cosmetics and the like.

That is, the present invention is represented by the following formula 2
-O-β-D-galactopyranosyl-L-ascorbic acid or a salt thereof, and 5,6-isopropylidene-L
-2-O-β-D-galactopyranosyl-L-ascorbic acid or a compound thereof, which comprises causing β-galactosidase to act on a solution containing ascorbic acid or a salt thereof and a compound containing a β-galactosyl group. Method for producing salt and 2-O-β-D-galactopyranosyl-L-
It is intended to provide foods and drinks, anti-susceptive disease agents and cosmetics characterized by containing ascorbic acid or a salt thereof as an active ingredient.

[0008]

[Chemical 2]

The present invention will be described in detail below. The 2-O-β-D-galactopyranosyl-L-ascorbic acid of the present invention has a structure represented by the above formula, and is L-ascorbic acid and galactose which are components used in medicines, foods, cosmetics and the like. When used as an active ingredient of the food or drink, anti-susceptive disease agent or cosmetic of the present invention, it may be a salt such as sodium salt, potassium salt or lithium salt.

The β-galactosidase used in the present invention is 2-O-β-D-galactopyranosyl by a transglycosylation reaction when it is allowed to act on a solution containing a β-galactosyl group-containing compound and L-ascorbic acid. Any source can be used as long as it synthesizes -L-ascorbic acid, and the origin and type are not limited, but from the viewpoint of yield, Lipomyces starkeyi (Ministry of Industrial Science and Technology No. 8948, ATCC12659, ATCC20
825, ATCC56304, ATCC56305
Etc.), Rhodotorula minuta (ATCC10658, AT
CC16731, ATCC16732, ATCC167
33, ATCC60108 etc.), Bullera singularis
(ATCC24193), Aspellgirus orizae (ATC
C1003, ATCC1011, ATCC7252, A
Β-galactosidase produced by TCC9376) is preferred.

As a method for producing β-galactosidase from these microorganisms, liquid culture or solid culture is usually used. In the case of liquid culture, the culture supernatant can be used as it is as an enzyme agent, and in the case of solid culture, the extract can be used as it is as an enzyme agent. In some cases, the bacterial cells can be used as they are as an enzyme agent. In addition, an enzyme produced by a known method can be used if necessary. It is also possible to immobilize these enzymes or bacterial cells producing the enzymes and use them in a continuous reaction or a batch reaction repeatedly.
Also, if necessary, 5,6-isopropylidene-L may be added during the growth of a microorganism having a β-galactosyl group transfer reaction ability.
-Coexistence of ascorbic acid and a compound containing a β-galactosyl group to give 2-O-β-D-galactopyranosyl-
It is also possible to produce L-ascorbic acid.

The 5,6-isopropylidene-L-ascorbic acid used in the present invention is not limited to free 5,6-isopropylidene-L-ascorbic acid, as well as its alkali metal salt or alkaline earth salt, as long as no inconvenience occurs. It means a metal salt of 5,6-isopropylidene-L-ascorbic acid such as a metal salt, or a mixture thereof. Further, the compound containing a β-galactosyl group used as a substrate for β-galactosidase may be a natural product, an organic synthesis product or an enzymatic reaction product regardless of the origin, and galactose, lactose or the like is used.

In the present invention, the concentration of 5,6-isopropylidene-L-ascorbic acid during the reaction is 0.2
~ 20% (W / V) is suitable, 0.3 ~ 10% (W / V)
Is preferable, and 0.5 to 5% (W / V) is more preferable.
The concentration of β-galactosidase is suitably 1 to 30 mg / ml, preferably 2 to 15 mg / ml, more preferably 3 to 10 mg / ml. Lactose concentration is 5-50%
(W / V) is suitable, 10 to 40% (W / V) is preferable, and 20 to 35% (W / V) is more preferable.

The pH during the reaction is 3.0 to 7.
0 is suitable, 3.5 to 6.0 is preferable, and 4.0 to
5.5 is more preferable, but depending on the enzyme used, pH
Should be selected.

The reaction temperature is suitably in the range of 10 to 90 ° C, preferably 30 to 80 ° C, and 40 to
70 ° C. is more preferable.

The reaction time is not particularly limited and may be selected such that the amount of L-ascorbic acid derivative produced is maximized, but 15 to 120 minutes is suitable, and 30 to 100 minutes is preferable. , 40 to 90 minutes are more preferable. After completion of the reaction, this reaction solution may be heated in a boiling water bath to inactivate the enzyme, and at the same time, the isopropylidene group which is a protective group of 5,6-isopropylidene-L-ascorbic acid may be eliminated.

The L-ascorbic acid derivative produced by such a method can be separated by usual separation means such as membrane separation, ion exchange column chromatography, activated carbon column chromatography, liquid chromatography, silica gel column chromatography and the like. it can. At this time, unreacted 5,6-isopropylidene-L separated
-A compound containing ascorbic acid and a β-galactosyl group can be reused as a raw material for the transglycosylation reaction.

2-O-β-D- which is the active ingredient of the present invention
Galactopyranosyl-L-ascorbic acid is not only a stable and safe vitamin C enhancer, but also a taste improver, a sour agent, a stabilizer, a quality improver, an antioxidant, an ultraviolet absorber, etc. , 0.001% by weight or more, depending on the purpose, can be advantageously used in cosmetics such as foods and drinks, favorite foods, feeds, preventive agents and therapeutic agents for susceptive diseases, and skin-beautifying agents and skin-whitening agents.

Further, 2-O-β-D-galactopyranosyl-L-ascorbic acid is well harmonized with various substances having tastes such as sourness, saltiness, astringency, umami and bitterness,
Since it has great acid resistance and heat resistance, it can be used advantageously for ordinary food and drink and favorite foods as a vitamin C enhancer, taste improver, acidulant, quality improver, stabilizer, antioxidant, etc. You can

Examples of the food and drink and the favorite foods include alcoholic beverages such as synthetic liquor, fruit liquor, Western liquor, Chuhai, tea, juice, carbonated beverages, lactic acid beverages, lactic acid bacteria beverages, soft drinks such as sports drinks, instant juices, Instant tea, instant soup and other food and drink, soy sauce, powdered soy sauce, miso, powdered miso, moromi, sauce, ketchup, mayonnaise, dressing, lemon juice, vinegar, vinegar, vinegar, sushi vinegar, powdered sushi vinegar, mirin, mirin seasoning, Tempura soup, noodle soup,
Chinese sauce, dumpling sauce, roasted meat sauce, curry roux, stew, soup sauce, dashi sauce, various seasonings such as complex seasonings, oils such as salad oil and margarine, fruit syrup pickles, ice honey etc. Syrups, butter creams, custard creams, peanut pastes, flower pastes, spread pastes such as fruit pastes, jams, marmalades, syrup pickles, fruits such as sugar and fruits, and processed foods of vegetables.

Also, various Japanese sweets such as rice cracker, hail, rice cake, manju, uiro, mochi, bean paste, yokan, jelly, castella, candy etc., bread, cookies, crackers, pies, biscuits, pudding, cakes, cream puffs, Waffles, donuts, chocolate, chewing gum, caramel, candies, various Western confectionery such as snacks, ice cream, ice confections such as sherbet, processed foods such as breads and cooked rice, pickles, meat products,
Examples include fish meat products, boiled vegetables, prepared foods, milk drinks, dairy products, meat, fish meat, fruits, vegetables and other bottles and canned products.

Further, vitamin C enhancers, taste improvers, quality improvers, stabilizers, antioxidants, and palatability are also applied to feeds and feeds for domestic animals such as livestock, poultry, bees, silkworms and fish. It can be blended for the purpose of improvement.

In addition, troche, liver oil drop, complex vitamin preparation, tube feeding, oral medicine, injection, toothpaste paste, mouthwash, mouthwash, lipstick, eye shadow,
Advantageously blended in lotions, foundations, emulsions, hand creams, sun creams, face soaps, shampoos, rinses, hair preparations, other solids, pastes, liquids, cosmetics such as skin beautifiers and skin lightening agents. Can be implemented. Further, it can be used as an ultraviolet absorber, a deterioration preventing agent, etc. by blending it with a plastic product or the like.

2-O-β-D-galactopyranosyl-L
-A disease that is prevented or treated by ascorbic acid is a susceptible disease in the present invention, and includes, for example, viral disease, bacterial disease, traumatic disease, immune disease, allergic disease, diabetes, cataract, malignant tumor, arteriosclerosis, etc. It may be. The shape of the prophylactic or therapeutic agent for 2-O-β-D-galactopyranosyl-L-ascorbic acid-sensitive disease can be freely selected according to the purpose. For example, sprays, eye drops, nasal drops, mouthwashes, injections and other liquids,
Ointment, dusting agent, paste, powder, granule, capsule,
Examples include solid agents such as tablets and suppositories. When necessary, one or two other components such as therapeutic agents, physiologically active substances, antibiotics, adjuvants, bulking agents, stabilizers, coloring agents, flavoring agents, excipients, etc. It is also possible to use in combination with the above.

2-O-β-D-galactopyranosyl-L
The dose of ascorbic acid can be appropriately adjusted depending on the content, administration route, administration frequency and the like. It is preferably 0.0005 to 100 g, preferably 0.005 to 50 g, and more preferably 0.01 to 10 g per day for an adult.
A range of g is suitable.

2-O-β-D-galactopyranosyl-L
As a method of incorporating ascorbic acid, a known method such as mixing, dissolving, dipping, spraying, coating, spraying or pouring can be appropriately selected in the steps until the products are completed. In addition, 2-O-β-D-galactopyranosyl-
When L-ascorbic acid is a free acid, it is reacted with an aqueous solution of metal hydroxide, metal carbonate or the like, if necessary, to give 2-O-β-D-galactopyranosyl-L-ascorbic acid. It is also possible to use salt as a pH adjuster and a substance having both vitamin C action and minerals, which can be used as a nutrient enhancer, a chemical, or the like.

[0027]

EXAMPLES The present invention will be described in more detail below with reference to examples. In addition, in Examples 5 to 15, 2 obtained in Example 1 was used.
-O-β-D-galactopyranosyl-L-ascorbic acid was used. Example 1 1.25 g of 5,6-isopropylidene-L-ascorbic acid (Ishizu Pharmaceutical Special Reagent) and 30 g of lactose (Ishizu Pharmaceutical Special Reagent) were dissolved in distilled water to 100 ml, and then the pH was adjusted to 4.5. . Aspellgirus oryzae-derived β-galactosidase (Sumilacto GLL, manufactured by Shin Nippon Kagaku Kogyo Co., Ltd.) was added to this to 7.5 mg / ml, and the mixture was added at 60 ° C. at 60 ° C.
Reacted for minutes. After the reaction, boil for 5 minutes to inactivate the enzyme,
The isopropylidene group was removed.

The obtained reaction product was analyzed using a reverse phase system (octadecyl silylated silica gel) column for high performance liquid chromatography manufactured by Waters. At this time, as an eluent, 0.1 M KH ₂ PO ₄ /0.1 MH ₃ PO ₄
(PH 2) was used to elute the reaction product at a flow rate of 0.7 ml / min. The detection at that time was performed with UV ₂₄₅ . As a result, 6.25 mg of L-ascorbic acid derivative was produced. The reaction yield at this time was 0.5%.

Next, 50 ml of the above reaction product was applied to a 45 ml activated carbon column (for chromatography by Wako Pure Chemical Industries) and eluted with water at 10 ml / min. At this time, L-
Since ascorbic acid and L-ascorbic acid derivative were eluted in this order, only the eluted target L-ascorbic acid derivative was collected and concentrated. Lyophilize the concentrate to 2.08
mg of L-ascorbic acid derivative was obtained.

When the ¹³ C-NMR analysis was performed on the obtained L-ascorbic acid derivative, the following results were obtained. ¹³ C-NMR (σ value ppm) C-1 104.715 of galactose C-2 72.513 of galactose C-3 74.332 of galactose C-4 70.430 of galactose C-5 76.915 galactose of galactose C-6 62.835 L-ascorbic acid C-1 104.886 L-ascorbic acid C-2 185.253 L-ascorbic acid C-3 174.951 L-ascorbic acid C-4 77. 948 C-5 of L-ascorbic acid 71.946 C-6 of L-ascorbic acid 64.266 Further, this L-ascorbic acid sugar derivative does not show a direct reducing property, and 2,6-dichlorophenolindophenol It was found that it did not undergo reductive bleaching and had no cytochrome C reducing activity. Furthermore, by hydrolyzing the obtained L-ascorbic acid sugar derivative with β-galactosidase, the structure of the L-ascorbic acid sugar derivative is 2-O-β.
It was found to be -D-galactopyranosyl-L-ascorbic acid.

Example 2 1.25 g of 5,6-isopropylidene-L-ascorbic acid (Ishizu Pharmaceutical Special Grade Reagent) and 30 g of lactose (Ishizu Pharmaceutical Special Grade Reagent) were dissolved in distilled water to 100 ml, and the pH was adjusted to 4.5. Was prepared. To this, solution type β-galactosidase derived from Lipomyces starkeyi (Microtechnology Research Institute, Microbial Co. No. 8948) was added at 7.5 mg / ml and reacted at 40 ° C. for 60 minutes. After the reaction, the enzyme was inactivated by boiling for 5 minutes to remove the isopropylidene group. Solution-type β-galactosidase is Lipomyces starkeyi (Microtechnology Research Institute No. 8948),
Lactose 10%, glycerol 2%, yeast extract 0.05%
, Ammonium sulfate 0.5%, K ₂ HPO ₄ 0.3%, KH
100 ml of sterile agar medium (pH 5.6) containing 0.1% of ₂ PO _{4 and} 0.05% of MgSO ₄ .7H ₂ O was shake-cultured in a 500 ml Sakaguchi flask at 30 ° C. for 3 days, and the culture solution was centrifuged by a usual method. And extracted from the obtained bacterial cells. 18 g of wet cells obtained from 1 L of culture broth were suspended in 100 ml of 20 mM phosphate buffer (pH 6.0), crushed with a French press, and the supernatant of the crushed solution was purified by DEAE Sepharose column chromatography to obtain microbial cells. 1 g
Therefore, 0.36 units of β-galactosidase could be obtained. The activity was measured with 250 u of a 5 mM ortho-nitrophenylgalactosyl (ONPG) aqueous solution.
l, Mackleben buffer (pH 5.0) 500ul, β
-Mix 250ul of galactosidase solution and mix at 40 ℃ for 1
Reaction for 5 minutes or 30 minutes, 1M Na ₂ CO ₃
The reaction was stopped by adding 250 ul, and the absorbance was measured at 420 nm. The activity was calculated by setting the molecular extinction coefficient of ONPG = 21300.

When analyzed by high performance liquid chromatography in the same manner as in Example 1, 6.25 mg of L-ascorbic acid derivative was formed. The reaction yield at this time was 0.5%.

Next, the L-ascorbic acid derivative was purified in the same manner as in Example 1 to obtain 2.08 mg of the L-ascorbic acid derivative.

Example 3 1.25 g of 5,6-isopropylidene-L-ascorbic acid (Ishizu Pharmaceutical Special Grade Reagent) and 30 g of lactose (Ishizu Pharmaceutical Special Grade Reagent) were dissolved in distilled water to 100 ml, and the pH was adjusted to 4.5. Was prepared. Rhodotorula minuta (AT
Solution type β-galactosidase derived from CC10658) was added at 7.5 mg / ml and reacted at 40 ° C. for 60 minutes. After the reaction, the enzyme was inactivated by boiling for 5 minutes to remove the isopropylidene group. Solution type β-galactosidase is Rhodotorula minuta (ATCC10658) with lactose 10%, glycerol 2%, yeast extract 0.05%.
, Ammonium sulfate 0.5%, K ₂ HPO ₄ 0.3%, KH
₂ PO ₄ 0.1%, the MgSO ₄ · 7H ₂ O sterile liquid medium (pH 7.0) containing 0.05% 100 ml in 500ml Erlenmeyer flasks, 30 ° C., shaking cultured for 2 days, which in the pre-culture, the same composition Inoculate into a 3 L jar containing 2 L of culture medium, 30 ° C, aeration 1 vvm, stirring 500 rpm, pH
The culture broth cultured at 6.7 or more for 2-3 days was centrifuged by a usual method and extracted from the obtained bacterial cells. Wet cells 13g
Was crushed in the same manner as in Example 2 and the crushed liquid was purified to obtain 0.078 units of β-galactosidase per 1 g of the bacterium.

When analyzed by high performance liquid chromatography in the same manner as in Example 1, 6.25 mg of L-ascorbic acid derivative was formed. The reaction yield at this time was 0.5%.

Next, the L-ascorbic acid derivative was purified in the same manner as in Example 1 to obtain 2.08 mg of the L-ascorbic acid derivative.

Example 4 1.25 g of 5,6-isopropylidene-L-ascorbic acid (Ishizu Pharmaceutical Special Grade Reagent) and 30 g of lactose (Ishizu Pharmaceutical Special Grade Reagent) were dissolved in distilled water to 100 ml and the pH was adjusted to 4.5. Was prepared. In addition to this, Bullera singularis (AT
Solution type β-galactosidase derived from CC24193) was added so as to have a concentration of 7.5 mg / ml, and the mixture was reacted at 40 ° C. for 60 minutes. After the reaction, the enzyme was inactivated by boiling for 5 minutes to remove the isopropylidene group. Solution type β-galactosidase was prepared by using Bullera singularis (ATCC24193) with lactose 10% and yeast extract (DIFCO) 0.75%.
100 ml of sterile agar medium (pH 3.75) containing
Shake culture in a 00 ml Erlenmeyer flask at 24 ° C for 2 days,
As a preculture, a 3 L jar containing 2 L of medium having the same composition was inoculated and cultured at 22 ° C., aeration 0.3 to 1 vvm, agitation 375 rpm, pH 3.5 or more for 2 to 3 days. Was centrifuged by a usual method and extracted from the obtained bacterial cells. 0.125 per 1 g of bacterial cells was obtained by crushing 13 g of wet bacterial cells in the same manner as in Example 2 and purifying the disrupted liquid.
A unit of β-galactosidase could be obtained.

When analyzed by high performance liquid chromatography in the same manner as in Example 1, 6.25 mg of L-ascorbic acid derivative was formed. The reaction yield at this time was 0.5%.

Next, the L-ascorbic acid derivative was purified in the same manner as in Example 1 to obtain 2.08 mg of the L-ascorbic acid derivative.

Example 5 Stability test of 2-O-β-D-galactopyranosyl-L-ascorbic acid Using the following components, a soft drink containing grapefruit juice was obtained according to a conventional method. Ingredients wt% Grapefruit juice 20.0 Sugar 12.0 Citric acid 1.0 2-O-β-D-Galactopyranosyl-L-ascorbic acid 0.1 Water Prepared to 100%

The obtained soft drinks were stored at 40 ° C. for 120 days and a stability test was conducted. That is, the vitamin C remaining in the soft drink water was analyzed by high performance liquid chromatography (LC-2 manufactured by Shimadzu Corporation, column: Bio-Rad AMI).
NEX HPX-87H, flow rate: 0.6 ml / min, solvent solution: 0.01 MH ₂ SO ₄ , detection wavelength: 245 nm)
Was quantified at. The results are shown in Table 1. The numbers in Table 1 show the residual rate of vitamin C.

Next, a vitamin C activity test of the soft drink water obtained in Example 5 was conducted. That is, non-fat dry milk, non-fat soybeans and various basic vitamins other than vitamin C were used as a basic feed, and Hartley male guinea pigs (body weight of about 250) were used.
g, 10 fish), the resulting soft drink (2-O-β-D-galactopyranosyl-L-ascorbic acid concentration: 1.7 mg / ml) was orally administered in an amount of 1 ml each day. After administration, body weight change was measured. As a control group, 1 ml of distilled water was orally administered daily. The test results are shown in Table 2. The numbers in Table 2 show changes in the average body weight of guinea pigs.

Comparative Example 1 A soft drink was prepared in the same manner as in Example 5 except that 2-O-glycosyl-L-ascorbic acid was used instead of 2-O-β-D-galactopyranosyl-L-ascorbic acid. Water was obtained and a stability test was conducted in the same manner as in Example 5. The results of the stability test are shown in Table 1.

Comparative Example 2 A soft drink was prepared in the same manner as in Example 5 except that 6-O-stearyl-L-ascorbic acid was used instead of 2-O-β-D-galactopyranosyl-L-ascorbic acid. Water was obtained and a stability test was conducted in the same manner as in Example 5. The results of the stability test are shown in Table 1.

Comparative Example 3 A soft drink was prepared in the same manner as in Example 5 except that L-ascorbic acid was used instead of 2-O-β-D-galactopyranosyl-L-ascorbic acid. The stability test and the vitamin C activity test were conducted in the same manner as in 5. The results of the stability test are shown in Table 1, and the results of the vitamin C activity test are shown in Table 2.

[0046]

[Table 1]

As is clear from Table 1, the soft drink containing L-ascorbic acid (Comparative Example 3) and 6-O-stearyl-L-ascorbic acid as components (Comparative Example 2) is very unstable, It decreased with the passage of time. Also, 2
The soft drink (Comparative Example 1) containing -O-glycosyl-L-ascorbic acid as a component was relatively stable, but the stability was not so excellent as compared with the soft drink of the present invention. On the other hand, the soft drink (Example 5) containing 2-O-β-D-galactopyranosyl-L-ascorbic acid as a component of the present invention was very stable even at 40 ° C for a long period of time.

[0048]

[Table 2]

As is clear from Table 2, in the subject group, basic feed was given but vitamin C was deficient, so almost no weight gain was observed after the 7th day, and 3 out of 10 fish died after the 14th day. did. On the other hand, the group to which the soft drink containing 2-O-β-D-galactopyranosyl-L-ascorbic acid as a component (Example 5) was administered was a soft drink containing L-ascorbic acid as a component. Similar to the group to which (Comparative Example 3) was administered, an average daily weight gain of 4 to 5 g was exhibited, and no abnormality was observed during the administration period. That is, the soft drink containing the 2-O-β-D-galactopyranosyl-L-ascorbic acid of the present invention as a component (Example 5) exhibits the same vitamin C activity as L-ascorbic acid in vivo. It was a thing.

Example 6 Antioxidant test of 2-O-β-D-galactopyranosyl-L-ascorbic acid 1 g of 2-O-β-D-galactopyranosyl-L-ascorbic acid was added to 10 ml of distilled water. , And the whole amount was sprayed on 100 g of fried beans and dried to obtain a bean confectionery.

The obtained bean confectionery was stored at 40 ° C., 5 g was weighed out with time, and the solvent (chloroform: glacial acetic acid = 2:
After the extraction in 3), potassium iodide was added, the liberated iodine was titrated, and the increase in the peroxide value was measured to carry out the antioxidant capacity test. As a control group, non-sprayed bean confectionery was obtained, and the antioxidant ability test was conducted in the same manner. The test results are shown in Table 3. The numbers in Table 5 show the peroxide value.

Comparative Example 4 The procedure of Example 6 was repeated except that 2-O-glucosyl-L-ascorbic acid was used instead of 2-O-β-D-galactopyranosyl-L-ascorbic acid. Bean confectionery was obtained, and the antioxidant ability test was conducted in the same manner as in Example 6. The results of the antioxidant capacity test are shown in Table 3.

Comparative Example 5 A bean confectionery product was obtained in the same manner as in Example 6 except that BHA was used instead of 2-O-β-D-galactopyranosyl-L-ascorbic acid. Similarly, the antioxidant ability test was conducted. The results of the antioxidant capacity test are shown in Table 3.

[0054]

[Table 3]

As is apparent from Table 3, the confectionery prepared by spraying 2-O-glucosyl-L-ascorbic acid in which the hydroxyl group at the 2-position of L-ascorbic acid, which is considered to be an antioxidant activity center, was sprayed (Comparative Example 4). The peroxide value of () showed almost the same increase as that of the control group. On the other hand, in the bean confectionery (Example 6) sprayed with 2-O-β-D-galactopyranosyl-L-ascorbic acid of the present invention, the increase in peroxide value was suppressed to about 60% of the control group. Compared with the case of spraying BHA (butylhydroxyanisole) known as an antioxidant (Comparative Example 5), the increase was only about 30%, showing a high antioxidant activity. That is,
The bean confectionery (Example 6) sprayed with 2-O-β-D-galactopyranosyl-L-ascorbic acid of the present invention shows high antioxidant activity. In addition, the bean confectionery obtained in Example 6 has the same stability as in Example 5 and in vivo vitamin C.
Had activity.

Example 7 Preparation method of chewing gum Ingredient Weight g Gum base 22.0 Calcium carbonate 20.0 Powdered sugar 40.0 Glucose 20.0 Water candy 15.0 2-O-β-D-galactopyranosyl-L- Ascorbic acid 5.0 Fragrance 1.0 Using these components, a chewing gum was obtained according to a conventional method.

Example 8 Method for Making Sweet Bread Ingredients Weight g Wheat flour 100.0 Yeast 3.0 Yeast food 0.2 Salt 1.0 Sugar 15.0 Glucose 6.0 Shortening 3.5 Lecithin 0.5 Condensed milk 4.0 Vitamin B ₁ 0.5m 2-O-β-D-galactopyranosyl-L-ascorbic acid 0.12 Using these components, a confectionery bread was obtained according to a conventional method.

Example 9 Production Method of Mayonnaise Ingredient Weight g Egg yolk 17.0 Salad oil 68.0 Vinegar 9.4 Sugar 2.2 Salt 1.3 2-O-β-D-galactopyranosyl-L-ascorbic acid 1 0.0 Spice 1.1 Using these ingredients, mayonnaise was obtained according to a conventional method.

Example 10 Preparation Method of Eye Drops and Eye Irritation Test An eye drop was prepared according to a conventional method according to the following formulation. Ingredient weight g Propylene glycol 0.5 Chlorobutanol 0.01 Disodium hydrogen phosphate 0.01 Boric acid 1.3 Sodium chloride 0.9 2-O-β-D-galactopyranosyl-L-ascorbic acid 0. 1 Distilled water Remainder For comparison, an eye drop containing 0.05% by weight of L-ascorbic acid instead of 2-O-β-D-galactopyranosyl-L-ascorbic acid (Comparative Example 6). Was prepared. Next, a panel of 50 people evaluated the eye irritation of the eye drop having the above formulation. As a result, most did not complain of irritation.

Example 11 Preparation Method of Toothpaste and Storage Colorability Test A toothpaste was prepared according to a conventional method according to the following formulation. Ingredients Weight g Aluminum hydroxide 45.0 Sorbit 25.0 Sodium carboxymethylcellulose 1.0 Sodium lauryl sulfate 1.5 Gelling silica 2.0 Ethanol 0.1 Distilled water 23.7 Fragrance 1.0 Sodium saccharin 0.2 2 -O-β-D-galactopyranosyl-L-ascorbic acid 0.5

COMPARATIVE EXAMPLE 6 Instead of 2-O-β-D-galactopyranosyl-L-ascorbic acid (Example 11), a toothpaste containing the same amount of L-ascorbic acid (Comparative Example 7) and these products were prepared. A control toothpaste was prepared without the addition of. Each of these toothpastes is 30 ℃
6 months, 40 ° C. for 3 months, and 50 ° C. for 3 months, and the coloring degree was evaluated according to the following criteria. -: No coloring +: Minor coloring ++; Heavy coloring

The results are shown in Table 4.

[0063]

[Table 4]

As is clear from Table 4, the toothpaste containing L-ascorbic acid (Comparative Example 6) was markedly colored, but 2-O-β-D-galactopyranosyl-L-ascorbic acid was added. The toothpaste prepared (Example 11) showed no coloration.

Next, the toothpaste of Example 11 and Comparative Example 6 was used by 30 patients with degree 1 alveolar pyorrhea for 6 months, and redness of the gingiva, disappearance of pus, improvement of the blind sac, and improvement of swaying of the teeth. And the effectiveness was judged using the improvement of gingiva as an index. During the test, the patient was asked to brush his teeth twice a day in the morning and evening. Two judgments
It was conducted monthly according to the following criteria. 5 points: 5 items in the index were improved. 4 points: 4 items in the index were improved. 3 points: 3 items in the above index were improved. 2 points: Two items in the above index were improved. 1 point: One item in the index was improved. 0 point: No improvement or deterioration. Table 5 shows the total score of each dentifrice use group divided by the number of patients on each determination day.

[0066]

[Table 5]

2-O-β-D-galactopyranosyl-L
-No case was worsened by the use of a toothpaste containing ascorbic acid (Example 12), and abnormalities in the oral cavity due to the use thereof,
There was no sense of discomfort.

Example 12 Method for Producing Oral Pasta An oral pasta was obtained according to a conventional method according to the following formulation. Ingredients Weight g Ethanol 10.0 Squalene 20.0 Precipitable silica 5.0 Polyoxyethylene hydrogenated castor oil 0.1 Sodium lauryl sulfate 0.2 EDTA calcium 0.1 Perfume 0.6 Sorbitan monooleate 1.0 Glycyrrhizin Acid 0.1 Saccharin sodium 0.6 2-O-β-D-galactopyranosyl-L-ascorbic acid 0.5 The oral pasta obtained in Example 12 was stable and improved in identification degree as compared with Example 11. Showed the effect.

Example 13 Method for Producing Oral Lozenge By the following formulation, the ingredients were well mixed in accordance with a conventional method, and an oral troche was obtained by direct compression. Ingredient Weight g Lactose 97.0
Polyoxyethylene monostearate 0.2 Sodium lauryl sulfate 0.05 Chlorhexidine gluconate 0.02 Stevia extract 0.2 Perfume 0.1 Hydroxyethyl cellulose 1.93 2-O-β-D-galactopyranosyl- L-Ascorbic acid 0.5 The oral lozenge obtained in Example 13 showed stability of the degree of identification and the effect of improvement as in Example 11.

Example 14 Preparation Method of Mouthwash The following formulation was mixed well according to a conventional method to obtain a mouthwash. Ingredients g Solbit 10.0 Ethanol 5.0 Polyoxyethylene hydrogenated castor oil 0.1 Sucrose monopalmitate 0.2 Sodium lauryl sulfate 0.05 Saccharin sodium 0.2 Sodium hydrogen carbonate 0.05 EDTA calcium 0.05 Perfume 0.6 Distilled water 83.65 2-O-β-D-galactopyranosyl-L-ascorbic acid 0.1 The mouthwash obtained in Example 14 was stable with respect to Example 11 in terms of stability and improvement effect. showed that.

Example 15 Method for producing creamy cosmetic product A creamy cosmetic product was obtained by a conventional method using the components of the following formulation. Ingredient Weight g Squalane 25.0 Wax 5.0 Olive oil 3.0 Xanthan gum 1.0 Monoammonium glycyrrhizinate 0.3 Methylparaben 0.2 Distilled water 61.5 Perfume Suitable amount 2-O-β-D-galactopyranosyl- L-ascorbic acid 4.0

The above cream was applied to the face of 20 panelists (women) suffering from dark blacks, stains, and freckles 2 times a day.
The panelist himself evaluated the effect on three levels: effective, moderately effective, and ineffective. Also, instead of 2-O-β-D-galactopyranosyl-L-ascorbic acid, the same amount of L-ascorbic acid was added,
A creamy cosmetic was obtained and tested in the same manner, and the results are shown in Table 6.

[0073]

[Table 6]

Further, the above cream was stored at 40 ° C. for 6 months and examined for changes in appearance and presence of odor, and Table 7 shows the experimental results.

[0075]

[Table 7]

As is clear from Tables 6 and 7, the skin cosmetics of the present invention were found to be effective in terms of storage stability, whitening effect and rough skin improving effect.

[0077]

INDUSTRIAL APPLICABILITY The 2-O-β-D-galactopyranosyl-L-ascorbic acid or its salt which is the L-ascorbic acid sugar derivative of the present invention has excellent stability and is easily hydrolyzed in vivo. Therefore, it is non-toxic and exhibits sufficient vitamin C activity in vivo, and is used as a stabilizer, quality improver, antioxidant, bioactive agent, ultraviolet absorber, etc. as a beverage, processed food, seasoning, and taste. It can be advantageously used by including it in foods and drinks such as foods, preventive agents and therapeutic agents for susceptive diseases, and in cosmetics such as skin-beautifying agents and skin-whitening agents. Further, according to the production method of the present invention, the above L-ascorbic acid sugar derivative is 2-
O-β-D-galactopyranosyl-L-ascorbic acid or a salt thereof can be produced in good yield and at low cost.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location A61K 7/16 7252-4C 7/42 7252-4C 7/48 9051-4C 31/70 ADK 8314- 4C C09K 15/06 C12P 19/60 7432-4B

Claims (5)

[Claims] 1. 2-O-β-D-galactopyranosyl-L-ascorbic acid represented by the following formula or a salt thereof. [Chemical 1] 2. The β-galactosidase is allowed to act on a solution containing 5,6-isopropylidene-L-ascorbic acid or a salt thereof and a compound containing a β-galactosyl group. 2-O-β-D-
A method for producing galactopyranosyl-L-ascorbic acid or a salt thereof. 3. A food or drink comprising the 2-O-β-D-galactopyranosyl-L-ascorbic acid or a salt thereof according to claim 1 as an active ingredient. 4. An anti-susceptive disease agent containing the 2-O-β-D-galactopyranosyl-L-ascorbic acid or salt thereof according to claim 1 as an active ingredient. 5. A cosmetic comprising the 2-O-β-D-galactopyranosyl-L-ascorbic acid or the salt thereof according to claim 1 as an active ingredient.