.alpha.-Glycosyl-L-ascorbic acid, and its preparation and uses

.alpha.-Glycosyl-L-ascorbic acid exhibiting no direct reducing activity is formed in a solution containing L-ascorbic acid and an .alpha.-glucosyl saccharide when subjected to the action of a saccharide-transferring enzyme. .alpha.-Glycosyl-L-ascorbic acid is superiorly stable, and readily hydrolyzable in vivo to exhibit the activities inherent to L-ascorbic acid. Thus, .alpha.-glycosyl-L-ascorbic acid is favorably useful as a stabilizer, quality-improving agent, antioxidant, physiologically active agent and uv-absorbent in food pharmaceutical and cosmetic industries.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to an .alpha.-glycosyl-L-ascorbic acid 
exhibiting no direct reducing activity, and its preparation and uses. 
More particularly, the present invention relates to a novel substance, an 
.alpha.-glycosyl-L-ascorbic acid exhibiting no direct reducing activity. 
The present invention also relates to a biochemical process to prepare the 
same. 
Further, the present invention relates to foodstuffs, tobaccos, 
pharmaceuticals for susceptive diseases and cosmetics such as beverages, 
processed foods, preventive and remedy for susceptive diseases, 
skin-refining agent and skin-whitening agent which all contain the 
.alpha.-glycosyl-L-ascorbic acid. 
2. Description of the Prior Art 
L-Ascorbic acid, which has the chemical structure shown by the formula [I]: 
##STR1## 
is not synthesized in vivo in human, monkey and guinea pig, therefore is 
listed as an essential nutritive element, i.e. vitamin C. 
L-Ascorbic acid takes part in some physiological activities in vivo; for 
example, in the hydroxylation of proline and lysine which are necessary to 
synthesize collagen as the main element of living connective tissues; the 
oxidation-reduction reaction of cytochrome C wherein Fe.sup.+++ is reduced 
into Fe.sup.++ ; and in the immunopotentiation via the increase of 
leukocyte. These are because vitamin C plays a significant role in the 
maintenance and promotion of health in living body. 
Scurvy has been known long as a condition due to deficiency of L-ascorbic 
acid, and is marked by weakness of the skin, petechial hemorrhage, 
ecchymosis, and hemorrhages in the gingiva and marrow. To prevent scurvy 
for the maintenance of health, a recommended daily administration (RDA) is 
established for L-ascorbic acid; in particular, 60 mg for adult male and 
50 mg for adult female. 
Nowadays the use of L-ascorbic acid is not limited to agents which enrich 
vitamin C as an essential nutritive element, but is extending in various 
applications. More particularly, because of the chemical structure and 
physiological activities, L-ascorbic acid is useful as a souring agent, 
reductant, antioxidant, bleaching agent and stabilizer in various chemical 
reagents, foods and beverages: in pharmaceuticals for susceptive diseases 
such as preventive and remedy for viral diseases, bacterial diseases and 
malignant tumors; and further as a reductant, uv-absorbent and 
melanin-formation inhibitor in cosmetics including skin-refining agent and 
skin-whitening agent. 
The major drawback of L-ascorbic acid is that it readily looses the 
physiological activities because of its direct reducing activity, poor 
stability and high susceptibility to oxidation. 
To stabilize L-ascorbic acid, some saccharide derivatives of L-ascorbic 
acid have been proposed. For example, we disclosed in Vitamin, Vol.43, 
pp.205-209 (1971), ibid., Vol.47, pp.259-267 (1973), and Japanese Patent 
Publication No.38,158/73 a biochemical synthesis of L-ascorbic acid 
glucosides. 
Because of the facts that the glucosides are prepared by similar methods; 
that the formation of an ether bond at the primary alcohol group which is 
located at the number six carbon atom in L-ascorbic acid leads to the 
glucosides as described in the Japanese Patent Publication, for example, 
on the 2nd column, lines 14-16: that the saccharide-transfer reaction from 
maltose to an .alpha.-glucosyl group is responsible for the formation of 
glucosides; and that the glucosides exhibit a direct reducing activity, 
their chemical structure would be shown by the formula [II]: 
##STR2## 
As obvious from the results in the Japanese Patent Publication, the table 
in Example 1, the stability of the glucosides is superior to that of 
L-ascorbic acid, but is not enough for their commercialization. 
While Ishido et al. disclose in Japanese Patent Publication No.5,920/83 an 
organic chemical process to synthesize saccharide derivatives of 
L-ascorbic acid. 
These derivatives are, however, those wherein all the D-glucoses are bound 
in the .beta.-fashion because up to 21 .beta.-D-glucopyranosyl type 
derivatives of L-ascorbic acid including 
2,3-di-O-(.beta.-D-glucopyranosyl)-L-ascorbic acid are listed for 
explanation on the 7th column, line 6 to the 8th column, line 11. 
Masamoto et al. disclose in Japanese Patent Publication No.198,498/83 an 
organic chemical process to synthesize saccharide derivatives of 
L-ascorbic acid which are also of .beta.-glucosyl type. 
Studies on the .beta.-D-glucopyranosyl type derivatives of L-ascorbic acid 
confirmed that they hardly exhibit desired physiological activities in 
living body, especially, in human. Furthermore, conventional organic 
chemical processes have the drawbacks that they are inferior in economical 
efficiency because the reaction is very complicated and low in yield, as 
well as that the establishment of non-toxicity and safeness for the 
resultant derivatives is very difficult. 
As described above, the proposals of saccharide derivatives of L-ascorbic 
acid in the prior art have proved unsatisfactory in view of stability, 
safeness, physiological activity and economical efficiency, and not been 
practiced hitherto. 
SUMMARY OF THE INVENTION 
Accordingly, the realization of a saccharide derivative of L-ascorbic acid 
which is free from the drawbacks of conventional saccharide derivatives, 
i.e. superiorly stable, usable without a fear for toxicity, and capable of 
exhibiting the physiological activities of L-ascorbic acid in vivo, has 
been in great demand. 
The present invention is to overcome the drawbacks of conventional 
saccharide derivatives of L-ascorbic acid. More particularly, we studied a 
novel saccharide derivative of L-ascorbic acid which is obtainable by a 
biochemical process utilizing a saccharide-transfer reaction. 
As the result, we discovered a novel saccharide derivative of L-ascorbic 
acid which is superiorly stable, readily hydrolyzable in vivo, and 
excellently high in physiological activity, as well as developing its 
preparation and uses in foods, beverages, cosmetics and pharmaceuticals 
for susceptive diseases. Thus, we accomplished the present invention. 
Since when L-ascorbic acid is ingested with an .alpha.-glucosyl saccharide, 
the .alpha.-glycosyl-L-ascorbic acid is readily synthesized and then 
metabolized, it can be deemed to be a biosubstance, and this is ideally 
convenient in safeness. 
While the .beta.-D-glucosyl type derivatives obtainable by organic chemical 
processes are not synthesized in vivo, so they would be extraneous to 
living body.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is feasible with any .alpha.-glycosyl-L-ascorbic 
acid, regardless of its preparation process such as biochemical and 
organic chemical processes. 
In view of safeness and economical efficiency, desirably, 
.alpha.-glycosyl-L-ascorbic acid is formed by a biochemical process 
wherein a saccharide-transferring enzyme is allowed to act on a solution 
containing L-ascorbic acid and an .alpha.-glucosyl saccharide. 
The wording "exhibiting no direct reducing activity" means that unlike 
L-ascorbic acid, its saccharide derivative does not reduce and decolor 
2,6-dichlorophenolindophenol intact. 
The wording "L-ascorbic acid" as referred to in the invention means 
L-ascorbates such as alkaline metal salts, alkaline earth metal salts and 
mixtures thereof, and should not be restricted to free L-ascorbic acid, as 
far as the present invention is feasible therewith. Thus, if necessary, 
such as sodium L-ascorbate and calcium L-ascorbate are suitably usable in 
the saccharide-transfer reaction, as well as free L-ascorbic acid. 
The wordings ".alpha.-glycosyl-L-ascorbic acid" and 
"2-O-.alpha.-D-glucosyl-L-ascorbic acid" mean those in salt form, in 
addition to those in free acid form, as far as the present invention is 
feasible therewith. 
The .alpha.-glucosyl saccharides usable in the invention are those which 
permit a saccharide-transferring enzyme to form from L-ascorbic acid an 
.alpha.-glycosyl-L-ascorbic acid exhibiting no direct reducing activity. 
For example, maltooligosaccharides such as maltose, maltotriose, 
maltoteraose, maltopentaose, maltohexaose, maltoheptaose and maltooctaose 
are suitably chosen, as well as partial starch hydrolysates such as 
dextrin, cyclodextrin and amylose, liquefied starch, gelatinized starch, 
and solubilized starch. 
Consequently to facilitate the formation of .alpha.-glycosyl-L-ascorbic 
acid, one should choose an .alpha.-glucosyl saccharide which is 
susceptible to the saccharide-transferring enzyme to be used. 
For example, when .alpha.-glucosidase (EC 3.2.1.20) is used as the 
saccharide-transferring enzyme, maltooligosaccharides such as maltose, 
maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and 
maltooctaose are suitable, as well as partial starch hydrolysates and 
dextrins with a DE (Dextrose Equivalent) of about 5-60. When 
cyclomaltodextrin glucanotransferase (EC 2.4.1.19) is used as the 
saccharide-transferring enzyme, partial starch hydrolysates such as 
gelatinized starches with a DE below 1 and dextrins with a DE up to 60 are 
suitable. When .alpha.-amylase (EC 3.2.1.1) is used as the 
saccharide-transferring enzyme, partial starch hydrolysates such as 
gelatinized starch with a DE below 1 and dextrins with a DE up to about 30 
are suitable. 
The concentration of L-ascorbic acid during the reaction is generally 1 w/v 
% or higher, preferably, about 2-30 w/v %, while the concentration of an 
.alpha.-glucosyl saccharide is generally about 0.5- to 30-fold higher than 
that of L-ascorbic acid. 
The saccharide-transferring enzymes usable in the invention are those which 
transfer one or several .alpha.-glucosyl groups at least to the number two 
carbon atom in L-ascorbic acid without decomposing it when allowed to act 
on a solution containing L-ascorbic acid and an .alpha.-glucosyl 
saccharide which has an adequate susceptivity to the enzyme. 
For example, .alpha.-glucosidases derived from animals, plants and 
microorganisms such as those from mouse kidney, rat intestinal mucosa, dog 
small intestine, pig small intestine, rice seed, maize seed, and those 
from a culture which is obtainable by cultivating in a nutrient culture 
medium yeasts and bacteria of the genera Mucor, Penicillium and 
Saccharomyces; cyclomaltodextrin glucanotransferases from a culture of 
bacteria such as those of the genera Bacillus and Klebsiella; and 
.alpha.-amylase from a culture of bacteria such as those of the genus 
Bacillus are suitably chosen. 
Such a saccharide-transferring enzyme should not necessarily be purified 
prior to its use, as long as it fulfills the above requirements. 
Generally, the present invention is feasible with a crude enzyme. If 
necessary, saccharide-transferring enzymes can be purified by conventional 
method, prior to its use. Of course, commercialized 
saccharide-transferring enzymes can be used in the invention. The amount 
of a saccharide-transferring enzyme and reaction time are closely 
dependent each other. With an economical viewpoint, 
saccharide-transferring enzyme is used in an amount which completes the 
reaction within about 3-80 hours. 
Immobilized saccharide-transferring enzymes are favorably usable batchwise 
and in continuous manner. 
The reaction process according to the invention is usually carried out by 
adding a saccharide-transferring enzyme to a solution containing the above 
described L-ascorbic acid and an .alpha.-glucosyl saccharide, and keeping 
the mixture under conditions where the enzyme is substantially active; 
usually, at a pH in the range of about 3-9 and a temperature in the range 
of about 30.degree.-80.degree. C. Since during the reaction L-ascorbic 
acid tends to cause an oxidative decomposition, it is desirable to keep 
the mixture under conditions which shield aeration and light as far as 
possible so that L-ascorbic acid is in its reducing form. The reaction is 
favorably carried out in the presence of such as thiourea and hydrogen 
sulfide, if necessary. 
The desired substance can be obtained by incorporating L-ascorbic acid and 
an .alpha.-glucosyl saccharide in the culture of a growing microorganism 
which is capable of producing a saccharide-transferring enzyme. 
The .alpha.-glycosyl-L-ascorbic acid exhibiting no direct reducing activity 
will be explained hereinafter. Such a .alpha.-glycosyl-L-ascorbic acid 
bears an .alpha.-D-glucosyl group consisting of 1-7 glucosyl groups linked 
via the .alpha.-1,4 fashion, and such an .alpha.-D-glucosyl group is bound 
at least to the primary alcohol group which is located at the number two 
carbon atom. Particular substances are, for example, 
2-O-.alpha.-D-glucosyl-L-ascorbic acid, 2-O-.alpha.-D-maltosyl-L-ascorbic 
acid, 2-O-.alpha.-maltotriosyl-L-ascorbic acid, 
2-O-.alpha.-D-maltotetraosyl-L-ascorbic acid, 
2-O-.alpha.-D-maltopentaosyl-L-ascorbic acid, 
2-O-.alpha.-D-maltohexaosyl-L-ascorbic acid and 
2-O-.alpha.-D-maltoheptaosyl-L-ascorbic acid. Although .alpha.-glucosidase 
generally forms only 2-O-.alpha.-D-glucosyl-L-ascorbic acid, 
2-O-.alpha.-D-maltosyl-L-ascorbic acid and 
2-O-.alpha.-D-maltotriosyl-L-ascorbic acid can be formed in mixture, if 
necessary. 
In the case of using either cyclomaltodextrin glucanotransferase or 
.alpha.-amylase, .alpha.-glycosyl-L-ascorbic acids with a higher 
.alpha.-D-glucosyl group are formed in mixture. Dependently on the 
.alpha.-glucosyl saccharide, cyclomaltodextrin glucanotransferase yields 
an .alpha.-D-glucosyl group with a polymerization degree distributing in 
the range of 1-7, while .alpha.-amylase yields a slight narrower 
distribution. Such a mixture can be partially hydrolyzed with either of 
.alpha.-amylase (EC 3.2.1.1), .beta.-amylase (EC 3.2.1.2) and glucoamylase 
(EC 3.2.1.3) to reduce the polymerization degree of the .alpha.-D-glucosyl 
group, if necessary. For example, 2-O-.alpha.-D-maltosyl-L-ascorbic acid 
and higher polymers are hydrolyzed to accumulate 
2-O-.alpha.-D-glucosyl-L-ascorbic acid when subjected to glucoamylase. 
.beta.-Amylase predominantly hydrolyzes 
2-O-.alpha.-D-maltotetraosyl-L-ascorbic acid and higher polymers to 
accumulate 2-O-.alpha.-D-glucosyl-L-ascorbic acid, 
2-O-.alpha.-D-maltosyl-L-ascorbic acid and 
2-O-.alpha.-D-maltotriosyl-L-ascorbic acid in mixture. 
Although a reaction mixture obtained by either of the above methods usually 
contains the remaining L-ascorbic acid and .alpha.-glucosyl saccharide 
together with an .alpha.-glycosyl-L-ascorbic acid which exhibits no direct 
reducing activity, it can be prepared into final product without no 
further special treatment. Usually, such a reaction mixture is heated to 
inactivate the remaining enzyme, filtered and concentrated into a syrupy 
product which may be then dried and prepared into a powdery product. 
When a purified .alpha.-glycosyl-L-ascorbic acid product is needed, one can 
easily isolate an .alpha.-glycosyl-L-ascorbic acid in its possible purest 
form from the contaminants such as remaining L-ascorbic acid, D-glucose 
and .alpha.-glucosyl saccharides by one or more purification methods 
utilizing the difference in molecular weight and/or affinity; for example, 
membrane separation, gel filtration chromatography, column chromatography, 
high-performance liquid chromatography (HPLC) and ion exchange 
chromatography. In this case, the separated L-ascorbic acid and 
.alpha.-glucosyl saccharide can be favorably reused as a starting material 
in the saccharide-transfer reaction. If necessary, after completion of the 
saccharide-transfer reaction but before separation by such as 
chromatography, the reaction mixture can be treated by one or more 
methods; for example, a method wherein the reaction mixture is heated and 
the insolubilized substances are removed by filtration; another method 
wherein the reaction mixture is treated, for example, with activated 
carbon to adsorb the proteinaceous and coloring substances for their 
removal; and one another method wherein the reaction mixture is 
demineralized with cation exchange resin (H.sup.+ -form), and treated with 
anion exchange resin (OH-form) to remove anions and salts by adsorption. 
The .alpha.-glycosyl-L-ascorbic acid obtained in this way is characterized 
by: 
(1) It exhibits no direct reducing activity, and extremely stable. Unlike 
L-ascorbic acid, it scarcely causes the Maillard reaction. Because of 
these, it causes no undesired reaction when mixed with such as protein, 
lipid, saccharide and physiologically-active substance, but stabilizes 
these substances. 
(2) It is susceptible to hydrolysis to form L-ascorbic acid, and this 
elicits the same reducing activity as L-ascorbic acid. 
(3) It is readily hydrolyzable by the in vivo enzyme system into D-glucose 
and L-ascorbic acid, and the latter exhibits the physiological activities 
inherent to L-ascorbic acid. 
(4) It is highly safe because it is synthesized and then metabolized in 
vivo when L-ascorbic acid is ingested together with an .alpha.-glucosyl 
saccharide. 
(5) When an .alpha.-glycosyl-L-ascorbic acid product additionally contains 
an .alpha.-glucosyl saccharide, the .alpha.-glycosyl-L-ascorbic acid 
exhibits its inherent activities, while the .alpha.-glucosyl saccharide 
exhibits shape-imparting, filling and sweetening effects. Although a 
product free from .alpha.-glucosyl saccharide is low in shape-imparting 
and filling effects, the inherent effect is attainable with a less amount 
of the product. 
Because of these, the .alpha.-glycosyl-L-ascorbic acid exhibiting no direct 
reducing activity can be favorably incorporated as a stabilizer, 
quality-improving agent and uv-absorbent, desirably, in an amount of 0.001 
w/w % or more, in foods, beverages, preventives and remedies for 
susceptive diseases including viral diseases, bacterial diseases and 
malignant tumors, and cosmetics such as skin-refining agents and 
skin-whitening agents, as well as in agents directed to enrich a highly 
safe and stable, natural vitamin C. 
Since .alpha.-glycosyl-L-ascorbic acid is highly resistant to acid, heat 
and light, and well harmonizes with various substances which taste sour, 
salty, bitter, delicious and astringent, it is favorably usable as a 
vitamin C-enriching agent, taste-improving agent, quality-improving agent, 
stabilizer and antioxidant in foods and beverages in general, for example, 
seasonings such as soy sauce, say sauce powder, miso, miso powder, 
"moromi", "hishio", "furikake", mayonnaise, dressing, vinegar, 
"sanbai-zu", "funmatsu-sushi-su", "chuka-no-moto", "tentsuyu (soup for 
tenpura)", "mentsuyu (soup for Japanese-style noodles)", Worcester sauce, 
ketchup, "yakiniku-no-tare (soup for grilled meat)", curry roux, stew 
premix, soup premix, "dashi-no-moto", mixed seasoning, "mirin (heavily 
sweetened sake)", "shin-mirin (synthetic mirin)", table sugar and coffee 
sugar; Japanese-style confectioneries such as "senbei (rice crackers)", 
"arare (pellet-shaped senbei)", "okoshi (millet-and rice cracker)", 
"karinto (fried dough cookie)", "gyuhi (starch paste)", rice paste, "manju 
(bun with a bean-jam filling)", "uiro (sweet rice jelly)", "an (bean 
jam)", "yokan (sweet jelly of beans)", "mizu-yokan (soft adzuki-bean 
jelly)", "kingyoku", jelly, castella and "amedama (Japanese-style 
toffee)"; Western-style confectioneries such as bun, biscuit, cracker, 
cookie, pie, pudding, cream puff, waffle, sponge cake, doughnut, 
chocolate, chewing gum, caramel and candy; frozen desserts such as ice 
cream and sherbet; syrups such as those for fruit preserve and "kaki-gori 
(shaved ice)"; spreads and pastes such as butter cream, custard cream, 
flour paste and fruit paste; processed fruits such as jam, marmalade, 
syrup-preserved fruit and crystallized fruit; processed foods such as 
those of fruits and vegetables; cereals such as bakery product, noodle, 
vermicelli, boiled rice and synthetic meat; fatty food substances such as 
salad oil and margarine; pickled products such as "fukujin-zuke (sliced 
vegetables picked in soy sauce)", "bettara-zuke (fresh radish pickles)", 
"senmai-zuke" and "rakkyo-zuke (pickled shallots)"; premixes for pickled 
products such as "takuan-zuke-no-moto" and "hakusai-zuke-no-moto"; meat 
products such as ham and sausage; fish meat products such as fish meat 
ham, fish meat sausage, "kamaboko (boiled fish paste)", "chikuwa 
(literally bamboo wheels)" and "hanpen"; relishes such as "uni-no-shiokara 
(salted guts of sea urchin)", "ika-no-shiokara (salted guts of squid)", 
"su-konbu", "saki-surume" and "fugu-no-mirinboshi"; "tsukudani (food 
boiled down in soy sauce)" such as those of "nori (dried seaweed)", 
"sansai (mountain vegetables)", "surume (dried squid)", small fish and 
shellfish; daily dishes such as "nimame (cooked beans)", potato salad, 
"konbu-maki (tangle roll)" and "tenpura (deep-fried foods)"; egg and milk 
products such as "kinshi-tamago", milk beverage, butter and cheese; 
bottled and canned products such as those of meat, fish meat, fruit and 
vegetable; alcoholic drinks such as synthetic sake, "zojo-shu", liqueur, 
wine and whisky; beverages such as coffee, cocoa, juice, carbonated 
beverage, lactic acid beverage and lactobacillus beverage; and premixes 
and instant foodstuffs such as pudding premix, hot cake premix, instant 
juice, instant coffee, "sokuseki-shiruko (premix of adzuki-bean soup with 
rice cake)" and instant soup. Furthermore, .alpha.-glycosyl-L-ascorbic 
acid can be favorably incorporated in feeds and pet foods for domestic 
animals and poultries including honey bee, silkworm and pet fish for the 
enrichment of vitamin C, the improvement of their taste qualities and the 
prevention of oxidation. 
Also, .alpha.-glycosyl-L-ascorbic acid can be favorably incorporated in 
special foods and beverages, preventives and remedies for susceptive 
diseases, cosmetics including skin-refining agent and skin-whitening 
agent, for example, cigar, cigarette, troche, cod-liver oil drop, vitamin 
compound, oral refreshing agent, cachou, gargle, intubation nutrient, 
internal medicine, injection, dentifrice, lipstick, eye shadow, milky 
lotion, moisture liquid, cosmetic cream, foundation, sunscreen agent, 
cleansing soap, shampoo and rinse, in addition to the uses as uv-absorbent 
and deterioration-preventing agent for plastics and also as a substrate 
for assaying glycoside hydrolases. 
The wording "susceptive diseases" as referred to in the invention means 
those which are prevented and/or treated with .alpha.-glycosyl-L-ascorbic 
acid; for example, viral diseases, bacterial diseases, traumatic diseases, 
immunopathies, allergy, diabetes, cataract and malignant tumors. The shape 
and form of pharmaceuticals for susceptive diseases can be freely chosen 
to meet to their final use; for example, liquid pharmaceuticals such as 
nebula, collyrium, collunarium, collutory and injection, paste 
pharmaceuticals such as ointment, cataplasm and cream, and solid 
pharmaceuticals such as powder, granule, capsule and tablet. In the 
preparation of such a pharmaceutical, one or more ingredients, for 
example, remedy, biologically-active substance, antibiotic, adjuvant, 
filler, stabilizer, coloring agent and flavoring agent, can be suitably 
used in combination, if necessary. 
The dose is adequately changed dependently on the 
.alpha.-glycosyl-L-ascorbic acid content, administration route and 
administration frequency; usually, in the range of about 0.001-100 
g/day/adult as .alpha.-glycosyl-L-ascorbic acid. 
Cosmetics can be prepared similarly as in pharmaceuticals. 
.alpha.-Glycosyl-L-ascorbic acid is incorporated in products by 
conventional method, for example, mixing, kneading, dissolving, soaking, 
spreading, applying, spraying and injecting, before completion of their 
processing. 
When .alpha.-glycosyl-L-ascorbic acid and 2-O-.alpha.-D-glucosyl-L-ascorbic 
acid are in free acid form, they can be, if necessary, converted, for 
example, into sodium salt, calcium salt, magnesium salt, iron salt, copper 
salt and zinc salt by allowing them to react with an aqueous solution of 
such as metal hydroxide and metal carbonate so that the resultant 
substance is capable of adequately adjusting pH and also exhibiting the 
activities of minerals and vitamin C. Such a substance is favorably usable 
in nutritive fortifiers and chemical agents. 
The following experiments will explain in detail a typical 
.alpha.-glycosyl-L-ascorbic acid exhibiting no direct reducing activity 
according to the invention. 
EXPERIMENT 1 
Preparation of .alpha.-glucosidase 
A fresh rat small intestine was added to 0.1M phosphate buffer (pH 7.0) to 
20% by weight, fed to a homogenizer, and centrifuged at 4,000.times.g for 
10 minutes, after which the supernatant was added with a trypsin 
commercialized by Merck & Co., Inc., Rahway, N.J., USA, to give a final 
concentration of 0.1 g/l, allowed to stand at ambient temperature for 4 
hours, added with 2 volumes of a chilled ethanol, and centrifuged. The 
sediment was dissolved in 0.01M phosphate buffer (pH 7.0), and the 
solution was placed in a semipermeable membrane tube, and dialyzed for 15 
hours against a fresh preparation of the same buffer. 
Thereafter, the liquid inside the tube was chromatographed successively on 
columns of DEAE-cellulose and hydroxyapatite in usual manner to recover an 
.alpha.-glucosidase-active fraction which was then lyophilized to obtain 
an .alpha.-glucosidase specimen. 
The specimen had a specific activity of 40.7 units/mg protein, and the 
purification degree and activity yield were 357-folds and about 47% 
respectively. 
One unit of .alpha.-glucosidase is defined as the amount of enzyme that 
releases 1 micromole glucose at 37.degree. C. over a time period of 1 
minute when assayed under the following conditions. After appropriately 
diluting, 100 microliters of an enzyme solution is added to a mixture 
solution of 250 microliters of 4 w/v % maltose and 750 microliters of 0.1M 
acetate buffer (pH 6.0) containing 1.35 mM EDTA, and the mixture is 
allowed to react at 37.degree. C. for 30 minutes, incubated in boiling 
water for 3 minutes to suspend the reaction, and centrifuged. Thereafter, 
20 microliters of the supernatant is sampled, added with 1 ml of "GLUCOSE 
B TEST", a coloring reagent for the glucose oxidase method commercialized 
by Wako Pure Chemical Industries, Ltd., Osaka, Japan, incubated at 
37.degree. C. for 20 minutes for color development, and assayed for 
absorbance at 505 nm. 
EXPERIMENT 2 
.alpha.-D-Glucosyl-L-ascorbic acid 
Experiment 2(1) 
Saccharide-transfer reaction 
In 100 parts by weight of 0.2M acetate buffer (pH 5.3) was dissolved 7.04 
parts by weight of L-ascorbic acid, 12.8 parts by weight of maltose and 
0.2 parts by weight of thiourea, and the solution was added with 0.5 
units/g maltose of a partially-purified .alpha.-glucosidase specimen 
prepared by the method in Experiment 1, and allowed to react at 50.degree. 
C. for 5 hours under light-shielding conditions. The reaction was 
suspended by adding 4 volumes of 1.06 w/v % metaphosphatic acid solution 
to the reaction mixture to inactivate the enzyme. 
HPLC analysis of the reaction mixture revealed that about 30% of the 
starting L-ascorbic acid was converted into a saccharide derivative. 
Experiment 2(2) 
Purification 
The reaction mixture was then subjected to a gel filtration chromatography 
on a column of "Bio-Gel P-2", a gel product of Bio-Rad, Richmond, Calif., 
USA, using water for elution to recover an .alpha.-D-glucosyl-L-ascorbic 
acid-rich fraction which was then subjected to HPLC on "Shim-Pack ODS", a 
column product of Shimadzu Seisakusho Ltd., Kyoto, Japan, using 0.3% 
acetic acid for elution. The .alpha.-D-glucosyl-L-ascorbic acid-rich 
fraction was recovered, concentrated in vacuo and pulverized by 
lyophilization to obtain a high-purity .alpha.-D-glucosyl-L-ascorbic acid 
specimen, purity of 99.9%, in the yield of about 80% against the 
.alpha.-D-glucosyl-L-ascorbic acid in the reaction mixture. 
Experiment 2(3) 
Physicochemical properties 
A typical .alpha.-glycosyl-L-ascorbic acid specimen, prepared by the method 
in Experiment 2(2), was determined for the following physicochemical 
properties. 
Another typical .alpha.-glycosyl-L-ascorbic acid with a higher 
.alpha.-D-glucosyl group, obtained by the method in Example A-1, was 
characterized by as far as possible, and its properties are given in the 
brackets. 
(1) Elemental analysis Found: C=42.6%, H=5.36% Calculated; C=42.3%, 
H=5.38%, N&lt;0.01% (for chemical formula C.sub.12 H.sub.18 O.sub.11) 
(2) Molecular weight FD mass spectrometric analysis with "M-80B", a mass 
spectrometry commercialized by Hitachi Ltd., Tokyo, Japan, revealed a 
(M+H).sup.+ peak at 339 (molecular weight for chemical formula C.sub.12 
H.sub.18 O.sub.11 is 338). 
(3) uv-Absorption spectrum Exhibiting an absorption peak at 260 nm when at 
pH 7.0, while exhibiting an absorption peak at 238 nm when at pH 2.0. 
[Exhibiting substantially the same property] 
(4) Infrared absorption spectrum The KBr tablet method was used. The result 
was as shown in FIG. 1. 
[Exhibiting substantially the same property] 
(5) NMR spectrum The nmr spectrum was determined with "JNMGX400", an nmr 
spectrometry commercialized by Japan Electron Optics Laboratory Co., Ltd., 
Tokyo, Japan. 
The solvent was D.sub.2 O, and the pH during the measurement was 2.8. 
TSP (sodium 3-trimethyl-silylpropionate-2,2,3,3-d.sub.4) was used as the 
internal standard. 
.sup.1 H-NMR .delta.ppm (in D.sub.2 O); 
3.50 (1H, dd, J=9.5 Hz, J=9.7 Hz) 
3.56 (1H, dd, J=3.4 Hz, J=9.5 Hz) 
3.75 (2H, d, J=6.4 Hz) 
3.78 (2H, d, J=3.0 Hz) 
3.86 (1H, dd, J=9.5 Hz, J=9.5 Hz) 
4.02 (1H, dt, J=9.7 Hz, J=3.0 Hz) 
4.08 (1H, td, J=6.4 Hz, J=1.5 Hz) 
4.91 (1H, d, J=1.5 Hz) 
5.52 (1H, d, J=3.4 Hz) 
These data confirm that the alcohol group which is located at the number 
two carbon atom forms together with D-glucose a glucoside via an ether 
bond. 
(5) Dissociation constant The pKa is 3.0. Comparison of this to those for 
various derivatives of L-ascorbic acid in Table 1 in J.Jernow et al., 
Tetrahedron, Vol.35, pp.1,483-1,486 (1979) and in Table 2 in Pao-Wen Lu et 
al., Journal of Agricultural Food Chemistry, Vol.32, pp.21-28 (1984) 
suggests that in the substance of the invention, the alcohol group which 
is located at the number two carbon atom in L-ascorbic acid is responsible 
for the .alpha.-D-glucosyl bond, while the alcohol group which is located 
at the number three carbon atom is in free form. 
(6) Methylation analysis The substance was methylated by the method 
described in Pao-Wen Lu et al., Journal of Agricultural Food and 
Chemistry, Vol.32, pp.21-28 (1984) wherein L-ascorbic acid was methylated 
with diazomethane to predominantly form 3-O-methyl-L-ascorbic acid. 
Hydrolysis of the resultant led to the formation of 3-O-methyl-L-ascorbic 
acid and D-glucose as the predominant products. 
The nmr spectrum, dissociation constant and methylation analysis suggest 
that the alcohol group which is located at the number two carbon atom 
forms with D-glucose an .alpha.-glucoside linkage via an ether bond. 
(7) Solubility in solvents Readily soluble in water, 0.1N sodium hydroxide 
and 0.1N acetic acid; soluble in methanol and ethanol; and insoluble in 
ether, benzene and chloroform. 
[Exhibiting substantially the same property] 
(8) Coloring reaction Exhibiting no direct reducing activity, and not 
reducing and decoloring 2,6-dichlorophenolindophenol. Negative to the 
2,4-dinitrophenylhydrazine reaction. Turning green on the 
anthrone-sulfuric acid reaction. 
[Exhibiting substantially the same property] 
(9) Stability 
(a) Hydrolyzable by .alpha.-glucosidase or by treatment with 1N 
hydrochloric acid at 100.degree. C. for 5 minutes to form L-ascorbic acid 
and D-glucose at a molar ratio of 1:1. [Hydrolyzable by glucoamylase to 
form 2-O-.alpha.-D-glucosyl-L-ascorbic acid and D-glucose] 
(b) Unhydrolyzable by .beta.-glucosidase. [Exhibiting substantially the 
same property] 
(c) 2-O-.alpha.-D-Glucosyl-L-ascorbic acid was compared respectively to the 
6-O-.alpha.-D-glucosyl-L-ascorbic acid disclosed in Japanese Patent 
Publication No.38,158/73, and L-ascorbic acid for their stability in 
aqueous solution. More particularly, each sample was adjusted to a 
concentration of 70 micromoles and to pH 7.0 or 2.0, placed in a cuvette, 
and measured for its absorbance at either 260 nm and pH 7.0 or at 245 nm 
and pH 2.0 while keeping the solution at 20.degree. C. The remaining ratio 
(%) was calculated with the absorbance. The results were as shown in Table 
I. As obvious from the results in Table I, unlike 
6-O-.alpha.-D-glucosyl-L-ascorbic acid and L-ascorbic acid, 
2-O-.alpha.-D-glucosyl-L-ascorbic acid is extremely stable in aqueous 
solution. 
[Exhibiting substantially the same property as that of 
2-O-.alpha.-D-glucosyl-L-ascorbic acid] 
TABLE I 
______________________________________ 
Time (hour) 
pH 0 0.25 0.5 1.0 21.0 
______________________________________ 
7 2GAsA 100% 100% 100% 100% 100% 
6GAsA 100% 58% 36% 17% 8% 
AsA 100% 47% 20% 8% 2% 
2 2GAsA 100% 100% 100% 100% 100% 
6GAsA 100% 99% 98% 91% 55% 
AsA 100% 99% 97% 87% 10% 
______________________________________ 
Note: 
2GAsA is the symbol for 2O-D-glucosyl-L-ascorbic acid of the invention; 
6GAsA, for 6O-D-glucosyl-L-ascorbic acid as a control; and AsA, for 
Lascorbic acid as one another control. 
(10) Physiological activities 
(a) Reducing activity on cytochrome C 2-O-.alpha.-D-Glucosyl-L-ascorbic 
acid, 6-O-.alpha.-D-glucosyl-L-ascorbic acid and ascorbic acid were 
compared for their reducing activity on cytochrome C. To a mixture of 0.5 
ml of 0.2 mM EDTA in 0.1M potassium phosphate buffer (pH 7.8) and 0.1 ml 
of 0.1 mM cytochrome C was added an prescribed amount of water to give a 
final volume of 1 ml which was then added with 10 microliters of 10 mM of 
either sample, and determined for its change in absorbance at 550 nm and 
ambient temperature with a photospectrometry. The difference in absorbance 
(.DELTA.A/minute/10 microliters) was determined with the initial reaction 
rate, and the reducing activity was estimated with the difference in 
absorbance. As the result, it was confirmed that unlike 
6-O-.alpha.-D-glucosyl-L-ascorbic acid and L-ascorbic acid, 
2-O-.alpha.-D-glucosyl-L-ascorbic acid exhibited no reducing activity. 
Also was confirmed that 2-O-.alpha.-D-glucosyl-L-ascorbic acid exhibited a 
reducing activity when hydrolyzed by an .alpha.-glucosidase specimen 
prepared by the method in Experiment 1. 
(b) Collagenic activity 2-O-.alpha.-D-Glucosyl-L-ascorbic acid, 
6-O-.alpha.-D-glucosyl-L-ascorbic acid and L-ascorbic acid were tested for 
their collagenic activity. 
A human fibroblast cell, cell density of 7.times.10.sup.4 cells/plate, was 
cultured in Eagle's minimal essential medium supplemented with 10% FCS for 
1 week, added with 4 .mu.Ci/ml .sup.3 H-proline, 20 .mu.g/ml of 
.beta.-aminopropionitrile and 0.25 mM of either sample, and cultured for 
an additional 24 hours. The resultant was then added with 10 w/v % 
trichloroacetic acid to recover the collagenous element in the culture, 
followed by lyophilization. The resultant specimen was dissolved, and the 
solution was adjusted to an appropriate pH, treated with a collagenase 
(Type III) at 37.degree. C. for 90 minutes, and centrifuged. Collagenic 
activity was estimated by determining the radio activity in the 
supernatant. 
As the result, it was confirmed that 2-O-.alpha.-D-glucosyl-L-ascorbic acid 
had the same collagenic activity as that of L-ascorbic acid. 
Also was confirmed that 6-O-.alpha.-D-glucosyl-L-ascorbic acid was slightly 
inferior in collagenic activity to that of 
2-O-.alpha.-D-glucosyl-L-ascorbic acid. 
The above physicochemical properties confirm that the 
.alpha.-glycosyl-L-ascorbic acid exhibiting no direct reducing activity 
prepared in this Experiment has the chemical structure shown by the 
formula [III]: 
##STR3## 
wherein n is an integer from 0 to 6. 
2-O-.alpha.-D-Glucosyl-L-ascorbic acid, a typical 
.alpha.-glycosyl-L-ascorbic acid, has the chemical structure shown by the 
formula [IV]: 
##STR4## 
EXPERIMENT 3 
Synthesis in vivo 
Rats were orally administered with 1 g L-ascorbic acid and 500 mg maltose 
as in the form of 5 ml of 10 w/v % solution, and their blood was sampled 
at intervals and centrifuged. HPLC of the supernatants or plasmas 
confirmed that the peaks of .alpha.-D-glucosyl-L-ascorbic acid and a small 
amount of .alpha.-D-maltosyl-L-ascorbic acid appeared about 30 minutes 
after the administration, reached their maxima at 180 minutes, thereafter 
suddenly disappeared, and disappeared from blood for 360 minutes. 
The substance which exhibited one of these peaks and corresponded to 
.alpha.-D-glucosyl-L-ascorbic acid as the predominant element was isolated 
and studied in detail, confirming that its physicochemical properties were 
identical to those of 2-O-.alpha.-D-glucosyl-L-ascorbic acid. 
Accordingly, it would be concluded that .alpha.-glycosyl-L-ascorbic acid is 
highly safe because it is a biosubstance which is synthesized, metabolized 
and disappeared in vivo. 
EXPERIMENT 4 
Acute toxicity 
A high-purity .alpha.-D-glucosyl-L-ascorbic acid specimen, prepared by the 
method in Experiment 2(2), was orally administered to 7 week-old dd mice 
for acute toxicity test. As the result, no mouse died when administered 
with up to 5 g of the specimen, and higher dose was difficult. 
These confirmed that the specimen was extremely low in toxicity. An 
.alpha.-glycosyl-L-ascorbic acid prepared by the method in Example A-1 was 
tested similarly as above to obtain the same result, confirming that the 
toxicity of this specimen was extremely low. 
The following Examples A and Examples B will illustrate the 
.alpha.-glycosyl-L-ascorbic acid exhibiting no direct reducing activity 
and its uses respectively. 
EXAMPLE A-1 
.alpha.-Glycosyl-L-ascorbic acid 
Nine parts by weight of .alpha.-cyclodextrin was dissolved in 20 parts by 
weight of water by heating, and the solution was added with 3 parts by 
weight of L-ascorbic acid under reducing conditions, thereafter while 
keeping the solution at pH 5.5 and 60.degree. C., added with 150 units/g 
.alpha.-cyclodextrin of cyclomaltodextrin glucanotransferase 
commercialized by Hayashibara Biochemical Laboratories, Inc., Okayama, 
Japan, and allowed to react for 40 hours. The reaction mixture was fed to 
"AQ-303 ODS" HPLC system, a product of Yamamura Chemical Laboratories Co., 
Ltd., Kyoto, Japan, equipped with "LC-6" column, a product of Shimadzu 
Seisakusho Ltd., Kyoto, Japan, and eluted with 0.1M KH.sub.2 PO.sub.4 
--H.sub.3 PO.sub.4 buffer (pH 2.0) at a flow rate of 0.5 ml/minute while 
monitoring with "MULT-340" detector system, a product of Japan 
Spectroscopic Co., Ltd., Tokyo, Japan. As the result, L-ascorbic acid 
appeared at a retension time of 9.5 minutes, while the newly formed 
.alpha.-D-glucosyl-L-ascorbic acid, .alpha.-D-maltosyl-L-ascorbic acid, 
.alpha.-D-maltotriosyl-L-ascorbic acid, 
.alpha.-D-maltotetraosyl-L-ascorbic acid, 
.alpha.-D-maltopentaosyl-L-ascorbic acid, 
.alpha.-D-maltohexaosyl-L-ascorbic acid and 
.alpha.-D-maltoheptaosyl-L-ascorbic acid appeared at respective retension 
time of 11.2 minutes, 15.7 minutes, 20.6 minutes, 24.9 minutes, 28.1 
minutes, 32.1 minutes and 38.6 minutes. About 50% of the L-ascorbic acid 
was converted into .alpha.-glycosyl-L-ascorbic acid. Thereafter, the 
reaction mixture was heated to inactivate the remaining enzyme and 
filtered, and the filtrate was purified in accordance with the method in 
Experiment 2(2) with a slight modification to isolate particular 
.alpha.-glycosyl-L-ascorbic acid elements. The elements were then mixed, 
concentrated in vacuo and pulverized to obtain a powdery 
.alpha.-glycosyl-L-ascorbic acid product in the yield of about 90% against 
the starting ascorbic acid, on the dry solid basis (d.s.b.). 
The product exhibits no direct reducing activity, but exhibits 
satisfactorily high stability and physiological activity. Thus, the 
product is favorably usable as a stabilizer, quality-improving agent, 
physiologically active agent and uv-absorbent in foods, beverages, 
pharmaceuticals for susceptive diseases and cosmetics, as well as in the 
agents directed to enrich vitamin C. 
EXAMPLE A-2 
.alpha.-Glycosyl-L-ascorbic acid 
Forty parts by weight of dextrin (DE about 6) was dissolved in 50 parts by 
weight of water by heating, and the solution was added with 13 parts by 
weight of L-ascorbic acid under reducing conditions, thereafter while 
keeping the solution at pH 5.6 and 65.degree. C., added with 270 units/g 
dextrin of cyclomaltodextrin glucanotransferase, and allowed to react for 
40 hours. After analyzing the reaction mixture by HPLC similarly as in 
Example A-1, about 65% of the L-ascorbic acid was converted into 
.alpha.-glycosyl-L-ascorbic acids such as .alpha.-D-glucosyl-L-ascorbic 
acid, .alpha.-D-maltosyl-L-ascorbic acid, 
.alpha.-D-maltotriosyl-L-ascorbic acid, 
.alpha.-D-maltotetraosyl-L-ascorbic acid, 
.alpha.-D-maltopentaosyl-L-ascorbic acid and 
.alpha.-D-maltohexaosyl-L-ascorbic acid similarly as in Example A-1. 
Thereafter, the reaction mixture was heated to inactivate the remaining 
enzyme and filtered, after which the filtrate was purified in usual manner 
by the decoloration with activated carbon, and concentrated to obtain a 
syrupy .alpha.-glycosyl-L-ascorbic acid product additionally containing 
.alpha.-glucosyl saccharides in the yield of about 90% against the weight 
of the starting materials, d.s.b. 
The .alpha.-glycosyl-L-ascorbic acid in the product exhibits no direct 
reducing activity, but exhibits satisfactorily high stability and 
physiological activity. Thus, the product is favorably usable as a 
seasoning, moisture-retaining agent, quality-improving agent, 
physiologically active agent and uv-absorbent in foods, beverages, 
pharmaceuticals for susceptive diseases and cosmetics, as well as in 
agents directed to enrich vitamin C. 
EXAMPLE A-3 
2-O-.alpha.-D-Glucosyl-L-ascorbic acid 
One part by weight of a syrupy .alpha.-glycosyl-L-ascorbic acid product 
additionally containing .alpha.-glucosyl saccharides, prepared by the 
method in Example A-2 with a slight modification, was dissolved in 4 parts 
by weight of water, and the solution was added with 100 units/g syrup 
solid of glucoamylase (EC 3.2.1.3) commercialized by Toyobo Co., Ltd., 
Osaka, Japan, and allowed to react at 50.degree. C. for 50 hours. HPLC 
analysis of the reaction mixture revealed that particular 
.alpha.-glycosyl-L-ascorbic acids were converted into 
2-O-.alpha.-D-glucosyl-L-ascorbic acid. 
Thereafter, the reaction mixture was heated to inactivate the remaining 
enzyme and filtered, and the filtrate was purified by the method in 
Experiment 2(2) with a slight modification to recover a 
2-O-.alpha.-D-glucosyl-L-ascorbic acid-rich fraction which was then 
concentrated in vacuo and pulverized to obtain a high-purity 
2-O-.alpha.-D-glucosyl-L-ascorbic acid, purity of 99% or higher, in the 
yield of about 80% against the starting L-ascorbic acid, d.s.b. 
Characterization of the product confirmed that its physicochemical 
properties were substantially the same as those of the 
2-O-.alpha.-D-glucosyl-L-ascorbic acid in Experiment 2(3). 
The 2-O-.alpha.-D-glucosyl-L-ascorbic acid is favorably usable as a 
stabilizer, quality-improving agent, physiologically active agent, 
uv-absorbent, chemical and pharmaceutical material in foods, beverages, 
pharmaceuticals for susceptive diseases, cosmetics and reagents, as well 
as in agents directed to enrich vitamin C which exhibits no direct 
reducing activity, but exhibits satisfactorily high stability and 
physiological activity. 
EXAMPLE A-4 
.alpha.-Glycosyl-L-ascorbic acid 
Twenty parts by weight of dextrin (DE 18) was dissolved in 70 parts by 
weight of water by heating, and the solution was added with 10 parts by 
weight of L-ascorbic acid under reducing conditions, further added with 4 
units/g dextrin of a partially-purified .alpha.-glucosidase prepared by 
the method in Experiment 1, and allowed to react at pH 5.0 and 50.degree. 
C. for 8 hours under light-shielding conditions. The reaction mixture was 
purified, concentrated and pulverized by the method in Example A-2 with a 
slight modification to obtain a powdery product in the yield of about 90%. 
The product contained about 10 w/w % .alpha.-glycosyl-L-ascorbic acid. 
The .alpha.-glycosyl-L-ascorbic acid exhibits no direct reducing activity, 
but exhibits satisfactorily high stability and physiological activity. 
Thus, the product is favorably usable as a seasoning, moisture-retaining 
agent, quality-improving agent, physiologically active agent and 
uv-absorbent in foods, beverages, pharmaceuticals for susceptive diseases 
and cosmetics, as well as in agents directed to enrich vitamin C. 
EXAMPLE A-5 
.alpha.-Glycosyl-L-ascorbic acid 
Ten parts by weight of maltose was dissolved in 80 parts by weight of water 
by heating, and the solution was added with 10 parts by weight of 
L-ascorbic acid and 4 units/g maltose of a rice seed .alpha.-glucosidase 
commercialized by Sigma Chemical Co., Saint Louis, Mo., USA, and allowed 
to react at pH 6.0 and 45.degree. C. for 6 hours under light-shielding 
conditions. The reaction mixture was purified, concentrated and pulverized 
by the method in Example A-2 with a slight modification to obtain a 
powdery product in the yield of about 90%. The product contained about 15% 
.alpha.-glycosyl-L-ascorbic acid. 
The .alpha.-glycosyl-L-ascorbic acid in the product exhibits no direct 
reducing activity, exhibits satisfactorily high stability and 
physiological activity. Thus, the product is favorably usable as a 
sweetener, seasoning, moisture-retaining agent, quality-improving agent, 
physiologically active agent and uv-absorbent in foods, beverages, 
pharmaceuticals for susceptive diseases and cosmetics, as well as in 
agents directed to enrich vitamin C. 
EXAMPLE A-6 
.alpha.-Glycosyl-L-ascorbic acid 
EXAMPLE A-6(1) 
Preparation of .alpha.-glucosidase 
Mucor javanicus IFO 4570 was inoculated and cultivated at 30.degree. C. for 
44 hours under aeration-agitation conditions in 500 parts by weight of a 
liquid culture medium which contained water together with 4.0 w/v % 
maltose, 0.1 w/v % potassium phosphate monobasic, 0.1 w/v % ammonium 
nitrate, 0.05 w/v % magnesium sulfate, 0.05 w/v % potassium chloride, 0.2 
w/v % polypeptone and 1 w/v % calcium carbonate which had been sterilized 
by heating and sterilely added to the water immediately before the 
inoculation. After completion of the culture, the mycelia was recovered 
and immobilized in usual manner. 
EXAMPLE A-6(2) 
Preparation of .alpha.-glycosyl-L-ascorbic acid 
Forty parts by weight of "SUNMALT.RTM." a crystalline maltose 
commercialized by Hayashibara Co., Ltd., Okayama, Japan, was dissolved in 
70 parts by weight of water by heating, and the solution was added with 10 
parts by weight of L-ascorbic acid and 10 units/g maltose of an 
immobilized .alpha.-glucosidase prepared by the method in Example A-6(1) 
under light-shielding conditions, and allowed to react at pH 5.5 and 
50.degree. C. for 3 hours. 
The reaction mixture was filtered to remove the immobilized 
.alpha.-glucosidase which was then reused in another reaction batch. After 
heating, the filtrate was purified, concentrated and pulverized by the 
method in Example A-2 with a slight modification to obtain a powdery 
product in the yield of about 95%. 
The product contained about 7 w/w % .alpha.-glycosyl-L-ascorbic acid. 
The .alpha.-glycosyl-L-ascorbic acid in the product exhibits no direct 
reducing activity, but exhibits satisfactorily high stability and 
physiological activity. Thus, the product is favorably usable as a 
sweetener, seasoning, moisture-retaining agent, quality-improving agent, 
physiologically active agent and uv-absorbent in foods, beverages, 
pharmaceuticals for susceptive diseases and cosmetics, as well as in 
agents directed to enrich vitamin C. 
EXAMPLE B-1 
Chewing gum 
Twenty-five parts by weight of gum base and 20 parts by weight of a powdery 
.alpha.-glycosyl-L-ascorbic acid obtained by the method in Example A-6 
were kneaded at 60.degree. C. with a mixer, and the mixture was added with 
50 parts by weight of "MABIT.RTM.", an anhydrous crystalline maltitol 
commercialized by Hayashibara Shoji Inc., Okayama, Japan, 1.5 parts by 
weight of calcium phosphate and 0.1 part by weight of an L-menthol 
including .beta.-cyclodextrin, and further mixed with a small amount of 
seasoning, rolled and cut to obtain the captioned product. The product is 
a vitamin C-enriched, low-cariogenic and low-caloric chewing gum. 
EXAMPLE B-2 
"Gyuhi (starch paste)" 
One part by weight of waxy rice starch was mixed with 1.2 parts by weight 
of water, and the mixture was mixed to homogeneity with 1.5 parts by 
weight of sucrose, 0.7 parts by weight of "SUNMALT.RTM.", a crystalline 
.beta.-maltose commercialized by Hayashibara Co., Ltd., Okayama, Japan, 
0.5 parts by weight of a syrupy .alpha.-glycosyl-L-ascorbic acid obtained 
by the method in Example A-2 while gelatinizing by heating. Thereafter, 
the resultant was molded and packaged in usual manner to obtain "gyuhi". 
The product is a vitamin C-enriched, Japanese-style confectionery with 
excellent flavor and biting properties, which looks like "kibi-dango 
(millet dumpling)". The product exhibits a long shelf life because its 
retrogradation is effectively suppressed. 
EXAMPLE B-3 
Mixed sweetener 
A mixed sweetener was obtained by mixing 100 parts by weight of honey, 50 
parts by weight of isomerized sugar, 2 parts by weight of "kurozato 
(unrefined sugar)" and 1 part by weight of a high-purity 
2-O-.alpha.-D-glucosyl-L-ascorbic acid powder obtained by the method in 
Example A-3. 
The product is a vitamin C-enriched sweetener, and suitable for health 
food. 
EXAMPLE B-4 
Chocolate 
Forty parts by weight of cacao paste, 10 parts by weight of cacao butter, 
50 parts by weight of anhydrous crystalline maltitol and 1 part by weight 
of an .alpha.-glycosyl-L-ascorbic acid powder obtained by the method in 
Example A-1 were mixed to homogeneity, and the mixture was fed to a 
refiner to reduce the particle size, transferred to a conche, and kneaded 
therein at 50.degree. C. for 2 days. In the kneading step, 0.5 parts by 
weight of lecithin was added and dispersed to homogeneity. Thereafter, the 
content was adjusted to 31.degree. C. with a thermoregulator, and placed 
in a mold immediately before the solidification of the butter, deaerated 
with a vibrator, and solidified by passing it through a 10.degree. C. 
cooling tunnel over a period of 20 minutes. The content was removed from 
the mold, and packaged to obtain the captioned product. 
The product is free of hygroscopicity and excellent in color, gloss and 
texture, as well as smoothly melting in the mouth to exhibit a moderate 
and mild sweetness and flavor. The product is a vitamin C-enriched, 
low-cariogenic and low-caloric chocolate. 
EXAMPLE B-5 
Cream filling 
A cream filling was obtained by mixing in usual manner 1,200 parts by 
weight of "FINETOSE.RTM.", a crystalline .alpha.-maltose commercialized by 
Hayashibara Co., Ltd., Okayama, Japan, 1,000 parts by weight of 
shortening, 10 parts by weight of an .alpha.-glycosyl-L-ascorbic acid 
powder obtained by the method in Example A-2, 1 part by weight of 
lecithin, 1 part by weight of lemon oil and 1 part by weight of vanilla 
oil to homogeneity. 
The product is a vitamin C-enriched cream filling which is excellent in 
taste, flavor, melting and biting properties, and the oxidation of the 
fatty substances is effectively suppressed. 
EXAMPLE B-6 
Tablet 
Twenty parts by weight of a high-purity 2-O-.alpha.-D-glucosyl-L-ascorbic 
acid powder obtained by the method in Example A-3 was mixed to homogeneity 
with 13 parts by weight of crystalline .beta.-maltose, 4 parts by weight 
of cornstarch, 1 part by weight of rutin and 0.5 parts by weight of 
riboflavin, and the resultant was tableted to obtain the captioned 
product, 150 mg each. 
The product is a stable and easily swallowable vitamin compound of vitamin 
C, vitamin P and vitamin B.sub.2. 
EXAMPLE B-7 
Capsule 
Ten parts by weight of calcium acetate monohydrate, 50 parts by weight of 
magnesium L-lactate trihydrate, 57 parts by weight of maltose, 20 parts by 
weight of an .alpha.-glycosyl-L-ascorbic acid powder obtained by the 
method in Example A-2, and 12 parts by weight of a .gamma.-cyclodextrin 
inclusion compound containing 20% eicosapentaenoic acid were mixed to 
homogeneity, and the mixture was fed to a granulator, and then 
encapsulated in gelatine to obtain capsules, 150 mg each. 
The product is favorably usable as a high-quality blood cholesterol 
lowering agent, immunopotentiator and skin-refining agent in preventive 
and remedy for susceptive diseases, as well as in foodstuffs directed to 
the maintenance and promotion of health. 
EXAMPLE B-8 
Ointment 
One part by weight of sodium acetate trihydrate, 4 parts by weight of 
DL-calcium lactate and 10 parts by weight of glycerine were mixed to 
homogeneity, and the mixture was added to another mixture of 50 parts by 
weight of vaseline, 10 parts by weight of vegetable wax, 10 parts by 
weight of lanolin, 14.5 parts by weight of sesame oil, 1 part by weight of 
an .alpha.-glycosyl-L-ascorbic acid obtained by the method in Example A-4 
and 0.5 parts by weight of peppermint oil, and mixed to homogeneity to 
obtain an ointment. 
The product is favorably usable as a high-quality sunscreen agent, 
skin-refining agent, skin-whitening agent and promoter for healing injury 
and burn. 
EXAMPLE B-9 
Injection 
A high-purity .alpha.-D-glucosyl-L-ascorbic acid powder obtained by the 
method in Experiment 2(2) was dissolved in water, neutralized and 
sterilely filtered in usual manner to obtain a pyrogen-free solution which 
was then distributed to 20 ml glass vials to give an 
.alpha.-D-glucosyl-L-ascorbic acid content of 500 mg, dried in vacuo and 
sealed to obtain the captioned product. 
The product is intramuscularly and intravenously administrable alone or in 
combination with vitamins and minerals. The product requires no cold 
storage, and exhibits an excellently high solubility in saline when in 
use. 
The product, which exhibits in vivo an about 2-10-fold longer residence 
time than L-ascorbic acid, is gradually hydrolyzed to release L-ascorbic 
acid which then elicits its inherent physiological activities. 
Besides supplementing vitamin C, the product acts as an antioxidant to 
exert both activated oxygen-removing and lipoperoxide formation-inhibiting 
effects when hydrolyzed. Thus, the product is favorably usable in 
preventive and remedy for various susceptive diseases such as viral 
diseases, bacterial diseases, traumatic diseases, immunopathies, allergy, 
diabetes, cataract, circulatory diseases and malignant tumors. 
EXAMPLE B-10 
Injection 
Six parts by weight of sodium chloride, 0.3 parts 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 a 
high-purity 2-O-.alpha.-D-glucosyl-L-ascorbic acid powder obtained by the 
method in Example A-3 were dissolved in 1,000 parts by weight of water, 
and sterilely filtered in usual manner, and 250 ml aliquots of the 
resultant pyrogen-free solution were distributed to sterilized plastic 
vessels to obtain the captioned product. 
The product is usable in the supplement of vitamin C, calorie and minerals. 
The product acts as an antioxidant to exert both activated oxygen-removing 
and lipoperoxide formation-inhibiting effects when hydrolyzed. Thus, the 
product is favorably usable in the restoration of health during and before 
suffering from diseases, as well as in preventive and remedy for 
susceptive diseases such as viral diseases, bacterial diseases, traumatic 
diseases, immunopathies, allergy, diabetes, cataract, circulatory diseases 
and malignant tumors. 
EXAMPLE B-11 
Intubation nutrient 
Twenty four gram aliquots of a compound consisting of 20 parts by weight of 
crystalline .alpha.-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 part 
by weight of magnesium carbonate, 0.1 part by weight of an 
.alpha.-glycosyl-L-ascorbic acid powder obtained by the method in Example 
A-5, 0.01 part by weight of thyamine and 0.01 part by weight of riboflavin 
were packed in laminated aluminum bags, and heat-sealed to obtain the 
captioned product. 
In use, one bag of the product is dissolved in about 300-500 ml of water, 
and the solution is favorably usable as an intubation nutrient directed to 
oral and parenteral administration to the nasal cavity, stomach and 
intestine. 
EXAMPLE B-12 
Bath liquid 
A bath liquid was obtained by mixing 21 parts of DL-sodium lactate, 8 parts 
by weight of sodium pyruvate, 5 parts by weight of an 
.alpha.-glycosyl-L-ascorbic acid powder obtained by the method in Example 
A-1 and 40 parts by weight of ethanol with 26 parts by eight of refined 
water and appropriate amounts of coloring agent and flavoring agent. 
The product is suitable for skin-refining agent and skin-whitening agent, 
which is diluted by 100-10,000-folds in bath water when in use. In this 
case, bath water is replaceable with cleansing liquid, astringent and 
moisture liquid. 
EXAMPLE B-13 
Milky lotion 
One half part by weight of polyoxyethylene behenyl ether, 1 part by weight 
of polyoxyethylene sorbitol tetraoleate, 1 part by weight of oil-soluble 
glyceryl monostearate, 0.5 parts by weight of pyruvic acid, 0.5 parts by 
weight of behenyl alcohol, 1 part by weight of avocado oil, 1 part by 
weight of a high-purity 2-O-.alpha.-D-glucosyl-L-ascorbic acid powder 
obtained by the method in Example A-3 and appropriate amounts of vitamin E 
and antiseptic were dissolved in usual manner by heating, and the solution 
was added with 1 part by weight of L-sodium lactate, 5 parts by weight of 
1 3-butylene glycol, 0.1 part by weight of carboxyvinyl polymer and 85.3 
parts by weight of refined water, emulsified with a homogenizer, added 
with an appropriate amount of flavoring agent, and mixed by stirring to 
obtained the captioned product. 
The product is favorably usable as a high-quality sunscreen agent, 
skin-refining agent and skin-whitening agent. 
EXAMPLE B-14 
Cosmetic cream 
Two parts by weight of polyoxyethylene glycol monostearate, 5 parts by 
weight of self-emulsifying glycerine monostearate, 2 parts by weight of a 
high-purity 2-O-.alpha.-D-glucosyl-L-ascorbic acid powder obtained by the 
method in Example A-3, 1 part by weight of liquid paraffin, 10 parts by 
weight of glyceryl trioctanate and an appropriate amount of antiseptic 
were dissolved in usual manner by heating, and the mixture was added with 
2 parts by weight of L-lactic acid, 5 parts by weight of 1,3-butylene 
glycol and 66 parts by weight of refined water, emulsified with a 
homogenizer, added with an appropriate amount of flavoring agent, and 
mixed by stirring to obtained the captioned product. 
The product is favorably usable as a high-quality sunscreen cream, 
skin-refining agent and skin-whitening agent. 
As described above, .alpha.-glycosyl-L-ascorbic acid, a novel substance of 
the invention, is free of direct reducing activity, superior in stability, 
and readily hydrolyzable in vivo to exhibit the antioxidant and 
physiological activities inherent to L-ascorbic acid. Furthermore, 
.alpha.-glycosyl-L-ascorbic acid is a highly safe substance because it is 
synthesized and metabolized in vivo. 
.alpha.-Glycosyl-L-ascorbic acid is easily formed by a biochemical process 
wherein a saccharide-transferring enzyme is allowed to act on a solution 
containing L-ascorbic acid and an .alpha.-glucosyl saccharide. Thus, 
.alpha.-glycosyl-L-ascorbic acid is superior in economical efficiency, and 
commercializable with an ease. 
Since the .alpha.-glycosyl-L-ascorbic acid exhibiting no direct reducing 
activity is satisfactorily high in stability and physiological activity, 
it is favorably usable as a stabilizer, quality-improving agent, 
antioxidant, physiologically active agent and uv-absorbent in foodstuffs 
including beverages and processed foods, preventive and remedies for 
susceptive diseases, and cosmetics including skin-refining agent and 
skin-whitening agent. Thus, the .alpha.-glycosyl-L-ascorbic acid of the 
invention has an extensive use, and is very significant in these 
industries.