JP3065170B2 - Method for producing 6-O-β-D-galactopyranosyl-L-ascorbic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to 6-O-β-D-galactopyranosyl-L, an ascorbic acid sugar derivative having excellent stability.
-It relates to a method for producing ascorbic acid in high yield.

[0002]

2. Description of the Related Art L-ascorbic acid is not only widely used as an essential vitamin or an antioxidant in the field of medicines and foods, but also has a melanin-reducing action, and is therefore used as a whitening cosmetic. It is.

However, L-ascorbic acid is a very unstable substance, and is known to be easily decomposed and denatured by heat, light, oxygen and the like. Many attempts have been made to stabilize such unstable L-ascorbic acid, and some derivatives have been developed. For example, ascorbic acid phosphate (JP-A-57-140789), glycosyl ascorbic acid (JP-A-58-5920), ascorbic acid fatty acid ester (JP-A-62-840)
No. 72 gazette) can be given as a specific example.

[0004] As a method for producing 6-O-β-D-galactopyranosyl-L-ascorbic acid, a mixture of L-ascorbic acid and lactose or a lactose-containing substance is mixed with Aspergillus oryzae (Japanese Unexamined Patent Publication No. No. 311490), enzymes derived from microorganisms such as Escherichia coli and Aspergillus nigar, enzymes derived from animal organs such as cattle liver, and β-galactosidase derived from plant seeds such as jack beans. There is known a method of producing the same by acting.

[0005]

As described above, various ascorbic acid derivatives have been developed, but their stability is still not sufficient, and their production methods also require expensive reagents. , The yield is low, and the reaction conditions are extremely severe. Therefore, an object of the present invention is to provide a method for producing 6-O-β-D-galactopyranosyl-L-ascorbic acid at a high yield and at low cost under mild conditions.

[0006]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that a mixture of L-ascorbic acid and lactose or a lactose-containing mixture can be treated with Bacillus.
When β-galactosidase derived from circulans (Bacillus circulans) is acted on, it is less susceptible to inhibition by galactose, is inexpensive and has a high yield under mild conditions, and has a stable 6-O-β-D-galactose represented by the following chemical formula: Pyranosyl-L
-It has been found that ascorbic acid can be synthesized, and the present invention has been completed.

[0007]

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[0008] That is, the present invention is characterized in that β-galactosidase derived from Bacillus circulans is allowed to act on a mixture of L-ascorbic acid and lactose or a lactose-containing substance. It is intended to provide a method for producing galactopyranosyl-L-ascorbic acid.

Hereinafter, the present invention will be described in detail. In the present invention, the concentration of L-ascorbic acid during the reaction is 0.1 to 30.
% (W / V) is appropriate, preferably 1 to 25% (W / V), and more preferably 15 to 20% (W / V). The lactose used in the present invention may be ordinary lactose or a lactose-containing material that has not been highly purified. The lactose concentration during the reaction is suitably 1 to 30% (W / V), but 5 to 20%.
(W / V) is preferable, and 10 to 15% (W / V) is more preferable. The pH at the time of the reaction is suitably from 3 to 9, preferably from 5 to 7, and more preferably from 5.5 to 6.5. The unit number of β-galactosidase (EC 3.2.1.23) derived from Bacillus circulans used for the reaction is 10 to 15 units.
0 unit / ml is appropriate. Next, the reaction temperature was 2
The range of 0-60 degreeC is suitable, the range of 30-50 degreeC is preferable, and 35-40 degreeC is more preferable. The reaction time is not limited, but 6-O-β-D-galactopyranosyl-L-
A time that maximizes the amount of ascorbic acid produced may be selected.

6-O-β-D- produced by such a method
Galactopyranosyl-L-ascorbic acid can be separated by ordinary separation means such as ion exchange column chromatography, activated carbon column chromatography, liquid chromatography, silica gel column chromatography and the like.

[0011]

Next, the present invention will be described specifically with reference to examples. Example 1 Sodium L-ascorbate (Ishizu Pharmaceutical special grade reagent) 19
g and lactose 15 g in 100 ml of distilled water,
The pH was adjusted to 6 with hydrochloric acid. To this, 7,500 units of β-galactosidase (manufactured by Daiwa Kasei) derived from Bacillus circulans were added, and 5
Allowed to react for hours.

The obtained reaction solution was used for Bio-Rad (BIO-RA).
D) AMINEX ION EXC column for high performance liquid column chromatography
LUSION) HPX-87H. At this time,
Using 0.01 N H ₂ SO ₄ as eluent, flow rate 0.6 ml
The reaction was eluted at / min. The detection at that time was performed with UV ₂₄₅ .

As a result, 9.5 g of 6-O-β-D-galactopyranosyl-L-ascorbic acid was produced. At this time, the reaction yield was 27.8%.

Next, 50 ml of the above reaction product was applied to a 450 ml activated carbon column (for chromatography by Wako Pure Chemical Industries), and eluted with water at 15 ml / min. At this time, L-ascorbic acid and 6-O-β-D-galactopyranosyl-L-ascorbic acid elute in this order.
Only β-D-galactopyranosyl-L-ascorbic acid was collected and concentrated. Transfer the concentrate to 5 ml of anion exchange resin Dowe
x 2 × 8 column (Cl ⁻ type), flow rate 1 ml / mi
After passing 100 ml of distilled water with n, 100 ml
Of 0.04 M NaCl was passed through the column to elute the desired 6-O-β-D-galactopyranosyl-L-ascorbic acid. This fraction is electrodialyzed (Micro-Acilizer manufactured by Asahi Kasei Corporation)
And lyophilized to obtain 4.2 g of 6-O-β-D-galactopyranosyl-L-ascorbic acid. The purification yield at this time was 82.5%.

Example 2, Comparative Example 1 1.9 g of sodium L-ascorbate, 1.5 g of lactose,
And an amount of galactose having a predetermined concentration [galactose concentration: 0, 1.0, 1.5, 2.5, 5.0, 10.
0.15.0% (W / V)] was dissolved in 10 ml of distilled water, and the pH of each solution was adjusted to 5 with 1N hydrochloric acid. To this solution, 750 units of β-galactosidase (manufactured by Daiwa Kasei) derived from Bacillus circulans was added, and reacted at 40 ° C. for 5 hours. 6-O-β-D-galactopyranosyl-L-ascorbic acid produced in each reaction solution was quantified by the above liquid column chromatography.

For comparison, instead of β-galactosidase from Bacillus circulans (Example 2), β-galactosidase from Aspergillus oryzae (Grade XI, manufactured by Sigma) ( Comparative Example 1) was added, reacted similarly, and quantified.

As a result, Aspergillus oryzae (As
pergillus oryzae),
Galactose concentration as low as 5% (W / V) and 50%
6-O-β-D-galactopyranosyl-L-ascorbic acid synthesis inhibition (IC ₅₀ ), whereas β-galactosidase from Bacillus circulans had a galactose concentration of 15%. % (W / V)
IC ₅₀ was observed in and high concentration.

As is apparent from the results, Bachiras
Β-galactosidase from Bacillus circulans is an Aspergillus oryzae
lusoryzae), it is less susceptible to galactose inhibition than β-galactosidase, and it is considered that the synthesis rate of 6-O-β-D-galactopyranosyl-L-ascorbic acid is high.

[0019]

According to the present invention, 6-O-β-D-galactopyranosyl-L-ascorbic acid can be produced inexpensively and with high yield under mild conditions.

────────────────────────────────────────────────── ─── Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C12P 19/00-19/64 C07H 1/00-23/00 C12N 9/00-9/99 BIOSIS (DIALOG) ) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A mixture of L-ascorbic acid and lactose or a lactose-containing substance is mixed with Bacillus circulans
A method for producing 6-O-β-D-galactopyranosyl-L-ascorbic acid, which comprises reacting β-galactosidase from irculans).