JPS5854799B2 - Manufacturing method of glycosyl vitamins - Google Patents

Description

Detailed Description of the Invention The present invention involves contacting a glycosyl vitamin aqueous solution containing a large amount of water-soluble saccharides with a porous synthetic adsorbent to adsorb the glycosyl vitamin onto the porous synthetic adsorbent. The present invention relates to a method for producing glycodyl vitamins, which is characterized by eluating and collecting glycodyl vitamins from a synthetic adsorbent.

Glycodyl vitamins are highly water-soluble vitamins in which sugars are bound to vitamins. Also known as riboflavinyl glycoside), glycodyl vitamin P (
Other names include rutin glycoside and esculin glycoside.

However, the glycosyl vitamin-containing reactant prepared in this way contains a large amount of water-soluble saccharides, such as saccharides with glycosidic bonds and their decomposition products, which were used as substrates (sugar donors) during the reaction together with glycosyl vitamins. They coexist.

As a method for producing highly pure glycodyl vitamins by separating and removing water-soluble saccharides from such glycodyl vitamin-containing reactants, for example, Japanese Patent Publication No. 48-19957
A method for producing glycosyl vitamin B by the hydrosulfide reduction precipitation method disclosed in Japanese Patent Publication No. 1982-32073, or a method for producing glycosyl vitamin P by the basic lead acetate precipitation method disclosed in Japanese Patent Publication No. 54-32073. And so on.

However, these methods not only consume a large amount of chemicals, but also require complicated operations, making it difficult to implement them industrially.

The present inventor has conducted intensive studies with the aim of easily collecting glycodyl vitamins from a glycodyl vitamin aqueous solution containing a large amount of water-soluble saccharides.

As a result, we found that porous synthetic adsorbents such as porous styrene-divinylbenzene polymer resin selectively adsorb glycodyl vitamins without adsorbing water-soluble sugars that coexist in large quantities. The present invention was completed by confirming that glycosyl vitamins can be easily eluted and collected by bringing an organic solvent into contact with a porous synthetic adsorbent that has adsorbed glycosyl vitamins.

However, the present invention was not effective for collecting ascorbic acid glycoside, which is described in Japanese Patent Publication No. 48-38158, among glycodyl vitamins.

Glycodyl vitamin B2 referred to in the present invention refers to vitamin B
A substance in which sugar is bound to 2, such as riboflavinyl glucoside, riboflavinyl maltoside, riboflavinyl isomaltoside, riboflavinyl galactoside, etc.

Furthermore, glycosyl vitamin P is a substance in which sugar is bound to vitamin P, and includes, for example, rutin glucoside, rutin maltoside, esculin glucoside, esculin maltoside, esculin maltotrioside, and the like.

The porous synthetic adsorbent referred to in the present invention is porous and has a large adsorption surface area, and is nonionic, such as styrene-divinylbenze copolymer, phenol-form, etc. Synthetic resins such as 1J resin, acrylate resin, methacrylate resin, etc., such as commercially available product names manufactured by Rohm & Haas Co., Ltd. - Light XAD-1, Amparite XA
D-2, Amberlight XAD-4, Amberlight X
AD-7, AMBERLITE Examples include Ion HP-30, Diaion HP-40, Diaion HP-50, and IMACTI 5yn-42, IMACTI 5yn-44, and IMACTI Syn-46.

In addition, in the present invention, the method of contacting these porous synthetic adsorbents may be a batch method, but in the case of mass production, a continuous method in which liquid is passed through a column packed with porous synthetic adsorbents is suitable. There is.

To describe the present invention specifically, for example, Japanese Patent Publication No. 52-28
As disclosed in Japanese Patent Publication No. 844 and Japanese Patent Publication No. 54-32073, cyclodextrin glucanotransferase (E, C, 2,4,1゜19), α-amylase (E,
C, 3, 2, 1, 1), α-glucosidase (E, C,
Glycosyltransferases such as 3, 2, 1, 20) are reacted to produce glycosyl vitamins, and if necessary, the reaction solution is treated with magnesium aluminate silicate, magnesium aluminate, etc. Impurities such as proteins are adsorbed and removed, and desalted by treatment with a strongly acidic ion exchange resin (H type) and a weakly basic ion exchange resin (OH type).

When the aqueous solution containing glycodyl vitamins prepared in this way is passed through a column packed with a porous synthetic adsorbent, glycodyl vitamins and unreacted vitamins are adsorbed, whereas a large amount The water-soluble saccharides coexisting in the water flow out without being adsorbed.

Next, after washing the porous synthetic adsorbent adsorbing the glycosyl vitamin and unreacted vitamins with dilute alkaline water, water, etc., a relatively small amount of an organic solvent or a mixture of an organic solvent and water, for example, When a liquid such as methanol water or ethanol water is passed through it, it is separated from the water-soluble saccharides, and the glycosyl vitamins are eluted first.If the flow rate is increased or the organic solvent concentration is increased, the unreacted vitamins are eluted. .

This glycosyl vitamin-rich eluate is distilled, an organic solvent is first distilled, and then concentrated to an appropriate concentration to obtain a sillage-like product containing glycosyl vitamins as the main component.

Furthermore, by drying and powdering this syllabary glycodyl vitamin product, a powdered product containing glycodyl vitamin as a main component can be obtained.

This elution operation of glycosyl vitamins and unreacted vitamins using an organic solvent also serves as an operation for regenerating the porous synthetic adsorbent used at the same time, thus making it possible to repeatedly use this porous synthetic adsorbent.

Furthermore, the present invention has the feature that not only water-soluble saccharides but also impurities such as salts can be removed at the same time.

The glycosyl vitamin thus obtained and manufactured according to the present invention has (1) extremely high water solubility.

(2) High light resistance and stability.

(3) Less bitterness.

(4) It is hydrolyzed into sugar and vitamins by enzymes in the body, and exhibits physiological activity as a vitamin.

Because of its characteristics, it can be used, for example, as a vitamin supplement for fortifying foods and drinks, feed, and feedstuffs, as a cosmetic compounding agent, and as a mouthwash, eye drops, nasal drops, oral medicine, etc.
It can be advantageously used as a compounding agent for pharmaceuticals such as injections.

In particular, in the case of glycosyl vitamin B2, it can be used as a water-soluble yellow food coloring, and furthermore, it can be used as a water-soluble food coloring compound, such as glycosylvitamin B2 and water-soluble food coloring blue No. 1. It can be easily blended with other water-soluble food pigments, such as easily preparing a water-soluble edible green pigment that has not been previously available.

Next, a few examples will be described.

Example 1 (1-1) Regulation of transferase (cyclodextrin glucanotransferase) Bacillus stearothermophilus
lus) FERM-PA2222, soluble starch 2 w/v%, ammonium nitrate 1 w/v%
v %, dipotassium phosphate 0.1 w/v %, magnesium sulfate heptahydrate 0.05 w/v %, corn staple liquor 0.5 w/v, calcium carbonate 1
The cells were inoculated into a sterilized liquid medium 101 consisting of w/v water and cultured with aeration at 50° C. for 3 days.

The obtained culture solution was centrifuged, and the supernatant was diluted with ammonium sulfate 0.7
Salting out at saturation, the activity of cyclodextrin glucanotransferase (E, C, 2, 4, 1, 19) is approximately so,
A crude enzyme preparation having ooo units was obtained.

1 unit of activity here means 5 units of 0.3 w/v % soluble starch solution containing pH 5, 5, 0,02M acetate buffer and 2X10-3M calcium chloride.
Add 0.2 rILl of an appropriately diluted enzyme solution (approximately 1 to 2 units per a) to rILl, react at 40°C for 10 minutes, then take 0.5 ml of the reaction solution and add 15 ml of a 0.02N aqueous sulfuric acid solution. to stop the reaction, and add 0.0% to this reaction stop solution. IN iodine potassium iodide solution 0.2
This is the amount of enzyme that completely eliminates the iodine coloration of soluble starch 15η by adding ml of soluble starch to develop color, then measuring the absorbance at 660 nm, and reacting at 40°C for 10 minutes.

1-2) Production of glycosyl vitamin B2 2.5 g of riboflavin, 500 g of dextrin D, E, 18
, 600 ml of water, the temperature was cooled to 60°C, and then 1,000 units of the crude cyclodextrin glucanotransferase preparation prepared in Example (1-1) were added, pi-16,0, 20 while keeping the temperature at 60℃
Allowed time to react.

When the reaction solution was analyzed by paper chromatography, it was found that riboflavin 65 was found to be riboflavinyl glycoside (
It was converted to glycosyl vitamin B2).

This reaction solution was kept at a temperature of 95°C for 10 minutes to inactivate the enzyme, then cooled to room temperature, and 1 g of magnesium aluminate silicate granules (trade name: Lycilin, manufactured by Fuji Chemical Industry Co., Ltd.) was added. The mixture was kept for 30 minutes with occasional stirring, and then filtered through suction to adsorb and remove impurities such as proteins derived from the enzyme preparation.

The obtained liquid was treated with a porous synthetic adsorbent (Rohm & Ha
The solution was passed through a glass column filled with Amberly (trade name) XAD-7) 51 manufactured by S Company at SV2.

As a result, riboflavinyl glycoside and unreacted riboflavin in the solution were adsorbed to the porous synthetic adsorbent, while dextrin, oligosaccharides, salts, etc. leaked out without being adsorbed.

The porous synthetic adsorbent adsorbing this riboflavinyl glycoside and unreacted riboflavin was then washed with 30 J of water, and then washed with 50 v/v % methanol 101
The solution was passed through to elute riboflavinyl glycoside and riboflavin.

This eluate was distilled under reduced pressure to remove methanol, further concentrated, and then dried under reduced pressure and powdered to obtain about 3.5 g of an orange-yellow powdered glycosyl vitamin B2 product.

Honjima contains a large amount of riboflavinyl glycoside, so it is extremely easily soluble in cold water, has excellent fire resistance, and has little bitterness, so it can be used as a vitamin B2 fortifier for various foods and drinks, cosmetics, pharmaceuticals, etc. can be suitably used as a water-soluble food coloring agent.

Example 2 (2-1) Adjustment of transferase (α-glucosidase) 7/L/dose 4 w/v%, monopotassium phosphate 0,1 w/v%, ammonium nitrate 0.1w/
v%, magnesium sulfate 0.05 w/v%, potassium chloride 0.05w/v%, polypeptone 0.2w/v%
Mucor javanicus (Mucor
cor javanicus) I FO4570 was cultured with shaking at a temperature of 30° C. for 44 hours.

After culturing, collect the bacterial cells and add 0 to 48 g of wet bacterial cells.
．． After adding 500 ml of 4M urea solution dissolved in 5M acetate buffer (pH 5,3) and standing at a temperature of 30°C for 40 hours,
Centrifuged.

This supernatant was dialyzed overnight in running water, brought to saturation with ammonium sulfate by 0.9, left overnight at a temperature of 4°C, the resulting salt precipitate was collected, and 50ml of 0.01M acetate buffer (PH5,3) was added. After suspension and dissolution, the supernatant was centrifuged and used as a crude α-glucosidase sample.

(2-2) Production of glycosyl vitamin B2 1 g of riboflavin, 100 g of dextrin of D, E, 10
After heating and dissolving in 0rfLl of water, the crude α-glucosidase preparation prepared in Example (2-1) was cooled to a temperature of 50° C. 40. d was added thereto, and the mixture was reacted for 400 hours while maintaining the pH at 8.9 and the temperature at 50°C.

When the reaction solution was analyzed by paper chromatography, 88% of riboflavin was found to be riboflavinyl glycoside (
It was converted to glycosyl vitamin B2).

This reaction solution was kept at a temperature of 95°C for 10 minutes to inactivate α-glucosidase, and then treated with magnesium aluminate silicate in the same manner as in Example (1-2) to remove contaminants such as proteins derived from the enzyme preparation. Adsorbed and removed things.

The obtained liquid was passed through a stainless steel column filled with a porous synthetic adsorbent (trade name: Diaion HP-20 manufactured by Mitsubishi Chemical Industries, Ltd.) at SV2.

As a result, riboflavinyl glycoside and unreacted riboflavin in the solution were adsorbed to the porous synthetic adsorbent, and water-soluble saccharides such as dextrins, oligosaccharides, and glucose and salts flowed out without being adsorbed.

Next, the porous synthetic adsorbent adsorbing riboflavinyl glycoside and unreacted riboflavin was washed with 1O1 of water, and then 20 v/v% ethanol 51 was passed through it to elute riboflavinyl glycoside and riboflavin. .

This eluate was distilled under reduced pressure in the same manner as in Example (1-2) to remove ethanol, and was further concentrated, dried, and powdered to obtain an orange-yellow powdered glycosyl vitamin B2 product of approximately 1.1 g.

Similar to the product obtained in Example (1-2), this product can be suitably used as a vitamin B2 fortifier for various foods and drinks, cosmetics, pharmaceuticals, etc., and also as a water-soluble food coloring agent.

Example 3 Production of glycosyl vitamin B2 After heating and dissolving 2 g of riboflavin and lactose Ion in 900 m of 0.2 M phosphate buffer (1) H6,8), it was cooled to a temperature of 30°C, and transferase solution 2 was added to this. .000m
1 and 2.000 rILl of β-galactosidase (manufactured by Nutritional Biochemicals) were mixed and reacted at a temperature of 30'C for 16 hours.

When the reaction solution was analyzed by paper chromatography, the 23rd group of riboflavin was found to be riboflavinyl glycoside (
It was converted to glycosyl vitamin B, ).

After concentrating this reaction solution to 1,000M, perchloric acid was added to give a final concentration of 2, and proteins were removed by centrifugation.

The supernatant was neutralized to pH 5.0 with IN-caustic soda solution and concentrated to 200 mA', then gradually cooled with stirring to a final temperature of 4°C and maintained for 16 hours.

The riboflavin crystallized in this solution was removed by centrifugation, and the resulting supernatant was collected using an ion exchange resin (Amberley) IR.
-200 (H type) and Amberlight IR-68 (O
H type) was used for ion exchange and desalting using 200a each.

This desalted solution is passed through a column filled with a porous synthetic adsorbent (trade name: IMACTI 5yn-42 manufactured by IMACTI) at SV2 to adsorb riboflavinyl glycoside and remaining dissolved riboflavin, lactose, After separating from water-soluble sugars such as glucose and galactose,
Riboflavinyl glycoside and boflavin were eluted with ethanol water in the same manner as in Example (2-2), concentrated, dried, and powdered to obtain an orange-yellow powdered glycosyl vitamin B2 product of approximately 0.0 sg.

Similar to the product obtained in Example (1-2), this product can be suitably used as a vitamin B2 fortifier for various foods and drinks, cosmetics, pharmaceuticals, etc., and also as a water-soluble food coloring agent.

Example 4 Production of glycosyl vitamin P Rutin 2g% D, E, 300g of dextrin of 18 was dissolved by heating in 1,800rrLl of water, and then the temperature was 60°C.
The crude cyclodextrin glucanotransferase preparation 1 prepared by the method of Example (1-1) was added to this.
,000 units were added thereto, and the mixture was reacted for 16 hours while maintaining the pH at 6.0 and the temperature at 60°C.

Analysis of the reaction solution by paper chromatography revealed that 93% of rutin had been converted to rutin glycoside (glycosyl vitamin P).

This reaction solution was heated in the same manner as in Example (1-2) to inactivate the enzyme, and then 1 g of magnesium aluminate powder (trade name: Adsorbent M-511, manufactured by Hokkaido Soda Co., Ltd.) was added and stirred. After keeping the mixture for 20 minutes, it was filtered to adsorb and remove impurities such as proteins derived from the enzyme preparation.

The resulting precipitate was treated with a porous synthetic adsorbent CRohrrz & Ha
The solution was passed through a glass column filled with Amberly (trade name) XAD-2) 41 manufactured by AS Corporation at SV3.

As a result, rutin glycoside and unreacted rutin in the solution were adsorbed to the porous synthetic adsorbent, while dextrin, oligosaccharides, salts, etc. leaked out without being adsorbed.

Next, after washing the porous synthetic adsorbent adsorbing rutin glycoside and unreacted rutin with water 201,
Rutin glycoside and rutin were eluted by passing ethanol 81 at a concentration of 0 v/v.
) was concentrated, dried, and powdered to obtain about 4 g of a powdered glycoside vitamin P product.

This product contains a large amount of rutin glycoside, so
It is extremely easily soluble in cold water, has excellent stability, and has little bitterness, making it suitable as a vitamin P fortifier for various foods and drinks, cosmetics, and pharmaceuticals, as well as a compounding agent in sunscreen cosmetics. It can be used.

Example 5 Production of glycosyl vitamin P Dextrif 4 of Aesculin 3 g, D, E, 20
After heating and dissolving 00g in 2,00OrrLl of water, it was cooled to a temperature of 60°C, and then 1,000 units of the crude cyclodextrin glucanotransferase preparation prepared by the method of Example (1-1) was added, and - 6.0, temperature 60℃
The reaction was carried out for 20 hours while maintaining the temperature.

When the reaction solution was analyzed by paper chromatography, 91% of esculin was converted to esculin glycoside (glycosyl vitamin P).

This reaction solution was heated in the same manner as in Example (1-2) to inactivate the enzyme, and 2 g of magnesium aluminate silicate granules (trade name: Neusilin, manufactured by Fuji Chemical Industry Co., Ltd.) were added and stirred for 30 minutes. After being stored, it was filtered to adsorb and remove impurities such as proteins derived from the enzyme preparation.

The obtained liquid E was passed through a column filled with a porous synthetic adsorbent (trade name Diaion HP-50 manufactured by Mitsubishi Chemical Industries, Ltd.) 41 at SV2.

As a result, esculin glycoside and unreacted esculin in the solution were adsorbed onto the porous synthetic adsorbent, while dextrin, water-soluble sugars such as oligosaccharides, and salts flowed out without being adsorbed.

Next, as in Example 4, esculin glycoside and esculin were eluted from the porous synthetic adsorbent adsorbed with esculin glycoside and esculin, concentrated, dried,
Approximately 5 g of powdered glycoside vitamin P product was obtained by pulverization.

This product contains a large amount of rutin glycoside, so
It dissolves very easily in cold water, has excellent stability, and has little bitterness, making it suitable as a vitamin P fortifier for various foods and beverages, cosmetics, and pharmaceuticals, and as a compounding agent in sunscreen cosmetics. It can be used for

Claims (1)

[Claims] 1. A glycosyl vitamin aqueous solution containing water-soluble saccharides is brought into contact with a porous synthetic adsorbent to adsorb the glycosyl vitamins on the porous synthetic adsorbent, and the glycosyl vitamins are eluted and collected from the porous synthetic adsorbent. A method for producing glycodyl vitamins characterized by the following.