JPS5933360B2 - Method for producing α-glycosyl steviol glycoside - Google Patents

Description

Detailed Description of the Invention The present invention provides a starch partial decomposition product obtained by allowing cyclodextrin glucanotransferase (EC2.4.1.19) to act on an aqueous solution containing starch and steviol glycosides. An aqueous solution containing water-soluble sugars such as - A method for producing α-glycosyl steviol glycosides, which comprises eluating and collecting glycosyl steviol glycosides.

α-Glycosyl stevioside is disclosed in JP-A-54-5070.
As disclosed in the publication, α-glucosyl sugar compounds such as cyclodextrin glucanotransferase and the like are added to an aqueous solution containing stevioside and α-glucosyl sugar compounds such as starchy substances.
- It is known that the sweetener produced by reacting glucosyltransferase has good sweet taste and is highly water-soluble. However, in this reaction product, in addition to α-glycosyl stevioside, a large amount of aqueous saccharides such as α-glucosyl sugar compounds such as starches used as substrates (sugar donors) during the reaction and their decomposition products coexist. Conventionally, in order to separate and remove water-soluble saccharides from such reactants and produce higher purity α-glycosyl stevioside,
An extraction and distribution method using n-butanol was employed.

However, this method not only requires a large amount of expensive n-butanol, but also requires complicated operations, making it extremely difficult to implement it industrially. The present inventors applied cyclodextrin glucanotransferase to an aqueous solution containing starch and steviol glycosides, and obtained α-
We have conducted extensive studies with the aim of easily collecting α-glycosyl steviol glycosides such as α-glycosyl stevioside from an aqueous solution of glycosyl steviol glycosides.

As a result, porous synthetic adsorbents such as the porous styrene-divinylbenzene polymer resin disclosed in JP-A-51-23300 are able to adsorb water-soluble sugars such as starch partial decomposition products that coexist in large quantities. First, we unexpectedly discovered that α-glycosyl steviol glycosides can be selectively adsorbed, and by bringing an organic solvent into contact with a porous synthetic adsorbent adsorbing α-glycosyl steviol glycosides, d- The present invention was completed by discovering that glycosylsteviol glycosides can be easily eluted and collected.

The α-glycosyl steviol glycoside as used in the present invention is
It is sufficient that it is a glycoside of steviol as genin and contains an α-bond in the binding mode of its sugar moiety.
For example, α disclosed in Japanese Patent Application Laid-Open No. 54-5070
- Glycosyl stevioside, α-glycosyl rebaudioside, etc. The porous synthetic adsorbent referred to in the present invention is a porous, nonionic synthetic adsorbent such as styrene-divinylbenzene polymer resin.
Examples include the Amberlight XAD series and the Diaion HP series manufactured by Mitsubishi Chemical Industries, Ltd.

In addition, in the present invention, the method of bringing these porous synthetic adsorbents into contact may be a batch method, but in the case of mass production, a continuous method in which liquid is passed through a column filled with porous synthetic adsorbents is used. Are suitable.

To describe the present invention more specifically, the α-glycosylsteviol glycoside aqueous solution containing the water-soluble saccharides used in the present invention can be prepared using stevioside, rebaudioside, cyclodextrin glucanotransferase is reacted with an aqueous solution containing steviol glycosides such as starch gelatinized starch, starch liquefied product, maltodextrin, etc., and after the reaction is completed, the enzyme is inactivated by heating. Strain and prepare.

If necessary, remove impurities such as proteins and pigments using magnesium aluminate silicate, or desalt using a strongly acidic ion exchange resin (H type) or a weakly basic ion exchange resin (0H type). It's okay. When the aqueous solution prepared in this way is passed through a column packed with a porous synthetic adsorbent, α-glycosylsteviol glycosides and unreacted steviol glycosides are adsorbed admirably, but a large amount of Water-soluble sugars such as starch partial decomposition products that coexist are not adsorbed and flow out as they are. Next, this porous synthetic adsorbent is washed with dilute alkaline water, water, etc., and then passed through a relatively small amount of an organic solvent or a mixture of an organic solvent and water, such as methanol water, ethanol water, etc., to remove starch. Highly purified α- from which water-soluble saccharides such as partial decomposition products have been separated and removed
Glycosyl steviol glycosides are eluted first, and then unreacted steviol glycosides are eluted by increasing the flow rate or increasing the organic solvent concentration. This eluate containing a high content of α-glycosylsteviol glycosides is distilled to first remove the organic solvent, and then concentrated to an appropriate concentration to produce a syrup-like liquid sweet mainly composed of α-glycosylsteviol glycosides. By further drying and powdering the ingredients, a white powdery powdered sweetener containing α-glycosyl steviol glycoside as a main component can be easily produced. This elution operation of α-glycosylsteviol glycosides using an organic solvent also serves as a regeneration operation for the porous synthetic adsorbent used at the same time, making it possible to repeatedly use this porous synthetic adsorbent.

Furthermore, the present invention is particularly advantageous because not only water-soluble saccharides but also impurities such as salts can be removed at the same time.
In this way, α-glycosyl steviol glycosides produced by separating and removing water-soluble saccharides such as starch partial decomposition products,
It is excellent in terms of sweetness, sweetness, and solubility in water, and is used in various foods, drinks, cosmetics, pharmaceuticals, etc., just like the sweetener disclosed in JP-A-54-5070. It can be advantageously used as a sweetener, a corrosive, etc.

Next, a few examples will be described.

Example 1 (1-1) Preparation of α-glucosyltransferase Bacillus
Stearothermophilus (Baci-11usstea)
r0therm0philus) FERM-P. 7l6
2222, solid blue starch 2w/v%, ammonium nitrate 1w/v%, dipotassium phosphate 0.1w
/v%, magnesium sulfate heptahydrate 0.05w/v%
, Corn Stablica 0.5w/? , 10t of sterilized liquid medium consisting of calcium carbonate 1w/v% and water
The cells were inoculated and cultured with aeration and agitation 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.l.l9) was about 80,
A crude enzyme preparation having 000 units was obtained. The term here is t.
! ! E1 unit means PH5.5, O. 0.3w containing O2M acetate buffer and 2 x 10-3M calcium chloride
0.2111 of an appropriately diluted enzyme solution (approximately 1 to 2 units per ml) was added to the solid blue starch solution 5WLe of /v%, and after reacting at 40°C for 10 minutes, the reaction solution 0.5
Take WL1, mix it with 15 d of 0.02N monosulfuric acid aqueous solution to stop the reaction, add 0.2 d of 0.1N iodine potassium iodide solution to this reaction stop solution to develop color, and then measure the absorbance at 660 nm. 1 at 40℃
Soluble starch 151 by reacting for 0 minutes
1! This refers to the amount of enzyme that completely eliminates the iodine coloring of fl. (1-2) Method for producing steviol glycosides Place 20 dried Stevia leaves and stems in a 500-ton plastic container.
kg and 240 tons of hot water, kept for 1 hour with occasional stirring, then put it in a basket-type centrifugal dehydrator, added 100 tons of hot water to the residue, stirred well, centrifuged it again after 30 minutes, and then removed the P fabric. The residue was washed with 50 tons of warm water, and all the F liquids and washing liquids were combined and the dark brown extract was transferred to a plastic column packed with 80 tons of magnesium aluminate silicate (trade name Tomictus S granules manufactured by Tomita Pharmaceutical Co., Ltd.). The liquid was passed through at SV2.

The obtained pale yellow effluent was passed through a column made from Plascitta filled with 50 t of porous synthetic adsorbent (product name: HP-20 manufactured by Mitsubishi Chemical Industries, Ltd.) at SV2. The unsweetened effluent was then discarded. After washing this column with 200 tons of 1w/v% ammonia water and then with 200 tons of water,
Steviol glycosides were eluted by passing 0v/v% methanol 2401 through SVl. This eluate was distilled under reduced pressure to remove methanol, further concentrated, and then spray-dried to obtain about 2.1 kg of steviol glycosides in the form of white powder. This product was easily dissolved in hot water and had a strong sweet taste.

Further, as a result of acid hydrolysis of this product and measurement using gas chromatography, the content of steviol glycosides was approximately 88% as stevioside. (1-3) Steviol glycoside powder 2009 obtained by the method (1-2) for producing α-glycosyl steviol glycoside and 1,0009 maltodextrin (D.E.3O) in 3,000 ml of water
After heating and dissolving it, it was cooled to 60°C, and then (1-1)
Add 1,000 units of crude cyclodextrin glucanotransferase preparation prepared by the method described above, and adjust the pH to 6. O
The mixture was reacted at 60° C. for 40 hours to produce d-glycosyl steviol glycosides containing d-glycosyl stevioside and α-glycosyl rebaudioside A as main components.

This reaction solution was kept at 95°C for 10 minutes to inactivate the transferase by heating, then cooled to room temperature, and magnesium silicate aluminate powder (trade name: Neusilin, manufactured by Fuji Chemical Industry Co., Ltd.) 109 was added thereto. After keeping the mixture for 30 minutes with occasional stirring, the mixture was filtered under suction to adsorb and remove impurities such as pigments and proteins derived from the enzyme preparation. The almost colorless P liquid obtained was treated with an ion exchange resin (Amberlite I).
RA-200C (H type) and Amberlight 1RA
-93 (0H type)) was desalted by passing it through S2. This desalted solution was passed through a glass column filled with 5 tons of porous synthetic adsorbent (trade name: Annojinlite XAD-7, manufactured by ROhm & Haas) at SV2. The α-glycosylsteviol glycoside in the solution was adsorbed to this synthetic adsorbent, and the dextrin flowed out without being adsorbed.

Next, after washing this synthetic adsorbent with 30 tons of water,
The solution was passed through with 20 t of v/v% methanol to elute the α-glycosyl steviol glycoside. This eluate was distilled under reduced pressure to remove methanol, further concentrated, and then dried under reduced pressure and powdered to obtain approximately 2709 α-glycosyl steviol glycosides in the form of white powder. This product is an α-glycosyl steviol glycoside containing a large amount of α-glycosyl stevioside, α-glycosyl rebaudioside A, etc. It is extremely easily soluble in water and can be dissolved at any concentration. Moreover, it has a mellow and strong sweetness, so it is suitable as a sweetener, and can be advantageously used to sweeten various foods and drinks. Example 2: 10 kg of dry matter potato starch at 20 w/w%, 16
．． 0. Commercially available liquefied enzyme agent is 0.2 per solid starch? In addition, the D. E. When the temperature reached approximately 15, the liquefaction enzyme was inactivated by keeping the temperature at 120°C for 10 minutes.

After heating and dissolving stevioside in this liquefied liquid so that the ratio of stevioside to liquefied liquid was 1:3 by solid weight,
After cooling to 60°C, 5 units of cyclodextrin glucanotransferase prepared by the method (1-1) in Example 1 was added per gram of starch, and the pH was adjusted to 6. O for 24 hours to produce α-glycosyl steviol glycoside containing α-glycosyl stevioside as the main component, and then this reaction solution was kept at 95°C for 10 minutes to stop the reaction.
There was a lot of suction. The obtained solution was passed through a plastic column filled with 5 tons of magnesium aluminate silicate (trade name: Column Light, manufactured by Fuji Chemical Industry Co., Ltd.) at S1 to remove pigments and proteins derived from starch, enzyme preparations, etc. Removed impurities such as

The resulting colorless effluent was passed through a plastic column packed with 50 t of porous synthetic adsorbent (product name HP-40 manufactured by Mitsubishi Chemical Industries, Ltd.) at SV2. Steviol glycosides were adsorbed to this column, and debostrines in the reaction solution flowed out as they were. This dextrin can be used again as a substrate for the α-glucosyl transfer reaction by cyclodextrin glucanotransferase. After washing this column with about 500t of water, 40v
250 t/v% ethanol was passed through the flask through SV1 to elute α-glycosylsteviol glycosides containing α-glycosylstevioside as a main component.

This eluate was distilled under reduced pressure to remove ethanol, further concentrated, and then dried under reduced pressure and powdered to obtain about 4.1 kg of α-glycosyl steviol glycoside in the form of a white powder. This product is a steviol glycoside containing a large amount of α-glycosyl stevioside and a small amount of stevioside, and is suitable as a sweetener because it is extremely easily soluble in water and has a mellow and strong sweetness. Therefore, it can be advantageously used to sweeten various foods and drinks.

Claims (1)

[Claims] 1 Add cyclodextrin glucanotransferase (EC) to an aqueous solution containing starch and steviol glycosides.
2.4.1.19), the resulting aqueous solution containing water-soluble saccharide and α-glycosyl steviol glycoside is brought into contact with a porous synthetic adsorbent, and the adsorbent is coated with α-glycosyl steviol glycoside. 1. A method for producing α-glycosylsteviol glycosides, which comprises adsorbing α-glycosylsteviol glycosides and then eluating and collecting them from the adsorbent.