KR20050061007A - Enzymatic transglycosylation of l-ascorbic acid in a heterogeneous enzyme reaction system using insoluble starch as the glycosyl donor - Google Patents

Abstract

In the present invention, L-ascorbic acid (L-ascorbic acid) using L-ascorbic acid (L-ascorbic acid-α-glycoside) which is a sugar transfer derivative with improved stability and solubility using various sugar transfer enzymes It relates to a bioconversion process to produce. The present invention is characterized in that the insoluble swelling starch (vaginal) modified by modifying the starch particle structure by extruding or amplifying the raw starch (quality) instead of the soluble starch such as soluble liquefied starch or dextrin used as a sugar donor in the conventional manufacturing method as a sugar donor. When used or raw starch (vaginal) is to use a heterogeneous enzymatic reaction system that is mixed with the crushed friction medium, stirred to react.

When the insoluble starch (quality) is used as a sugar donor, the sugar transition yield of L-ascorbic acid is greatly improved, and since the viscosity is lower than that of the soluble sugar donor, it can be added at a high concentration, so that a high concentration sugar transfer reaction is possible. You can get ascorbic acid before. In addition, the production of water-soluble maltooligosaccharides produced as a by-product of the sugar transition is greatly suppressed, and the remaining sugar donor after the completion of the reaction is insoluble, and can be removed by simple unit operations such as filtration and centrifugation. do.

Sugar transition using heterogeneous enzymatic reaction system using such insoluble starch (quality) as sugar donor Ascorbic acid production method is a new high-efficiency sugar transition with advantages such as reaction yield, reaction rate, product concentration, and simplification of separation and purification process. It can be said ascorbic acid manufacturing method.

Description

{Enzymatic transglycosylation of L-ascorbic acid in a heterogeneous enzyme reaction system using insoluble starch as the glycosyl donor} using a heterogeneous enzymatic reaction system using insoluble starch as a sugar donor

The present invention is L- ascorbic acid-2-O-α-glucoside (L-ascorbic acid-2-O-) derivative of L- ascorbic acid with improved stability and solubility than the raw material L- ascorbic acid (L-ascorbic acid) The present invention relates to a new method of bioconverting L-ascorbic acid-α-glycoside containing α-glucoside as a main component in high yield using various sugar transfer enzymes. In detail, a sugar transition such as cyclodextrin glucanotransferase, alpha-glycosyltransferase, alpha-glucosidase, or alpha-amylase at the carbon position (L) -ascorbic acid 2 of the structure of formula (I ) L-ascorbic acid-α-glycoside, which is a sugar transition derivative of (L) -ascorbic acid having a structure as shown in formula (II ), can be prepared by transferring glucose in a sugar donor by one or more molecules. Among the resulting sugar transfer derivatives, L-ascorbic acid-2-O-α-glucoside in which one molecule of glucose having a structure as shown in Formula III is transferred is mainly produced.

<Formula I>

       (L) -ascorbic acid

<Formula II>

     L-ascorbic acid-α-glycoside

<Formula III>

     L-ascorbic acid-2-O-α-glucoside

L-ascorbic acid, also known as vitamin C, has strong antioxidant activity. In addition, it has various physiological functions such as promoting collagen synthesis and skin whitening. It is also reported that it is a necessary ingredient of teeth, gums and bones, provides resistance and immunity to infections, prevents scurvy and cold, and helps to absorb iron to prevent anemia. Currently, L-ascorbic acid is used as an essential nutrient, vitamin C fortifying agent, as an acid, reducing agent, antioxidant, bleach and stabilizer in various chemicals and foods, and in medicine, susceptibility such as viral disease, bacterial disease and malignant tumor Used as a prophylactic and therapeutic agent for diseases. In addition, cosmetics such as skin cleansing agents and skin whitening agents are commonly used as reducing agents, ultraviolet (UV) absorbers, and melanogenesis inhibitors.

However, there is a problem that L-ascorbic acid is easily oxidized and decomposed in the presence of oxygen, heat and metal ions, so that the physiological activity is easily lost. Therefore, several methods have been devised to improve the stability of L-ascorbic acid. The result is magnesium-L-ascorbyl phosphate, ethyl-L-ascorbyl ether, and L-ascorbic acid-2-O-phosphate. Various chemical derivatives of L-ascorbic acid have been developed. In addition, L-ascorbic acid-α-1,6-glucoside (L-ascorbic acid-α-1,6-glucoside) and L-ascorbic acid- 2-O-α-glucoside (L-ascorbic acid-2-O- A sugar transfer derivative of L-ascorbic acid, such as α-glucoside), has been developed. Recently, α-1,6-maltosyl-L-ascorbate has been developed using sugar transfer amylases (Korean Patent Application No. 1020010051171).

Among them, L-ascorbic acid-2-O-α-glucoside, which is a sugar transfer derivative, is known to have high stability and good physiological activity in vivo ( Chem. Pharm. Bull. (Tokyo). 1990, 38 (11) ): 3020-3023, Int. J. Vitam. Nutr. Res. 1992, 62 (4): 318-323, J. Nutr. Sci. Vitaminol. (Tokyo). 1992, Special volume: 161-164). L-ascorbic acid-2-O-α-glucoside has been commercialized by Hayashibara Co., Japan, and its production method is described in Japanese Patent Publication No. 127,072 / 89 or Korean Patent No. 1001624950000. In addition, a technique for separating and purifying high purity of the material is described in Korean Patent Nos. 1001584550000, 1002449100000, and Patent Application No. 1020010032124.

For the sugar transfer reaction, various glycotransferases such as α-glucosidase, α-amylase, α-glucosyltransferase or cyclodextrin glucanotransferase, etc., are used as raw materials and sugar receptors in starch in L-ascorbic acid. It is synthesized by transferring glucose. In this case, as the sugar donor, various maltooligosaccharides, dextrins, cyclodextrins, amylose, liquefied starch, gelatinized starch and soluble starch may be used.

L-ascorbic acid-2-O-α-glucoside preparation method of YAYASHIBARA uses sugar starch, liquefied starch, gelatinized starch, dextrin, cyclodextrin and various maltooligosaccharides (Japanese Patent Publication No. 127,072 / 89) , Korean Patent No. 1001624950000), These sugar donors are soluble starch dissolved in water, the sugar transition proceeds in a homogeneous enzymatic reaction system in which the raw material L- ascorbic acid, sugar donor starch, and enzyme are all dissolved in water It has the characteristic of becoming. In this method, L-ascorbic acid, L-ascorbic acid-2-O-α-glucoside, as well as starch (vaginal) remaining after the reaction, and maltooligosaccharides remaining as by-products are dissolved and mixed in an soluble state. Therefore, in order to prepare L-ascorbic acid-2-O-α-glucoside with high purity, separation-purification must be carried out using various methods such as membrane separation, gel filtration chromatography, column chromatography, and ion exchange chromatography.

Conventional manufacturing methods using such soluble starch and starch as a sugar donor have the following problems. First, soluble starch or dextrin, which is a sugar donor, has a high viscosity, making it difficult to add high concentrations, making it difficult to obtain ascorbic acid in high concentrations. In addition, a large amount of water-soluble maltooligosaccharides are generated as by-products during the sugar transfer reaction and must be subjected to a complex separation and purification process to remove them. Above all, the soluble starch, a sugar donor, is mixed in a homogeneous phase with other reaction products, so it has to go through a complicated process to separate and remove it. Therefore, the development of a new high-efficiency Ascorbic acid production process that can solve the above problems is desired.

The present inventors have swelled starch prepared by extruding or crushing raw starch (vaginal), or swelled starch (quality) suspended in an insoluble state while maintaining a particle structure without melting when added to water, such as alpha starch heated or dried. Method for preparing cyclodextrin in heterogeneous enzymatic reaction system using donor (Korean Patent No. 1000648520000, Japanese Patent No. 2047572), Sugar Transfer Sweetener Production Method (Korea Patent No. 1000792490000), and Glucosyl Xylitol Production Method (Korea Patent No. 1003477890000) has been invented.

      In addition, the present inventors added and agitated solid grinding media such as glass spheres in various enzyme reactions using raw starch as sugar donor, and then stirring the grinding friction medium-containing enzyme, which is a heterogeneous enzyme reaction system that significantly increased the rate and yield of the enzyme reaction. The steamless glycosylation method (Korean Patent No. 1000273230000), maltose preparation method (Korean Patent No. 1000583290000), and the sugar transition sweetener manufacturing method (Korean Patent No. 1000827290000) of raw starch using a reaction system have been invented.

      Recently, a study on the preparation of L-ascorbic acid-α-glycoside using a sugar transfer enzyme was carried out using a structurally modified insoluble swelled starch (vaginal) or a mixture of raw starch (vaginal) and ground friction media. The heterogeneous enzyme reaction system was found to be able to be used very well for the production of ascorbic acid was completed the present invention. The production of L-ascorbic acid-α-glycoside in accordance with the method of the present invention not only significantly improved the sugar transfer yield, but also the residual starch remaining after the sugar transfer reaction was insoluble, and thus separated by a simple method such as centrifugation or filtration. It was easy to separate and purify, minimize the production of by-product malto oligosaccharides, and can add high concentration and high purity of L-ascorbic acid-α-glycoside by adding high concentration of sugar donor and L-ascorbic acid. There were many advantages that could be produced.

Referring to the present invention in more detail, any kind of starch (quality) such as corn, potato, sweet potato, rice, wheat, sorghum, and tapioca starch may be used as insoluble raw starch (quality) used as a sugar donor. . As a method for producing swollen starch (vaginal), (1) equilibrate the water content of raw starch (vaginal) to a low level of 15-30%, and swelling and alphalating by extruding by extruder under high pressure with an extruder. Method: (2) equilibrate the starch (vaginal) to a low water content level and rapidly depressurize and detonate it after heating, or (3) immerse the starch (vaginal) in water, deform the structure with heated alpha starch, and dry. And swelling starch (vaginal) by grinding. The starch particles extruded, exploded, or heated and alpha-swelled to swell the structure have several times the available surface area for enzymatic activity compared to raw starch (enzyme) ( Enzyme and Microbial Technology 22: 217-222, 1998). Therefore, the contact, adsorption, and enzymatic action of the sugar transfer enzyme are very easy.

In addition, when raw starch (quality) is used as a sugar donor, the ground friction medium is mixed and stirred to overcome the low sugar transfer reaction rate. At this time, the structure of the raw starch is rapidly fragmented due to the interaction of the ground friction with the enzyme, thereby enzymatic reaction. This is done smoothly ( Kor. J. Appl. Microbiol. Biotechnol. 21: 461-467, 1994, Kor. J. Appl. Microbiol. Biotechnol. 22: 252-258, 1994). As a solid grinding friction medium, various types of solid media such as spherical, elliptical, amorphous, such as glass spheres, Teflon, ceramics, and new materials are used, and sizes of about 0.1 to 2.0 cm are appropriate. For the stirring and mixing of the reaction liquid including the grinding friction medium, if the reactor has a structure capable of smoothly crushing the reaction liquid, such as mechanical stirring using a rotating shaft and a rotor blade, a rotating agitator stirring the reactor itself, and a fluidized bed reactor using a fluid, do. Stirring strength should be used to change the structure of the starch so as to facilitate the enzymatic reaction, and excessive stirring will adversely affect the enzyme.

      The enzyme used may be any of various well-known enzymes such as cyclodextrin glucanotransferase, alpha-glycosyltransferase, alpha-glucosidase, and alpha-amylase. In addition, the reaction yield may be increased by adding a small amount of starch degrading enzymes such as pullulanase, dextranschecraze, amyloshcraze and dextrindextranase.

      The concentration of L-ascorbic acid, which is a raw material for the production of L-ascorbic acid-α-glycoside, is 20-600 g / L, and the concentration of the structurally modified insoluble starch (quality) is generally L-ascorbic acid. Adding 0.5-10 times the concentration is appropriate in terms of reaction rate and yield. In addition, when insoluble starch is added more than 400g / L, it is advantageous to add the sugar donor in a cost-effective manner in order to prevent other yield decrease in viscosity increase.

      In the case of cyclodextrin glucanotransferase, an appropriate ratio of the appropriate glycotransferase per starch quality is about 10 to 1500 units per gram of starch. When raw starch (quality) is used as a sugar donor, the amount of glass spheres added to the crushed friction medium is preferably about 2-10 times the weight of the added starch (quality) and the size is preferably 0.1 to 2.0 cm. The reaction temperature is suitable at about 45-55 ° C., which is the optimum temperature for enzymatic reaction, but can be applied at any temperature within 80 ° C. where no insoluble starch is dissolved. The pH of the reaction solution depends on the type and origin of the sugar transfer enzyme, but in the case of cyclodextrin glucanotransferase, a pH of about 4.0 to 11.0 is appropriate. During the reaction, the stirring speed is reacted while stirring at a speed of 100 to 500 rpm.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 In the process of preparing L-ascorbic acid-α-glycoside using the cyclodextrin glucanotransferase which is a sugar transfer enzyme, insoluble swelling starch was used as a sugar donor according to the method of the present invention. Comparison of Ascorbic Acid Production Yields with Soluble Potassium Liquefied Starch or Dextrin as in the Previous Production Process

The insoluble swelling starch prepared according to the method of the present invention was suspended in 50 mM phosphate-NaCl buffer (pH 7.0) at 10% (w / v), and the soluble steamed liquefied starch used in the conventional method for comparison purposes. Dextrins were dissolved in the buffer solution at the same concentration, respectively, and the sugar receptor, L-ascorbic acid, was dissolved to 10% (w / v). The sugar transfer enzyme cyclodextrin glucanotransferase was added to an amount corresponding to 200 units per gram of starch, and the sugar transfer enzyme reaction was performed for 48 hours while stirring at 50 rpm at 200 rpm.

As a result, as shown in Fig . 1 , when the structurally modified insoluble swelling starch was used as a sugar donor, the yield of L-ascorbic acid-α-glycoside in which glucose was transferred to L-ascorbic acid by one or more molecules was L-ascorbic acid. It is up to 53% compared to 38% and 42% of the production yields using soluble liquefied starch and dextrin according to the existing manufacturing method. Thus, the production of L-ascorbic acid-α-glycoside using the insoluble swelling starch modified in accordance with the present invention as a sugar donor was able to obtain an effect of more than 11% increase in the production yield.

Example 2 In the process of preparing L-ascorbic acid-α-glycoside using the cyclodextrin glucanotransferase which is a sugar transfer enzyme, the mixed friction medium was mixed with raw starch according to the method of the present invention and stirred. Ascorbic acid yield in case of using soluble cooked liquefied starch or dextrin used in the conventional method compare

As a donor, raw starch was suspended in 50 mM phosphate-NaCl buffer (pH 7.0) to 10% (w / v) according to the method of the present invention, and 3 mm diameter glass spheres were pulverized with a friction medium of 400 grams per liter of reaction solution. Was added. For comparison purposes, the soluble cooked liquid starch and dextrin, which are used in the conventional manufacturing method, were dissolved in the buffer solution at the same concentration, respectively, and the sugar receptor L-ascorbic acid was dissolved to 10% (w / v). Then, the sugar transfer enzyme cyclodextrin glucanotransferase was added to the amount corresponding to 200 units per 1 g of starch, respectively, and the sugar transfer enzyme reaction was performed for 48 hours while stirring at a speed of 200 rpm at 50 ° C.

As a result, as shown in FIG . 2 , the yield of L-ascorbic acid-α-glycoside reached 51% compared to the amount of L-ascorbic acid added, and the yield of 38% when the soluble liquefied starch and dextrin used in the conventional manufacturing method was used. A significant improvement over 42%.

Example 3 According to the present invention, maltooligosaccharides produced as by-products in the reaction supernatant obtained by using an insoluble swelled starch as a sugar donor or by mixing and stirring raw starch and a ground friction medium were analyzed by high pressure liquid chromatography. Chromatogram

When the insoluble swelling starch is used as a sugar donor according to the method of the present invention, when the sugar starch is produced ascorbic acid while stirring by adding a ground friction medium to the raw starch, soluble dextrin is used as a sugar donor according to the conventional manufacturing method. In order to compare the content of maltooligosaccharides accumulated as by-products in the case of preparing ascorbic acid, the content of maltooligosaccharides generated as by-products in the sugar transition reaction solution obtained in Examples 1 and 2 was analyzed by high-pressure liquid chromatography. The results are shown in FIG. 3 .

      According to the conventional method, when a soluble dextrin was used as a sugar donor, a large amount of malto oligosaccharide was generated as an unwanted by-product, whereas a very small amount of malto oligosaccharide was detected when an insoluble starch was used as a sugar donor. This means that not only can the yield significantly improve, but also greatly simplify the separation and purification process that requires the removal of maltooligosaccharides.

Example 4 In the process of preparing L-ascorbic acid-α-glycoside while using insoluble swelled starch or mixing ground friction media with raw starch according to the method of the present invention, soluble dextrin Analysis of the amount of residual starch that can be recovered by centrifugation of the reaction solution obtained after the completion of the reaction and the content of maltooligosaccharides remaining in the supernatant

The sugar transfer enzyme reaction solution obtained in Examples 1 and 2 was treated with a centrifuge at 10,000 × g for 5 minutes to separate the unreacted starch and the reaction supernatant, followed by drying the centrifuged unreacted starch at 100 ° C. for 8 hours. Dry weight was measured. Maltooligosaccharides in the supernatant were measured by high pressure liquid chromatography and shown in Table 1.

TABLE 1

According to the method of the present invention, L-ascorbic acid-α-glycoside was prepared by using a sugar-transfer reaction of cyclodextrin glucanotransferase using insoluble starch as a sugar donor and using soluble dextrin as in the conventional method. Comparison of the amount of residual starch that can be separated and removed by simple unit operation such as centrifugation mixed in the reaction mixture after the completion of the reaction, and the maltooligosaccharide equivalent required for separation and purification as accumulated by reaction in the reaction solution

Donor type Starch amount added (g / L) Centrifugable residual starch (g / L) Maltooligosaccharide residual in the reaction solution (g / L) Soluble Dextrin 100 - 10.0 Insoluble Swelling Starch 100 42.8  1.2 Insoluble Raw Starch 100 44.6 0.8

      When dextrin was used as a donor according to the conventional method, dextrin was present in an available state, and thus, unreacted residual dextrin could not be recovered using a centrifuge. However, when the sugar transfer reaction was carried out according to the present invention, the unreacted residual starch could be separated and removed to 42.8 g / L and 44.6 g / L by simple centrifugation after the completion of the reaction. This is more than 95% of the residual starch is separated off. When insoluble starch is used as a sugar donor according to the method of the present invention, the separation of unreacted starch remaining after the completion of the reaction is very easy, and it can be shown that greatly simplifying the separation and purification process of L-ascorbic acid-α-glycoside. Giving.

      In addition, very small amounts of maltooligosaccharides were produced as unwanted products. This means that not only can significantly improve the yield, but also greatly simplify the separation and purification process for removing maltooligosaccharides.

Example 5 In the process of preparing L-ascorbic acid-α-glycoside by using insoluble swelled starch as a sugar donor or by mixing and stirring raw starch and ground friction medium, L- as a raw material Effect of L-ascorbic acid-α-glycoside concentration and sugar transition on the yield of ascorbic acid and insoluble starch at high concentrations

       As a sugar donor, insoluble swelling starch (vaginal) or raw starch (vaginal) is added at a concentration of 100, 200, 300, 400, 500, and 600 g / L, and the sugar acceptor L-ascorbic acid also has an insoluble starch and a mixing ratio of 1 Each was added at the same concentration so as to be 1: 1 and suspended in a buffer solution. In the case of using raw starch as a sugar donor according to the present invention, 400 grams of glass spheres with a diameter of 3 mm were added per liter of the reaction solution as a ground friction medium. Cyclodextrin glucanotransferase, a sugar transfer enzyme, was added to the unit corresponding to 200 units per 1 g of starch, and the sugar transfer enzyme reaction was performed for 48 hours while stirring at 50 rpm at 200 rpm.

As shown in FIG. 4 , the concentration of L-ascorbic acid-α-glycoside was also increased to 200 g / L as the concentration of insoluble starch, a sugar donor, was increased. The sugar conversion yield was 55% and 53% at the concentration of insoluble starch, a sugar donor, up to 200g / L, but was decreased slightly at higher concentrations. This is presumably because the fluidity of the reaction solution is lowered and thus affects the enzyme reaction. However, this problem can be easily solved by using the fed-batch method of insoluble starch which is a sugar donor.

      Therefore, when preparing sugar ascorbic acid using insoluble starch as a sugar donor according to the present invention, it is difficult to add a high concentration due to the viscosity of the soluble starch in the conventional manufacturing method, significantly improving the problem of difficult to produce ascorbic acid in high concentration Thus, it can be said that the high concentration production of ascorbic acid is possible.

The present invention uses an insoluble swelling starch (quality) in which the starch particle is modified by extruding or amplifying the starch particle structure instead of a soluble starch such as soluble liquefied starch or dextrin used as a sugar donor in the conventional manufacturing method, or as a sugar donor. Alternatively, in the case of using raw starch (vaginal), a heterogeneous enzymatic reaction system in which the mixed friction medium is mixed and stirred is reacted. When the insoluble starch (quality) is used as a sugar donor, the sugar transition yield of L-ascorbic acid is greatly improved, and since the viscosity is lower than that of the soluble sugar donor, it can be added at a high concentration, so that a high concentration sugar transfer reaction is possible. You can get ascorbic acid before. In addition, the production of water-soluble maltooligosaccharides produced as a by-product of the sugar transition is greatly suppressed, and the remaining sugar donor after the completion of the reaction is insoluble, and can be removed by simple unit operations such as filtration and centrifugation. do. Sugar transition using heterogeneous enzymatic reaction system using such insoluble starch (quality) as sugar donor Ascorbic acid production method is a new high-efficiency sugar transition with advantages such as reaction yield, reaction rate, product concentration, and simplification of separation and purification process. It can be called ascorbic acid manufacturing method, and industrial use is expected.

1 is a process for preparing L-ascorbic acid-α-glycoside using the cyclodextrin glucanotransferase which is a sugar transfer enzyme, according to the method of the present invention and the case of using an insoluble swelling starch as a sugar donor The graph shows a comparison of ascorbic acid yield in the case of using soluble cooked liquefied starch and dextrin used in the manufacturing method.

2 is a process for preparing L-ascorbic acid-α-glycoside using cyclodextrin glucanotransferase which is a sugar transfer enzyme, by adding a ground friction medium to an insoluble raw starch as a sugar donor according to the method of the present invention. In the case of stirring and when using the soluble cooked liquefied starch or dextrin used in the conventional manufacturing method is a graph comparing the yield of ascorbic acid,

FIG. 3 is a chromatogram of maltooligosaccharides produced as a by-product of the reaction supernatant obtained by using insoluble swelled starch as a sugar donor or by mixing raw starch and ground friction medium with stirring. to be.

Figure 4 according to the present invention using the insoluble swelling starch as a sugar donor or by mixing and stirring raw starch and crushed friction medium to react L- ascorbic acid-α-glycoside in the process of producing, L- ascorbic acid as a raw material It is a graph showing the effect of the resulting sugar in the reaction of the addition of insoluble starch to each concentration to a high concentration on the L- ascorbic acid-α-glycoside concentration and the yield.

Claims (4)

In the process of transferring glucose molecules from the starch molecule to L-ascorbic acid by using a sugar transfer enzyme to prepare L-ascorbic acid-α-glycoside, which is sugar ascorbic acid, as a sugar donor L-ascorbic acid-α using a heterogeneous enzymatic reaction system that uses insoluble starch (vagina) such as live starch (vagina) or swelled starch (vagina) instead of using soluble starch (vagina) such as oligosaccharide. -Enzymatic preparation of glycosides. The enzymatic preparation of sugar-transferred L-ascorbic acid-α-glycoside using a heterogeneous enzymatic reaction system according to claim 1, wherein the insoluble swelling starch (vaginal) extruded, pulverized, or heat alphaned with insoluble starch is used as a sugar donor. Way. The method of claim 1, wherein the mixing ratio of L-ascorbic acid and insoluble starch (vaginal) is approximately 1: 0.1 to 10 by weight, and the sugar-transferase per 1 g of starch is added in an amount of 10 to 1,500 units so that L-ascorbic acid- Enzymatic preparation method of sugar-transferred ascorbic acid using a heterogeneous enzymatic reaction system using insoluble starch (vaginal) as a sugar donor to prepare α-glycoside. The sugar sugar using a heterogeneous enzymatic reaction system according to claim 1, wherein raw starch (quality) is used as a sugar donor and solid sugar grinding media such as glass spheres, Teflon, or various ceramic materials are added and stirred to carry out the sugar transfer reaction. Enzymatic preparation of this L-ascorbic acid-α-glycoside.