JP2004229668A - alpha-D-GLUCOPYRANOSYLGLYCEROL, METHOD FOR PRODUCING THE SAME AND APPLICATION THEREOF - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide α-D-glucopyanosylglycerols, to provide a method for efficiently producing the α-D-glucopyanosylglycerols, and to provide applications of the α-D-glucopyanosylglycerols. <P>SOLUTION: The α-D-glucopyanosylglycerols have low Maillard reactivity, heating stability, noncariogenic property, indigestible property and high moisture retention. The α-D-glucopyanosylglycerols are produced by allowing α-glucosidase act on a mixture of glycerol with succharides containing glucose, maltose and the like so as to transfer glucosyl into the glycerol. And, by continuously adding saccharides to a reaction mixture, the concentration of α-D-glucopyanosylglycerols is increased and the glycerols are efficiently produced. The α-D-glucopyanosylglycerols are effectively applied to foods, chemicals and medicines. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention has clean sweetness, extremely low browning and Maillard reactivity, excellent heat stability, and has functions such as indigestibility, non-cariogenicity, and high moisture retention. The present invention relates to α-D-glucopyranosylglycerols, a method for efficiently producing the same, and foods, chemicals, and pharmaceuticals that effectively utilize the properties thereof.

Many filamentous fungi such as Aspergillus are known to produce α-glucosidase in addition to amylase such as α-amylase and glucoamylase. α-Glucosidase is an exo-type amylase, also called glucosyltransferase or transglucosidase, which converts α-1 from maltose and oligosaccharides.
, 4 has the effect of transferring the bound glucose residue to another substance. For example, when the acceptor of the glucosyl group to be transferred is water, ethyl alcohol, glucose, maltose, or isomaltose, glucose, ethyl-α-glucoside (Publication No. 4-112798), isomaltose, panose, isomaltose, respectively. Generate an aus. When water is used as the acceptor, it can be seen as hydrolysis similar to the action of glucoamylase and the like, and this is hereinafter referred to as hydrolysis.

  Although there have been many reports on the transfer effect of α-glucosidase even in sake mash, the present inventors have reported that α-D-glucopyranosylglycerols (hereinafter referred to as GG) whose glycerol transfer receptor is glycerol. Was newly discovered, and it was further found that among other brews using koji, for example, miso and mirin also contained GG.

GG in the present invention is (2R) -1-O-α-D-glucopyranosyl glycerol, (2S) -1-O-α-D-glucopyranosyl glycerol, 2-O-α-D- It is composed of three components of glucopyranosyl glycerol, of which only 2-O-α-D-glucopyranosyl glycerol inhabits blue-green algae (also referred to as cyanobacteria), particularly in a high salt environment such as the ocean. It has been reported that the enzyme is present in cells, is biosynthesized in the cells by an enzyme reaction different from the present invention, and is involved in osmotic pressure regulation (Carbohydr. Res., 73, 193-202, 1979; Science, 210). Mar. Biol., 73, 301-307, 1983; J. Gen. Microbiol., 130, 1-4, 1984; lanta, 163,424~429,1985; Arch.Microbiol, 148,275~279,1987;. Mikrobiologiya, 60,596~600,1991;. J.Gen.Microbiol, such as 140,1427～1431,1994). However, in these reports, the properties of 2-O-α-D-glucopyranosylglycerol itself are not described, and 1-O-α-D-glucopyranosylglycerols are disclosed by the present inventors. It was discovered for the first time in sake mash. The respective ring structures are shown below.

  The concentration of GG in sake is as low as 0.5% at most, but GG in sake brings so-called “broad taste” and “push taste” of sake, resulting in a refreshing sweetness. I understood that it was giving. The sweetness of GG is about 0.55 times that of sucrose, and GG is stable to heating, hard to brown, and hard to cause Maillard reaction. In addition, GG has non-cariogenic, indigestible, and high moisturizing effects. Sake is used not only as a beverage but also as a seasoning and cosmetics, not least because of the characteristics of these GGs. It is expected that.

  As mentioned above, the concentration of GG in sake is low, and the content of GG in sake lees, a by-product of sake brewing, is low, and extraction and purification from these are inefficient. As an organic synthesis method, isomaltose, maltitol, etc., a method in which glycol cleaved with lead tetraacetate or periodate is reduced, or after β-glucoside synthesized by the Koenigs-Knorr reaction is anomerized, There is a method of hydrolyzing β-glucoside with β-glucosidase, but the yield is extremely poor, and purification and the like become very complicated. As described above, there is no excellent general synthesis method for producing α-glucosides, and an efficient production technique is required.

  The present inventors have conducted intensive studies and found that when α-glucosidase of fungi is allowed to act on a specific substrate in a glycerol solution having a relatively high concentration, excellent reactivity is exhibited and GG is efficiently produced. Technology was established. That is, α-glucosidase effectively reacts even in a relatively high concentration of glycerol solution, for example, at a weight-to-volume percentage of 25% or more (hereinafter, each concentration is expressed as a weight-to-volume percentage), and under this condition, a substrate such as Maltose hydrolysis was found to be less likely to occur.

  According to the present invention, GG can be mass-produced at low cost. GG is stable to heating, hard to brown, and hard to cause Maillard reaction. In addition, non-cariogenic, indigestible, and high moisturizing properties are recognized, and GG present in brews using koji such as sake mash is expected to be widely used as a functional substance in addition to taste substances. You.

  Hereinafter, the present invention will be described in detail.

  As a substrate in the present invention, besides maltose, glucose, sucrose, a mixed solution of oligosaccharides, starch syrup, a starch decomposed product such as α-amylase, and a saccharide having a hydrogenated reducing terminal such as maltitol may be used. it can.

  An enzyme that can be used in this reaction is α-glucosidase called α-1,6-glucosyltransferase. Most of yeast-derived α-glucosidase can be used because α-1,4-glucosyltransferase is mostly used, but most of mold-derived α-glucosidase can be used. Among them, Asp. oryzae, Asp. α-Glucosidase produced by Niger or the like is preferred. Not only purified enzymes but also crudely purified enzymes can be used. In addition, koji can be used in supplying the substrate and the enzyme simultaneously.

  The operating conditions are not specified. For example, when the reaction temperature was 37.5% glycerol for 24 hours, the production of GG was maximum at 40 ° C., but even at 30 ° C., 70% of the GG was produced. Recognized and available. Further, when the reaction is carried out at a lower temperature, the reaction rate decreases, but there is no problem in the production of GG. The working pH is preferably in the range of 3-6. The amount of enzyme to be added was tested in the range of 0.6 to 50 U (international unit) per gram (g) of the substrate using 5% maltose as the substrate. However, the addition of 2.5 U / g sufficiently achieves the purpose. In addition, under these conditions, the yield per substrate (maltose) is as high as 66%, but the glycerol used in the reaction is small, and the concentration of GG in the reaction solution is increased in order to increase the efficiency of the subsequent purification process. Is more preferred. Then, when the substrate was added every 24 hours, the enzyme stably acted and the concentration of GG could be increased even if substrate was added ten times in a row. Such a method of increasing the production amount by continuous batch processing is effective from the viewpoint of cost and operability.

  In the solution after the reaction, glycerol, glucose, oligosaccharide and the like exist in addition to GG. It can be used as it is as a sweetener for foods, humectants, and taste improvers. When high-purity GG is used, a purification method such as activated carbon column chromatography is effective. If the eluate is concentrated, syrup-like GG can be obtained. Further, when a part of the purified GG is subjected to amino column chromatography using a relatively high concentration of acetonitrile, for example, 85% acetonitrile as an eluate, 2-O-α-D-glucopyranosyl glycerol and 1-O -Α-D-glucopyranosylglycerols can be separated and purified.

  The characteristics of the purified GG were examined. As a result, it was found that GG is stable to heating, has less browning, and is less likely to cause the Maillard reaction than glucose, maltose, and sucrose.

  FIG. 1 shows the relationship between the degree of coloring and the heating temperature of 10% aqueous solutions of glucose, maltose, sucrose, and GG after heating at pH 4 or 7 for 60 minutes. The horizontal axis shows the heating temperature, and the vertical axis shows the degree of coloring (the value obtained by subtracting the absorbance at 720 nm from the absorbance at 420 nm in a cell having a thickness of 1 cm and multiplying by the dilution factor).

  FIG. 2 shows the relationship between the degree of coloring and the pH of a 10% aqueous solution of glucose, maltose, sucrose, and GG each containing 0.5% glycine after heating at 100 ° C. for 60 minutes. The horizontal axis represents pH, and the vertical axis represents the degree of coloring (the value obtained by subtracting the absorbance at 720 nm from the absorbance at 420 nm in a cell having a thickness of 1 cm and multiplying by the dilution factor).

  FIG. 3 shows the results of HPLC measuring the residual ratio of 10% aqueous solutions of glucose, maltose, sucrose and GG after heating at pH 4 or 7 for 60 minutes. The horizontal axis shows the heating temperature, and the vertical axis shows the residual rate.

  When a sucrose concentration corresponding to the sweetness of a 5% GG aqueous solution in a 1.5 to 4% (0.5% increment) sucrose aqueous solution was subjected to a sensory test by eight panelists, a sucrose concentration of 2% was obtained. The result was that 4 people had a sweetness of about 0.5% and 4 people had a sweetness of about 3%. Therefore, it was found that the sweetness of GG was about 0.55 times that of sucrose, and that it had a clear sweetness and did not have the bitterness that is a problem with many sugar alcohols. In addition, GG is non-cariogenic and indigestible, and has a higher moisturizing property than glycerol and sorbitol, which are widely used as moisturizers at present.

  FIG. 4 is a diagram showing the non-cariogenicity of GG. According to the method of Miyamura (Dental Sciences, 60, 717-731, 1973), a solution obtained by adding 1 ml of heart infusion broth and 3 ml of fresh saliva to 1 ml of water or 1 ml of a 1.25% aqueous glucose or GG solution, respectively. The time-dependent change in pH at 37 ° C. was shown.

  FIG. 5 is a diagram showing the moisture retention of GG. It shows the weight change when glycerol, sorbitol, and GG, which are in an equilibrium state at a relative humidity of about 60%, are released at about 35% relative humidity or absorbed at about 75% relative humidity. The horizontal axis indicates time, and the vertical axis indicates weight change.

Table 1 is a table showing the digestibility of GG. According to the method of Okada et al. (Journal of the Japan Society of Nutrition and Food Science, 43, 23-29, 1990), it showed digestibility by human saliva, artificial gastric juice, porcine pancreatic α-amylase, and rat small intestinal mucosal enzyme.

  GG discovered for the first time in sake mash has many functions such as sweeteners, various seasonings, Japanese and Western confectioneries, alcoholic beverages, various beverages, fruit and vegetable processed foods, livestock meat and fish meat products, dairy products, instantaneous It can be effectively used as a sweetener, a taste improver, a corrigent, and a humectant for foods, frozen foods, therapeutic foods and other foods, toothpastes, cosmetics and other chemicals and gargles, internal medicines and other pharmaceuticals.

(Action)
2.5 U / g of α-glucosidase was added to a solution having a maltose concentration of 5% and a glycerol concentration of 37.5%, and GG, glucose, and oligosaccharides were separated and quantified by HPLC for a solution reacted at 40 ° C. for 24 hours. As a result, oligosaccharides such as disaccharides such as isomaltose and trisaccharides such as panose and isomalttriose were hardly produced, and glucose and GG were recognized as main products. That is, it was found that, in the glycerin solution having such a concentration, the transglycosylation reaction of α-glucosidase to glucose or maltose was suppressed, and the transglycosylation reaction to glycerol was newly performed.

Table 2 shows that the composition of a solution obtained by adding α-glucosidase to a solution having a maltose concentration of 5% and a glycerol concentration of 37.5% per 2.5 g of maltose and reacting at 40 ° C. for 24 hours was measured by HPLC. It is a table | surface which shows the result.

  Yoshikawa et al. (Journal of the Japan Food Industry Association, 41, 878-885, 1994) reported that in the production of ethyl-α-glucoside (hereinafter referred to as α-EG) using α-glucosidase, maltose was converted to two glucoses. It has been reported that when comparing the hydrolysis activity of glycerol with the glycosyltransferring effect of producing glucose and α-EG from maltose, the value of the α-EG / glucose ratio increases when the latter glycosyltransferring activity takes precedence. Therefore, similarly, in the production of GG by the α-glucosidase of the present invention, when the sugar transfer effect of producing GG has priority over the hydrolysis effect of the substrate, the GG / G ratio (weight ratio) (hereinafter referred to as GG / G ratio) To increase). Assuming that the hydrolysis action and the transglycosylation action take place at the same speed, 2 moles of maltose as a substrate acts on 1 mole of each of water and glycerol, 3 moles of glucose (molecular weight 180) and 3 moles of GG (molecular weight 254). One mole results and the GG / G ratio is 254 / (180 × 3) = 0.47. The GG / G ratio determined from Table 2 was about 1.3, exceeding 0.47, indicating that the transfer reaction to glycerol had priority over the hydrolysis of the substrate. The substrate concentration and glycerol concentration were examined using the GG / G ratio and the yield of GG per substrate as indices. As a result, the yield was good when the substrate concentration was low, but the GG / G ratio was high near the substrate concentration of 5%, and the yield decreased when the glycerol concentration exceeded 50%.

  FIG. 6 is a diagram showing the relationship between the substrate concentration and the GG yield and GG / G. The results obtained by quantifying GG and glucose by HPLC using a solution obtained by reacting 2.5 U / g of mold-derived α-glucosidase in a 25% glycerol solution at pH 5 at 50 ° C. for 24 hours using maltose at each concentration as a substrate are shown. Was. The horizontal axis shows the substrate concentration, the numerical value on the left vertical axis shows the yield of GG per substrate in a bar graph, and the right vertical axis shows the GG / G ratio in the reaction solution as a line graph.

  FIG. 7 is a graph showing the relationship between the glycerol concentration and the GG yield and GG / G. The results obtained by quantifying GG and glucose by HPLC of a solution obtained by reacting 5% of maltose as a substrate and 2.5 U / g of mold-derived α-glucosidase in a glycerol solution of each concentration at pH 5 at 50 ° C. for 24 hours were used. . The horizontal axis shows the glycerol concentration, the numerical value on the left vertical axis shows the GG yield per substrate in a bar graph, and the right vertical axis shows the GG / G ratio in the reaction solution as a line graph.

  The purified GG was not decomposed by emulsin, but was decomposed into glucose and glycerol by maltase, confirming that it was an α-anomer. Furthermore, when purified GG was converted into trimethylsilyl (hereinafter, referred to as TMS) and analyzed using a capillary gas chromatography-mass spectrometer (hereinafter, referred to as GC-MS), it was separated into three peaks. As described later, GG is 2-O-α-D-glucopyranosyl glycerol (hereinafter referred to as GG-II), (2R) -1-O-α-D-glucopyranosyl glycerol (hereinafter, referred to as GG-II). R-GG-I) and (2S) -1-O-α-D-glucopyranosylglycerol (hereinafter referred to as S-GG-I). Was 10:49:41, for example.

  The identification of the three peaks separated by the GC-MS was confirmed from the elution time and the mass spectrum using chemically synthesized GG. The method for synthesizing GG-II utilized short glycol cleavage of maltitol with periodate. That is, the hydroxyl groups bonded to carbons 2, 3, and 4 of the glucopyranosyl group of maltitol are each in a cis configuration, and the bond between the carbons of the aglycone sorbitol is free to rotate. Glycol cleavage by salts proceeds faster between the carbons of sorbitol. If the reaction proceeds too much, the glucopyranosyl group will also be cleaved, so the reaction time must be limited. Therefore, 1 ml of 2% sodium periodate was added to 100 μl of 4% maltitol, and reacted at room temperature for 4 minutes. After completion of the reaction, barium chloride was added, and the resulting precipitate of barium periodate was removed by filtration. After desalting with an ion exchange column, the mixture was reduced with sodium borohydride and purified by activated carbon chromatography and HPLC. When the synthesized GG-II was converted to TMS and analyzed by GC-MS, only the peak eluted first among the three peaks of the TMS derivative of GG was recognized, and the peak of the TMS derivative of GG matched the mass spectrum. For S-GG-I, Kaneda et al. (Phytochemistry, 23, 794-798, 1984) reported that lyrioside D ((2S) -1-O-β-D-glucopyrano by cleavage of gentiobiose with lead tetraacetate. With reference to the method of synthesizing (sylglycerol), glycol cleavage of isomaltose with lead tetraacetate was used. 5 ml of acetic acid and 140 mg of lead tetraacetate (2 molar equivalents of isomaltose) were added to 500 μl of 10% isomaltose and reacted, and when the reaction mixture was added to 200 μl of iodine starch and 20 μl of the reaction mixture, the color of the iodine starch mixture remained unchanged. This was the end point of the reaction. Most of the acetic acid in the reaction solution was removed with a rotary evaporator, and further desalted with an ion exchange column, and then reduced and purified in the same manner as GG-II. When the synthesized S-GG-I was converted to TMS and analyzed by GC-MS, only the last eluting peak among the three peaks of the TMS derivative of GG was recognized, and the peak of the TMS derivative of GG matched the mass spectrum. Similarly to the above S-GG-I, when trehalulose is glycol-cleaved with lead tetraacetate (1 molar equivalent of trehalulose), reduced and purified, TMS is formed and analyzed by GC-MS. Among the three peaks of the GG-I TMS derivative and the GG TMS derivative, a peak eluted immediately before the S-GG-I TMS derivative was observed, and the peak of the GG TMS derivative coincided with the mass spectrum. . The decomposition product of trehalulose, which gives these two peaks, with lead tetraacetate is one compound (3-O-α-D-glucopyranosyl-2-oxo-1-propanal). To give two compounds in which the carbon at the position is in the (R), (S) -configuration. Since there was no difference in the mass spectra of the TMS derivatives of these two compounds, it was found that the second peak among the three peaks of the GG TMS derivative was the R-GG-I TMS derivative.

  FIG. 8 is a total ion chromatogram obtained by analyzing a TMS derivative of GG by GC-MS. The vertical axis indicates the total ion intensity of the fragment ions, and the horizontal axis indicates the analysis time. Abbreviations of components corresponding to each peak (GG-II is 2-O-α-D-glucopyranosyl glycerol, R-GG-I is (2R) -1-O-α-D-glucopyranosyl glycerol , S-GG-I (2S) -1-O-α-D-glucopyranosylglycerol) and the elution time (min) in parentheses. The ratio of the three components (elution order) in this chromatogram was approximately 10:49:41.

  FIG. 9 is a mass spectrum of a TMS derivative of GG-II obtained from a peak corresponding to GG-II in FIG. The vertical axis represents the ion intensity, and the horizontal axis represents the mass number of the fragment ion.

  FIG. 10 is a mass spectrum of the TMS derivative of R-GG-I obtained from the peak corresponding to R-GG-I in FIG. The vertical axis represents the ion intensity, and the horizontal axis represents the mass number of the fragment ion.

  FIG. 11 is a mass spectrum of the TMS derivative of S-GG-I obtained from the peak corresponding to S-GG-I in FIG. The vertical axis represents the ion intensity, and the horizontal axis represents the mass number of the fragment ion.

  These three components were also present in the sake mash, but the ratio of the three components of GG was, for example, 6:66:28. When the difference in the ratio of these three components was examined, among the three components of GG generated from maltose and glycerol by the action of α-glucosidase, first, R-GG-I and GG-II increased, and after that, S- GG-I is generated. Further, when α-amylase (derived from Bacillus subtilis) or glucoamylase (derived from Rizopus sp.) Is allowed to act on purified GG, GG is not degraded. However, when α-glucosidase is acted on, R-GG-I, GG -II decomposes and S-GG-I degrades later. That is, when the maltose as the substrate disappears, GG apparently becomes a substrate for α-glucosidase, and the ratio of the three components of GG changes. Thus, it seems that the ratio of the three components of GG is slightly changed due to the difference in the selectivity of the α-glucosidase receptor binding site and the difference in substrate recognition. Therefore, in the present invention, when the amount of the substrate decreases during the production of GG, the ratio of the three components of GG changes. However, in order to suppress the decrease due to hydrolysis of GG, it is desirable to increase the glycerol concentration. In the sake mash of parallel compound fermentation, the sugar transfer reaction of α-glucosidase of koji to glycerol mainly produced by yeast is thought to occur to produce GG, but at the same time, isomaltose or ethyl-α from glucose or alcohol. It is considered that the ratio of the three components of GG is different from that of the present invention as a result of generation of glucoside and competition with generation of GG.

  Next, examples of the present invention will be specifically described by dividing into production methods and applications, but the present invention is not limited to these examples.

(Example 1 of production method) 53 g of maltose monohydrate (50 g as maltose) and 375 g of glycerol were dissolved in water to make 1000 ml, and a commercially available α-glucosidase preparation derived from mold (Asp.niger) (transglucosidase L-amano, 2.5 ml of 50 U / ml (manufactured by Amano Pharmaceutical Co., Ltd.) was added, and reacted at 40 ° C. for 24 hours. After the reaction solution was heated at 80 ° C. for 10 minutes to inactivate the enzyme, the resulting suspended matter was removed by filtration through filter paper (Toyo Roshi Kaisha No. 2).

  5 μl of the filtrate was analyzed by high performance liquid chromatography (hereinafter referred to as HPLC). HPLC conditions were Shim-pack SCR-101 (N) (inside diameter 7.9 mm, length 30 cm, manufactured by Shimadzu Corporation), the column temperature was 50 ° C., water was used as the eluent, and the flow rate was A differential refractometer was used as a detector for 0.6 ml per minute. The GG concentration was determined by preparing an aqueous solution of about 2% with purified GG described later, and a solution obtained by adding maltase (derived from yeast, oriental yeast) to the aqueous solution and reacting at 37 ° C. overnight with the above HPLC. A calibration curve was created and calculated from the glucose concentration generated by the decomposition of GG due to the action of maltase, the peak area of GG decomposed and reduced, and the respective molecular weights. As a result of the measurement, the reaction filtrate contained 3.8% of GG, 3.0% of glucose, and 29.5% of glycerol, and the amount of oligosaccharides equal to or higher than disaccharides was very small. The GG yield of the reaction filtrate was 38 g, and the yield was 76%.

  Also, 1 μl of the reaction filtrate was placed in a test tube with a screw cap, placed in a desiccator overnight, and 100 μl of a TMS agent (TMSI-C, manufactured by GL Sciences) was added. 1 μl of the solution reacted at 60 ° C. for 10 minutes was added. The analysis was performed by GC-MS (HP5890 series II gas chromatograph manufactured by Hewlett-Packard, model 5971A mass detector). The GC-MS conditions were as follows: DB-225 (length: 30 m, inner diameter: 0.25 mm, film thickness: 0.15 μm, manufactured by J & W Scientific) was used for the capillary column, helium was used as the carrier gas (column back pressure: 8 PSI), and the split method (split ratio: 1). : 50), the inlet temperature is 240 ° C, the interface temperature is 280 ° C, and the column temperature is raised from 100 ° C to 200 ° C at a rate of 5 ° C per minute, and then between 16 minutes and 20 minutes. The eluted components are ionized by an electron impact ionization method (electron beam energy: 70 eV), and the mass spectrum is scanned at a rate of about 1.5 times per minute in a range of mass number of 70 to 650 amu (atomic mass unit). Collected. Each component was confirmed from the elution time and the obtained mass spectrum. Also, the component ratio of GG-II, R-GG-I, and S-GG-I in the reaction filtrate was calculated from the measurement result of the peak area of the total ion chromatogram obtained at the same time, and was 11:41:48. .

  The reaction filtrate was passed through a glass column with an inner diameter of 1.5 cm filled with an ion exchange resin (Amberlite MB-2, manufactured by Organo) up to a length of 30 cm according to a conventional method to obtain a deproteinized and desalted solution. . This solution was concentrated on a rotary evaporator to approximately half the volume. Activated carbon column chromatography was performed on 10 ml of this concentrated liquid using a 5 cm inner diameter glass column filled with 100 g of activated carbon for chromatography and 100 g of Celite (No. 535) (both manufactured by Wako Pure Chemical Industries, Ltd.) according to a conventional method. While appropriately measuring the eluate by the above-mentioned HPLC, the eluate was first eluted with water, glucose and glycerin were removed, and then 2% alcohol was passed through the column to collect fractions eluted by GG alone. This activated carbon column chromatography was performed twice, and the GG eluted fraction was concentrated with a rotary evaporator to obtain about 1 g of a colorless and transparent syrup-like purified GG.

(Example 2 of Production Method) To the solution after reaction for 24 hours in Example 1, only 53 g of maltose monohydrate (50 g as maltose) was further added, and the reaction was repeated under the same conditions. α-glucosidase stably acted even after 10 days, and as a result of repeating for 10 days, the yield of GG was 164 g, which was about 5-fold increase from one reaction.

  FIG. 12 is a diagram showing that the yield of GG was increased by continuously adding the substrate. Maltose was added as a substrate at a concentration of 5% in a 37.5% glycerol solution, and 2.5 U / g of mold-derived α-glucosidase was reacted at pH 5 at 40 ° C. for 24 hours. Repeated. A part of the reaction solution was taken at the end of each reaction, and the result of quantifying GG by HPLC was shown. The horizontal axis is the number of maltose additions and the total amount of maltose added per 1000 ml of the initial reaction solution, the bar graph shows the yield of GG per 1000 ml of the initial reaction solution by the numerical value on the left vertical axis, and the yield on the substrate by the numerical value on the right vertical axis. Is shown by a line graph.

  When the reaction solution was similarly analyzed by GC-MS performed in Example 1 of the production method, the component ratio of GG-II, R-GG-I, and S-GG-I was 9:50:41. In addition, by repeating the activated carbon column chromatography and the concentration of the GG elution fraction performed in Example 1 of the production method, 140 g of colorless and transparent syrup-like purified GG was obtained.

(Example 3 of the production method) About 20 g of the purified GG in the syrup obtained in Example 2 of the production method was made up to 30 ml with water, and a part thereof was placed in a column with a YMC-Pack Polyamine II (inner diameter 1 cm, length 25 cm, HPLC). The HPLC conditions were as follows: elution was carried out at a column temperature of 35 ° C. and 85% acetonitrile at a flow rate of 3 ml / min, and detected by a differential refractometer. The two peaks eluted were collected, respectively, and concentrated on a rotary evaporator. When each eluted fraction was converted to TMS and analyzed by GC-MS performed in Example 1 of the production method, the first fraction was GG-II, the later fractions were R-GG-I, S-GG- I was a mixture. This separation by HPLC and concentration of each fraction were repeated to obtain about 1.5 g of purified GG-II and about 15 g of purified GG-I in the form of syrup.

(Application Example 1 Sake) 900 ml of 40% alcohol, 50 g of anhydrous glucose, 70 g of powdered starch syrup, 60 g of syrupy GG obtained in Example 2 of the production method, 0.4 ml of 75% lactic acid, 1.1 g of succinic acid, 1.1 g of sodium glutamate 0.2 g was dissolved in water to 1200 ml to prepare a GG-added seasoning alcohol solution having an alcohol concentration of 30%. As a control, 900 ml of 40% alcohol, 80 g of anhydrous glucose, 100 g of powdered starch syrup, 0.4 ml of 75% lactic acid, 1.1 g of succinic acid, and 0.2 g of sodium glutamate were dissolved in water to 1200 ml, and the seasoning at an alcohol concentration of 30% was performed. An alcohol solution was prepared. 1200 ml of GG-added seasoning alcohol solution and 500 ml of water or 1200 ml of control seasoning alcohol solution and 500 ml of water were added to 1250 ml of sake mash, respectively, and sake lees were separated by centrifugation to obtain a brewed sake of about 20% alcohol. Each of these was burned, and after slagging, water was added and heated to about 15% alcohol to prepare a GG-rich sake and a control sake. As a result of a sensory test conducted on these by five panelists, the sake with a high GG content was compared with the control sake, with "Koku", "Fine and soft," "Swelling," and "Clean sweetness." Is good. " As described above, since GG is not only digestible but also has a refreshing sweetness and a deep flavor, sake with a high GG content has slightly reduced calories because GG is an indigestible substance.

(Application Example 2 Toothpaste) 5 g of syrup-like GG obtained in Example 2 of the production method, 15 g of calcium diphosphate, 1 g of pullulan, 0.5 g of sodium lauryl sulfate, 7 g of glycerol, 0 g of polyoxyethylene sorbitan laurate .15 g, a preservative of 20 mg and water of 4 ml were mixed by a conventional method to prepare a toothpaste. This product, which makes use of the sweetness and non-cariogenicity of GG, is particularly suitable for toothpaste for children.

(Application Example 3 Cosmetic Cream) 2 g of syrup-like GG obtained in Example 2 of the production method, 2 g of polyoxyethylene glycol monostearate, 5 g of self-emulsifying glyceryl monostearate, 1 g of α-glucosyl rutin, 1 g of liquid paraffin , 10 g of glyceryl trioctanoate and 50 mg of a preservative are dissolved by heating in a conventional manner, and 5 g of 1,3-butylene glycol, 2 g of lactic acid and 66 ml of purified water are added. After emulsification with a homogenizer, an appropriate amount of flavor is added and mixed. A cream was made. This product is particularly suitable as a cosmetic cream for dry skin due to the moisturizing effect of GG.

(Example 4 of use: calcium agent) 2 g of calcium lactate was ground in a mortar, dissolved in 30 ml of hot water (purified water), and 4 g of syrupy GG obtained in Example 2 of the production method was added and mixed to prepare a calcium agent. . This product can be used as a calcium supplement during childhood development. GG is suitable as a sweetener having excellent heat stability in an internal solution which needs to be heated at the time of taking, such as this product, especially for children.

  It should be noted that glycerol has three condensed sites, as compared with a case where only one condensed site with glucose is present, as in ethanol, which is a receptor for the transglycosylation reaction in ethyl-α-glucoside. Stereoselective glycosylation, particularly α-glucosylation, has become one of the important research topics in the field of carbohydrate chemistry, and GG has the selectivity of α-glucosidase receptor binding site and substrate specificity. It becomes an enzymatically interesting substance.

  In addition, GG can be a substrate for the synthesis of glyceroglycolipid, which is a component of a microorganism cell membrane. Glyceroglycolipids of microorganisms differ from those of animals and plants in that their constituent monosaccharides and bonds are diverse, and only a part of their biosynthesis and degradation has been elucidated. It will lead to the development of the microbial utilization industry. In addition, 2-O-α-D-glucopyranosylglycerol, which is a component of GG, can be isolated and purified, and its involvement in osmotic pressure regulation in some cyanobacteria is clear. It is an interesting substance in the industrial field.

It is a figure which shows browning of GG. It is a figure which shows Maillard reactivity of GG. It is a figure which shows the heating stability of GG. It is a figure which shows the non-caries property of GG. It is a figure which shows the moisture retention of GG. It is a figure which shows the relationship between a substrate concentration, GG yield, and GG / G. It is a figure which shows the relationship between glycerol concentration, GG yield, and GG / G. It is a total ion chromatogram when the TMS derivative of GG was analyzed by GC-MS. 9 is a mass spectrum of a TMS derivative of GG-II obtained from a peak corresponding to GG-II in FIG. 9 is a mass spectrum of a TMS derivative of R-GG-I obtained from a peak corresponding to R-GG-I in FIG. 9 is a mass spectrum of a TMS derivative of S-GG-I obtained from a peak corresponding to S-GG-I in FIG. It is a figure which shows that the yield of GG increased by continuously adding a substrate.

Claims (6)

Equation 1, Equation 2 or Equation 3

(2R) -1-O-α-D-glucopyranosyl glycerol, (2S) -1-O-α-D-glucopyranosyl glycerol and 2-O-α-D-glucopyranosyl glycerol A seasoning characterized in that α-D-glucopyranosylglycerols containing at least one of the following are used as a main component.   A food or drink to which the seasoning according to claim 1 is added.   The food or drink according to claim 2, which is an alcoholic beverage. Equation 1, Equation 2 or Equation 3

(2R) -1-O-α-D-glucopyranosyl glycerol, (2S) -1-O-α-D-glucopyranosyl glycerol and 2-O-α-D-glucopyranosyl glycerol A moisturizer characterized by comprising α-D-glucopyranosylglycerols containing at least one of the following as main components:   A cosmetic to which the humectant according to claim 4 is added. The cosmetic according to claim 5, which is a cosmetic cream.

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