JPS6337638B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to Stevia rebaudiana bertoni, which belongs to the Asteraceae family.
The present invention relates to a method for producing Rebaudiosde A, a sweet component contained in the leaves and stems of P. BERTONI, by fermentation. More specifically, in an aqueous solution or aqueous suspension containing stevioside and β-1,3 glucosyl sugar compound, stevioside and β-glucosyl sugar compound belonging to the genus Streptomyces are added.
-β between 1,3 glucosyl sugar compounds-
The culture solution, cells, processed products of microorganisms, or the β isolated from the microorganisms that produce enzymes with 1,3 glucosyl transfer activity and have the ability to convert stevioside to rebaudioside A. -Relates to a method for producing rebaudioside A, characterized in that an enzyme having 1,3 glucosyl transfer activity is used. Rebaudioside A (β-1,3-monoglucosyl stevioside) is found in Stevia leaves and stems.
It contains about 6%, the second highest content after the main component stevioside (6-12%). Until now, a mixture of the two has been used as a stevia sweetener, but while stevioside has a bitter taste and leaves an aftertaste, rebaudioside A has a mellow sweetness without bitterness, and is in contrast to sucrose. The sweetness factor is also high. Many attempts have been made to improve the taste quality of stevia sweeteners, such as adding natural sugars and amino acids, but recently a new method has been developed that adds sugar to stevioside to eliminate bitterness without increasing calories. Stevia sweeteners have been developed. (Japanese Unexamined Patent Publication No. 54-5070) However, it had the disadvantage that the sweetness factor was lower than that of stevioside. For this reason, there is a strong desire to develop a stevia sweetener containing only rebaudioside A or containing a high content of rebaudioside A. Conventionally, the method for obtaining rebaudioside A is as follows:
Stevioside is extracted and purified from dried Stevia leaves, a mixture of stevioside and rebaudioside A is obtained, and stevioside is removed by crystallization, followed by repeated recrystallization. It is necessary to increase the content of Baudioside A during extraction. Attempts have been made to improve the breed of Stevia leaves with a higher content of rebaudioside A through breeding of Stevia, but this is currently impractical due to instability in maintaining traits due to genetics. Therefore, the present inventors have already completed an invention aimed at converting stevioside into rebaudioside A by adding glucose to stevioside, which is present in the largest amount in a stevia leaf extract, by a fermentation method or an enzymatic method. It has been filed as Japanese Patent Application No. 57-31479. In the prior invention, these β-
A culture solution or fungus of a microorganism having an activity capable of transferring glucose from a 1,3 glucosyl compound to the C3 position of β-D-glyose bonded to the _hydroxyl group of steviol, which is an aglycone of stevioside, that is, β-1,3 glucosyl transfer activity. The purpose of this method is to produce rebaudioside A by reacting cells or a processed product of bacterial cells. However, in the method of this prior invention, two or more types of β-1,3 glucosyl steviosides including rebaudioside A are produced, but the molar conversion rate to rebaudioside A is less than 20%, which is satisfactory. It wasn't something. Therefore, in order to further search for microorganisms that have the ability to highly convert stevioside to rebaudioside A with remarkable selectivity, the present inventors isolated many microorganisms from a wide range of natural soils, and among them, Streptomyces spp. We discovered a microorganism that belongs to the genus and has strong β-1,3 glucosyltransferase activity, and also discovered the ability to convert steviaside to only rebaudioside A at a high molar conversion rate, and the present invention I was able to complete it. That is, the present invention provides an aqueous solution or aqueous suspension containing stevioside and a β-1,3 glucosyl sugar compound, which belongs to the genus Streptomyces, and contains β- A culture solution of a microorganism that produces an enzyme having 1,3 glucosyl transfer activity and has the ability to convert stevioside to rebaudioside A;
The present invention provides a method for producing rebaudioside A, characterized in that the enzyme having β-1,3 glucosyl transfer activity isolated from bacterial cells, processed bacterial cells, or microorganisms thereof is allowed to act. The Streptomyces bacterium used in the present invention contains β-1,3-transferase,
Any bacteria that produces rebaudioside A from stevioside and β-1,3 glucosyl sugar compounds may be used, but Streptomyces sp. DIC-, which will be described in detail below, is preferable.
108, Streptomyces sp. DIC-146. Other preferred examples include artificial mutant strains obtained by artificially mutating these strains, ie, mutagenesis treatment by chemical or physical means, or natural mutant strains. The mycological properties of the aforementioned two strains preferably used in the present invention are as follows. [Mycological properties of Streptomyces sp. DIC-108] (1) Morphological characteristics The growth of vegetative mycelia on the medium used (including ISP medium) was excellent, and it Forms abundant aerial mycelium on agar medium. Spore-forming aerial hyphae are straight or curved. The spores are spherical and the size is
The short axis x long axis is 0.7 to 0.8 μ x 1.0 to 1.2 μ. When observed using a scanning electron microscope, the surface structure of the spores is warty. (2) Growth status on various media 1 Sucrose nitrate agar medium (37℃) Gray aerial hyphae are formed in the shape of light brown basal hyphae, and no soluble pigments are observed. 2 Glucose-asparagine agar medium (37
℃) Growth is pale yellowish white and no formation of aerial mycelium is observed. Also, no soluble pigments were observed. 3 Glycerin-asparagine agar medium (ISP
Medium - No. 5 (37℃) Growth is pale yellow and no aerial mycelium is observed. Also, no soluble dyes were observed. 4 Starch/inorganic salt agar medium (ISP medium 37
°C) Green-gray aerial mycelium grows on the colorless growth,
No soluble pigments are observed. 5. Tyrosine agar medium (ISP medium-7, 37°C) No aerial mycelium grows on the light brown growth on the 7th day of culture, but white-gray aerial mycelium grows on the 14th day. No soluble pigments are observed. 6 Nutrient agar medium (37°C) Light greenish-gray aerial mycelium grows on greenish-gray-black growth, and no soluble pigment is observed. 7 Yeast malt agar medium (ISP medium-2, 37
°C) Light greenish-gray aerial mycelium grows on the light brown growth. No formation of water-soluble pigment was observed. 8 Oatmeal agar medium (ISP medium-3, 37
°C) Green-brown-gray aerial mycelium grows on the colorless growth, and no production of water-soluble pigments is observed. (3) Physiological properties 1 Growth temperature range In a growth test using yeast extract/malt extract liquid medium, it grows at 30°C to 50°C, but the optimum temperature is 37°C to 45°C. 2 Liquefaction of gelatin: Negative 3 Coagulation of skim milk and peptonization of skim milk: Negative 4 Production of melanin pigment (peptone/yeast/iron agar medium, ISP medium 6): Negative 5 Degradability of starch: Positive (in the decomposition zone) (forms a white ring) 6 Availability of carbon sources (Pridham, Godelive agar - 9, 37°C) D-glucose, L-arabinose, D-
It grows using xylose, D-fructose, inositol, L-rhamnose, D-mannitol, and galactose, but does not use sucrose, raffinose, or salicin. 7 Cell wall composition The sugar composition type listed in ISP belongs to Type I. Based on the above characteristics, this strain belongs to the genus Streptomyces, and is included in the Gray Series due to the color tone of its aerial hyphae, making it a non-chromodienic type strain that does not produce melanin-like pigments. belong to. When searching for known bacterial species regarding the taxonomic position of this strain based on these properties, we found that Streptomyces arabicus [Int.J.of
Sys.Bacteriology (abbreviation for International Journal of Systematic Bacteriology) vol.18No.2 p.294′68], Streptomyces griseoflavus
griseoflavus)〔Int.J.of Sys.Bacteriology
vol.19 No.4p.433'69], Streptomyces macrosporeus
[Int.J.of Sys.Bacteriology vol.18 No.2
p.142'68], Streptomyces matensis [Int.J.of Sys.
Bacteriology vol.18 No.2 p.344'68], is DIC
- It is listed as a bacterial species closely related to 108. Therefore, we cultured Streptomyces arabicus IFO-14035, Streptomyces griseoflavus IFO-12372, Streptomyces macrosporeus IFO-12794, and Streptomyces matensis IFO-12889, which are stored at the Fermentation Research Institute, under the same conditions. In comparison, Streptomyces arabicus and Streptomyces griseoflavus clearly differ from this bacterium in that they do not have the ability to decompose starch. Streptomyces macrosporeus and Streptomyces matensis both have the ability to decompose starch, but Streptomyces matensis was found to form a white ring in the decomposition zone that is unique to this bacterium. Table 1 Comparison of Streptomyces matensis IFO-12889 and this bacterium
Although there are slight differences in the color tone of the aerial mycelia in each culture medium, other mycological properties are in very good agreement. Therefore, this strain is considered to be a species extremely closely related to Streptomyces matensis. however,
They differ greatly in the presence or absence of β-1,3-glucosyltransferase activity; in other words, this strain has the activity of transferring glucose from β-1,3-saccharide compounds to stevioside, whereas Streptomyces matensis has this activity. Due to differences such as no activity and low optimum growth temperature, this bacterium was identified as Streptomyces sp. DIC-108 as a new species closely related to Streptomyces matensis. This strain has been applied for deposit with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and has been entrusted with Microbiological Research Institute No. 6593.

[Table] [Mycological properties of Streptomyces sp. DIC-146] (1) Morphological characteristics The growth of vegetative mycelium on the medium used (including ISP medium) was excellent, and the growth of vegetative mycelia was excellent on the glucose-asparagine agar medium. Aerial hyphae are formed well on any medium other than the above. Spore-forming aerial hyphae are spiral-shaped. The size of the spores and cylindrical body is short axis x long axis 0.7-0.8μ x 1.2-1.4μ. When observed using a scanning electron microscope, the surface structure of the spores is smooth. (2) Growth status on various media 1 Sucrose nitrate agar medium (37℃) Gray-brown aerial mycelia are formed on colorless growth.
No soluble pigments are observed. 2 Glucose-asparagine agar medium (37
°C) Light yellow growth with no formation of aerial mycelia. Also, no soluble pigments were observed. 3 Glycerin-asparagine agar medium (ISP
Medium - No. 5 (37℃) It grows pale grayish brown and forms purple-white-gray aerial mycelia. No soluble pigments are observed. 4 Starch/inorganic salt agar medium (ISP medium 37
°C) Reddish-gray aerial mycelium grows on the light brown growth, and red pigment is produced on the underside of the colony around the 14th day of culture. 5 Tyrosine agar medium (ISP medium-7, 37°C) Brown-white-gray to black-gray aerial mycelium grows on the gray-black growth, and no diffusible pigments are observed. 6 Nutrient agar medium (37°C) White-gray aerial mycelium is formed on the gray-brown growth, and red to vermilion pigments are produced on the back of the colony on the 14th day of culture. 7 Yeast/malt agar medium (ISP medium-2,
(37℃) Light brownish-gray aerial mycelium grows on the brown growth. No formation of water-soluble pigment was observed. 8 Oatmeal agar medium (ISP medium-3, 37
°C) Red-gray aerial mycelium grows on the gray-brown growth, and red pigment is produced on the underside of the colony around the 14th day of culture. (3) Physiological properties 1 Growth temperature range In a growth test using yeast extract/malt extract liquid medium, it grows at 30°C to 50°C, but the optimum temperature is 37°C to 45°C. 2 Liquefaction of gelatin: Positive 3 Coagulation of skim milk and peptonization of skim milk: Positive 4 Production of melanin pigment (peptone/yeast/iron agar medium, ISP medium 6): Negative 5 Degradability of starch: Negative 6 Carbon source Usability (Pridham, Godlieb agar, ISP medium-9, 37℃) D-glucose, L-arabinose, D-
It grows using xylose, D-fructose, inositol, L-rhamnose, raffinose, D-mannitol, and galactose, and sucrose and salicin are either unavailable or questionable. 7 Cell wall composition The sugar composition type listed in ISP belongs to Type I. From the above characteristics, this strain belongs to the genus Streptomyces, and from the color tone of the aerial hyphae, it is classified as a non-chromodienic type strain that is included in the gray series and does not produce melanin-like pigments. belong to. When searching for known bacterial species regarding the taxonomic position of this strain based on these properties,
Streptomyces olivaceus [Int.J.of Sys.
Bacteriology vol.18 No.2 p.154'68] is the most closely related species. Therefore, Streptomyces olivaceus IFO, a preserved strain of the Fermentation Research Institute,
-3409 under the same conditions, as shown in Table 2, there are differences in the color of aerial mycelia on oatmeal agar and starch inorganic salt agar, but other mycological properties are very different. They match well. Therefore, this strain is considered to be a species extremely closely related to Streptomyces olivaceus. However, they differ greatly in the presence or absence of β-1,3-glucosyltransferase activity, that is, this strain has the activity of transferring glycose from β-1,3 sugar compounds to stevioside, but Streptomyces olivaceus Since this bacterium does not have this activity and there are differences such as a low optimum growth temperature, this bacterium has been classified as Streptomyces sp. DIC-146 as a new species closely related to Streptomyces olivaceus.
was identified. This strain has been applied for deposit with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and has been entrusted with Microbiological Research Institute No. 6594.

【table】

[Table] The β-1,3 transferase used in the present invention allows these preferred microorganisms to be grown in a liquid medium containing known nutritional sources such as carbon sources, nitrogen sources, and inorganic components that can be utilized by normal microorganisms. Obtained by culturing under aerobic conditions using conventional methods. Examples of carbon sources include polysaccharides such as starch, amylose, curdlan, pachyman, laminarin, and amylopectin, alcohols such as methanol, glycerin, and higher alcohols, organic acids and their salts such as succinic acid, acetic acid, and higher fatty acids, and starch. , maltose, glucose, rhamnose, etc., with pachyman, curdlan, and laminarin being preferred. As the nitrogen source, any organic or inorganic source can be used, such as ammonium sulfate, ammonium chloride, ammonium phosphorus, sodium nitrate, urea, peptone, and casein. Natural nutritional sources including carbon sources, nitrogen sources and other nutritional substances include, for example, various types of molasses, cornstarch, oatmeal, flavoring liquids, fishmeal,
Examples include meat extract, yeast, yeast extract, potato extract, and malt extract. Examples of inorganic substances include dipotassium phosphate, monosodium phosphate, magnesium sulfate, and trace metals.
In addition, vitamins and the like may be added as necessary. Usage concentration: 0.1 to 40% by weight
is used. Also to suppress foaming during fermentation.
Up to 1.0% by weight of antifoaming agent may be added. As the antifoaming agent, common antifoaming agents such as silicone and soybean oil are used. As for the culture method, aerobic liquid culture such as shaking culture and aeration culture is suitable, and pH5.0-8.0, 20℃-50℃
for 1 to 6 days, preferably PH6.5 to 7.5, 35℃ to 40℃
Culture for about 2 days. As described above, Streptomyces sp. DIC-108 and DIC-146, which are preferably used in the present invention, have a higher optimal growth temperature than ordinary Streptomyces microorganisms, so they can be cultivated at high temperatures and are industrially useful bacteria. It is a seed. In addition, the molar conversion rate of the strain used in the present invention is calculated as rebaudioside A produced/stevioside reduced x 1.2 x 100, (1.2 = molecular weight of rebaudioside A/molecular weight of stevioside). ) The molar conversion rate is clearly higher than the conventional value of 20%, and shows a value of about 50 to 99%. Furthermore, the strain of the present invention has a reduction rate of stevioside at the same level as conventional strains, and the reduced stevioside is converted to rebaudioside A at a high conversion rate. The stevioside used in the present invention is not limited to highly purified stevioside, but may also be a mixture of stevioside and rebaudioside A, or a crude product containing other impurities. The present invention can also be implemented in the same manner. As the β-1,3 glucosyl sugar compound used in the method of the present invention, rebaudioside A is produced from stevioside using Streptomyces sp. DIC-108 or Streptomyces sp. DIC-146, which are preferably used. Any kind is fine, but for example, Pakiman,
Curdlan, laminarin, yeast cell walls or partial decomposition products of yeast cell walls, yeast as used herein includes, for example, Satucharomyces, Candeida, Pichia, Schizosatucharomyces, Torulopsis,
It refers to the genus Hysenula, etc., and it is also obtained from lyucosin (cell wall of diatoms), callose (cell wall of higher plants Haseolus), paramylon (cell wall of unicellular algae), lichenan (extract of moss), and seed endosperm of grasses. sugars (Gramineae here refers to rice, barley, wheat, etc.),
Examples include β-1,3 glucosyl sugar compound-containing compounds such as. Among them, preferable ones include pachyman, curdlan, and laminarin, and these are more preferable because they are easily available. Curdlan is a water-insoluble β-glucan mainly composed of β-1,3-glucoside bonds, and is a general term for polysaccharides that form a hard, elastic, heat-irreversible gel when the suspension is heated. It is. At present, products produced by microorganisms are commercially available. (See Agr. Biol. Chem., 29 , 757, '65 or Fermentation and Industry 36 , 2, '78). The culture solution and bacterial cells of Streptomyces sp. DIC-108 or Streptomyces sp. DIC-146, which are preferably used in the present invention, may be reacted in a batch manner, or the bacterial cells may be immobilized by a known method. The conversion reaction can also be carried out continuously. The transfer reaction conditions of the present invention include stevioside and β
- a culture solution of Streptomyces sp. DIC-108 or Streptomyces sp. DIC-146 in an aqueous solution containing a 1,3-glucosyl sugar compound,
Bacterial cells, processed bacterial cells, or β isolated from these
-An enzyme having 1,3 glucosyl transfer activity may be used for the reaction. Stevioside used in the reaction is
In the case of purified stevioside, the concentration of stevioside in the reaction solution is about 0.1 to about 10% by weight, β-1,
The concentration of the 3-glucosyl sugar compound may be about 0.1 to about 30% by weight. The pH and temperature of these reaction solutions may be any conditions that allow β-1,3 glucosyltransferase to react and produce rebaudioside A, but usually the pH is 3-10, preferably 5-10.
8. Temperature 20-70°C, preferably 30-50°C is appropriate. The reaction solution in which rebaudioside A is produced in this way can be used as a sweetener as it is. If necessary, after inactivating the enzyme or bacterial cells by heating, use a polymeric adsorption resin of styrene and divinylbenzene such as Diaion HP-20.
(Product name, manufactured by Mitsubishi Kasei Corporation), Amberlight XAD
-2 (trade name, manufactured by Organo Co., Ltd.), or ion exchange resins (for example, H-type strongly acidic ion exchange resins and
It can also be desalted using an OH-type weakly basic ion exchange resin and concentrated to produce a syrupy sweetener, or dried and powdered to produce a powdery sweetener. Furthermore, the desalted reaction solution can be purified to separate and collect rebaudioside A, which can be used as a sweetener. At this time, concentration, drying, and powdering are performed using known methods.
For example, various methods such as vacuum concentration, membrane concentration, vacuum drying, spray drying, etc. are freely used. The sweetness of rebaudioside A obtained in this way varies depending on the sweetness measurement conditions, but in general, it is approximately 1.3 times the sweetness corresponding to the solid weight of the stevioside used in the reaction. strong. In addition, its sweet taste is mellow, similar to sugar, without unpleasant tastes such as bitterness or astringency, and has a good aftertaste. Rebaudioside A is an odorless, white powder with no bitterness, unpleasant taste, or acrid taste, and is soluble in water. can be done. Also, rebaudioside A
can be used in combination with well-known synthetic sweet substances such as saccharin and its salts, sodium cyclamate, dihydrochalcone, aspartame, etc. to effectively utilize its taste characteristics, and one of these synthetic sweet substances or By adding the present compound to two or more types, it becomes possible to improve unpleasant tastes such as bitterness and sarcastic taste peculiar to synthetic sweet substances. In addition, by adding rebaudioside A to well-known sugar sweeteners such as sugar, fructose, glucose, lactose, starch syrup, dextrin, and starch, which are used as excipients, diluents, and adsorbents, sweetness can be improved. has been increased, making it possible to significantly reduce usage compared to conventional usage. Furthermore, if this compound is added to low-calorie sweet substances such as sorbitol, maltetol, mannitol, and xylitol, which have a lower sweetness than sugar, the sweetness can be enhanced without sacrificing the advantages of the sweet substance, resulting in a high-quality low-calorie sweet taste. You can get a fee. It goes without saying that rebaudioside A can be used as a sweetening source for general foods, diet foods, medicines, quasi-drugs, tobacco, feeds, and the like. For example, soybean oil, powdered soybean oil, miso, powdered miso, mash, mayonnaise, dressing, vinegar, sambai vinegar, powdered sushi vinegar, Chinese stock, tempura soup, mentsuyu, sauce, ketchup, yakiniku sauce, curry roux, stew stock, soup. Dashi ingredients, Dashi ingredients,
Composite seasonings, various seasonings such as mirin, new mirin, and table syrup, various Japanese sweets such as rice crackers, arare, okoshi, rice cakes, manjiyu, uiro, bean paste, yokan, water yolk, jelly, castella, and candy, Bread, biscuits, kratska, kutsky, pie, pudding, butter cream, custard cream, cream puff, Watsuful, sponge cake, donut, chocolate cake, chewing gum, caramel, yellow cake, etc., various Western sweets, ice cream, sherbets, ice cream cake, etc. Frozen confections, fruit pickled in syrup, watertight syrups, pastes such as flower paste, peanut paste, fruit paste, etc., fruit and vegetable processed foods such as jam, marmalade, syrup pickles, sugar confectionery, Fukujinzuke, senmaizuke, etc. Pickles such as Ratsukiyo-zuke, meat products such as ham and sausages, meat products such as meat ham, fish sausage, fish meat products such as kamaboko, chikuwa, and tempura, sea urchin, salted squid, sakisurume,
Bottles of various delicacies such as dried puffer fish, seaweed, wild vegetables, dried plums, small fish, simmered dishes made from shellfish, vegetable foods such as boiled beans, potato salad, kelp rolls, fish, meat, fruits, and vegetables. Alcoholic beverages such as stuffed and canned goods, synthetic liquor, fruit liquor, and Western liquor, coffee, cocoa,
It can be used to sweeten various foods and beverages, such as soft drinks such as juice, carbonated drinks, lactic acid drinks, and lactic acid bacteria drinks, pudding mixes, hot cake mixes, instant drinks, instant coffee, instant shiruko, and other instant drinks. In addition, as pharmaceuticals and quasi-drugs, it may be freely used as a sweetener in toothpaste, lipstick, lip balm, oral medications, troches, cod liver oil drops, mouth fresheners, oral pastilles, gargles, etc. The method of the present invention and the sweetener obtained thereby will be specifically explained below using Examples, and the following percentages are based on weight. Example 1 (1) Preparation of β-1,3 glucosyltransferase Streptomyces sp. DIC-108 (Feikoken Bacteria No. 6593) was mixed with yeast extract 0.2W/V%, polypeptone 0.2W/V%, Glucose 0.5% W/V
%, MgSO ₄・7H ₂ O0.1W/V%, K ₂ HPO ₄ 0.2%
Medium 5 consisting of W/V% was inoculated, and at the same time 100 g of separately sterilized curdlan was added and cultured with aeration at 37° C. for 40 hours. The obtained culture solution was centrifuged, and the supernatant was salted out with 0.8 saturated ammonium sulfate.
A crude enzyme preparation [] having 3-glucosyltransferase activity was obtained. The enzyme activity at this time was about 1000 units. Note that 1 unit of enzyme activity here means PH7.0,
This is the amount of enzyme (protein) required to produce 1 micromole of rebaudioside A when reacted at 50°C for 30 minutes in a 0.1M phosphate buffer with a stevioside substrate concentration of 0.5%. (2) Transfer reaction (manufacture of rebaudioside A) Stevia sweetener (trade name, Stevia-DIC, manufactured by Dainippon Ink Chemical Co., Ltd.) 10 g, curdlan (manufactured by Wako Pure Chemical Industries, Ltd.) 20 g, previously prepared 10 units of crude enzyme preparation [] in 0.1M phosphate buffer (PH7.0) 1
and stirred at 50°C for 30 minutes to react. The reaction solution was filtered and then heated at 90°C for 10 minutes to inactivate the enzyme. This solution was applied to synthetic adsorption resin Diaion HP-20 (product name, manufactured by Mitsubishi Kasei Corporation) by SV.
= 2 to adsorb steviosides,
Desorbed with 95% ethanol. After the ethanol in the desorption solution was distilled off under reduced pressure, Amberlite IR-120B (H type, trade name, Rohm and Haas product), which is a strong acidic ion exchange resin, and Amberlite IRA-93, which is a weakly basic ion exchange resin, were added. (OH type, product name,
(Rohm & Haas) at SV=2 for desalting. This is then concentrated under reduced pressure at below 70°C.
Vacuum dry powder rebaudioside A at 2.7
Sample No. 2 (referred to as improved sweetener) containing 3.3 g of stevioside was obtained. On the other hand, the control product (sample no.
As for 1), a crude enzyme preparation that had been inactivated by heat in advance was reacted in the same way, and purified using an adsorption resin or an ion exchange resin, which resulted in rebaudioside A.
was 1.4g, and stevioside was 5.2g. Comparing this with Sample No. 2, stevioside was reduced by about 36% and rebaudioside A was reduced by about 93% by acting with the crude enzyme preparation of β-1,3 glucosyltransferase []. It was confirmed that it is increasing. The molar conversion of this reaction is 57%
It was hot. Note that the molar conversion rate is calculated as: produced rebaudioside A/1.2 x reduced stevioside x 100. (However, 1.2 = molecular weight of rebaudioside A/molecular weight of stevioside) Comparative Example 1 In place of the crude enzyme preparation [ ] of Example 1, Driselase (manufactured by Kyowa Hakko Co., Ltd.) was added at a concentration of 0.1% at 50°C for 3 hours. When the same procedure was performed except for the reaction, 3.6 g of stevioside and rebaudioside were obtained.
A mixture containing 1.6 g of A was obtained, with a decrease of 1.6 g (31%) in steviaside and an increase in rebaudioside A by 0.2 g (14%) compared to Sample 1 in which the reaction of Example 1 was not performed. ) was confirmed to be generated. The molar conversion of this reaction was 10%. (3) Sweetness test of improved sweetener Prepare 0.02% and 0.05% aqueous solutions of Samples No. 1 and No. 2, and create 13 standard solutions of 1 to 7% sugar solutions in 0.5% concentration steps. A sweetness test was then conducted on these. The test was a two-point comparison method between a sample solution and a standard solution, and was conducted by 20 panel members at room temperature 20℃.
The results are shown in Table 3.

[Table] From the results in (a) and (b) of Table 3, the sweetness level of sample No. 1 is 0.02% aqueous solution with a sugar concentration of 3% (150 times the sweetness level)
equivalent to 6% sugar concentration (sweetness level) in a 0.05% aqueous solution.
120 times). Similarly, the sweetness of sample No. 2 corresponds to 3.5% and 6.5% of the sugar concentration, respectively, so the sweetness of the improved sweetener is judged to be approximately 1.1 to 1.2 times the sweetness of the stevioside used. Ru. (4) Taste quality test of improved sweetener Sample No. 1, the control product, and Sample No. 2, the improved sweetener, were used to compare the differences in sweetness quality. Calculated from the sweetness level determined in the sweetness level test, each sample was
%, 6%, and 10% sugar solutions. And sample No. 1 for each sweetness level,
The taste quality of sample solution No. 2 was compared. The test was conducted at room temperature of 20°C by 20 panel members. The results are shown in Table 4.

[Table] From the results in Table 4, it is clear that the sweetness quality of the improved sweetener of Sample No. 2 is superior to the control product of Sample No. 1 at all sweetness levels. Example 2 (Separation and confirmation of rebaudioside A) (1) Synthesis from stevioside 10 g of pure stevioside, 20 g of curdlan, β-1,3 glycosyltransferase crude enzyme obtained in Example 1 (1) Suspend 10 units of the standard sample in 0.1M phosphate buffer (PH7.0) and incubate at 50℃ for 1 hour.
The reaction mixture was stirred for hours. After filtering the reaction solution, 90
The enzyme was inactivated by heating at ℃ for 10 minutes. It was also confirmed that stevioside decreased by 4.8g. This solution is synthesized with adsorption resin Diamond Ion HP-
20 to 200 ml to adsorb the steviosides, and then desorbed with 80% ethanol, the solvent was distilled off, and the mixture was dried under reduced pressure. This is chloroform: methanol: water = 30:
After dissolving in a 25:4 solvent and adsorbing it on a 600 ml silica gel column (Wakogel C-300, trade name, manufactured by Wako Pure Chemical Industries, Ltd.), it was developed with the above solvent and a fraction corresponding to rebaudioside A was collected. , After concentration to dryness under reduced pressure, white powder equivalent to rebaudioside A 3.5
I got g. (referred to as sample No. 3) The molar conversion rate of this reaction was 60%. (2) Extraction of natural rebaudioside A On the other hand, add 150g of dried stevia leaves to 3 parts warm water (50%
The mixture was extracted twice for 3 hours at 3°C and concentrated to a volume of 3. After removing proteins from the extract, a 400ml column was filled with synthetic adsorption resin Diaion HP-21 and S.
Steviosides were adsorbed through the column at V.=2.0. Then add 750ml of 95% ethanol SV =
I installed and removed it using 2.0. After ethanol in the desorption solution was distilled off under reduced pressure, the dried product was dissolved in 600 ml of water.
Transfer the solution to strong acidic ion exchange resin IR-120B (H
Type) 130ml packed column at SV = 2.0, and then IRA-93 (OH
Desalting was carried out by passing 900 ml through a mold (type) at SV=2.0. This was then concentrated under reduced pressure at a temperature below 70°C and dried under vacuum to obtain approximately 20 g of refined stevia powder (trade name: Stevia-DIC). Add 5 times the amount of purified stevia (W/
W) was dissolved in hot methanol (60°C), and after cooling, the precipitate was filtered. Dry the filtrate under reduced pressure and
10 g was subjected to column chromatography on Wakogel C-300. Chloroform:methanol:water=6.5:3:1
Elution was performed with a solvent composition of (V/V/V), and 1.16 g of stevioside and 0.7 g of rebaudioside A were isolated. Sample No. 3 had the same retention time as rebaudioside A when analyzed by high-performance liquid chromatography, and was spotted on a silica gel 60F plate (manufactured by Merck & Co., Ltd.) and mixed with chloroform:methanol:water=30:20: When developed with a solvent composition of 4, the Rf value of stevioside is 0.84 when the Rf value of stevioside is 1.
The value was consistent with that of rebaudioside A. Also
In structural confirmation by ¹³ C-NMR, an NMR spectrum consistent with the data of rebaudioside A described by Tanaka et al. (Tetrahedron Letters, No. 13, 1005'76) was shown. (See Table 5, Figures 2 and 3) As a result, sample No. 3 was obtained by using stevioside and curdlan as substrates and reacting the β-1,3 glucosyltransferase crude enzyme preparation. teeth,
It was confirmed to be rebaudioside A.
The melting point of this sample No. 3 was measured and was 237-239℃.
It was hot.

【table】

【table】

[Table] Comparative Example 2 Instead of the crude enzyme preparation [] of Example 2, 0.1% concentration of Driselase (Kyowa Hakko Co., Ltd. product) was used at 50°C.
When the same operation was performed except that the reaction was carried out for 2 and a half hours, it was confirmed that stevioside decreased by 5 g (50%) and rebaudioside A 0.39 g (increased by 3.9%) was produced. The molar conversion of this reaction was 6.5%. Example 3 Medium 5 having the same medium composition as Example 1(1)
was inoculated with Streptomyces sp. DIC-146 (Feikoken Bibori No. 6594), and cultured with aeration at 37° C. for 40 hours in the same manner as in Example 1. The obtained culture solution was centrifuged, and the supernatant was salted out with 0.8 saturated ammonium sulfate to obtain a crude enzyme preparation having β-1,3 glycosyltransferase activity. The enzyme activity at this time was approximately 910 units. Stevia DIC (contains 51% stevioside, 14.5% rebaudioside A) 10g, curdlan 10
g. 9.1 units of the above-prepared enzyme [] were suspended in 1 liter of 0.1M phosphate buffer (PH7.0) and reacted at 50°C for 2 hours. It was purified with an adsorption resin and dried in the same manner as in Example 1 (1), and as a result of analysis by high performance liquid chromatography, 11.5 g of a white solid containing 32% stevioside and 26% rebaudioside A was obtained. . The molar conversion rate from stevioside to rebaudioside A at this time was about 94%. Example 4 Stevia - 500 ml of aqueous extract of stevia leaves (containing about 74% steviosides and 14.5% rebaudioside A) and curdlan 100 instead of DIC
g was added to 0.1 M phosphate buffer (PH7.0) to make a total volume of 5.0. 50 units of the crude enzyme preparation prepared in Example 1-(1) were added thereto, and the mixture was stirred at 40°C for 4 hours to react. After filtering the reaction solution, incubate at 90℃ for 10
The enzyme was inactivated by heat treatment for 1 minute. This solution is mixed with ion exchange resin Eyebarite IR-120B and
The rebaudioside A content in the steviosides obtained by removing impurities from the stevia leaf extract through IRA-93 is 25%, which is higher than the rebaudioside A in the original stevia leaf extract. It was approximately 1.7 times larger.

[Brief explanation of the drawing]

In the drawings, Figure 1 shows rebaudioside A.
and the thin layer chromatogram of sample 3, and the second
Figure 3 shows rebaudioside A and sample 3.
^13C —NMR chart is shown.

Claims (1)

[Claims] 1. Stevioside and β-glucosyl sugar compound belonging to the genus Streptomyces are added to the aqueous solution or suspension containing stevioside and β-1,3 glucosyl sugar compound.
β- between 1,3 glucosyl sugar compounds
The above-mentioned β isolated from a culture solution, cells, processed product of microorganisms, or microorganisms that produce an enzyme having 1,3 glucosyl transfer activity and have the ability to convert stevioside to rebaudioside A.
-A method for producing rebaudioside A, which comprises using an enzyme having 1,3 glucosyl transfer activity.