JPH0679547B2 - Stevia sweetener and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to rebaudioside A among stevia sweeteners.
(1) Stevioside, rebaudioside C, rebaudioside D, rebaudioside E, which is obtained by selectively decomposing a sweet component other than stevioside having a strong bitterness or residual taste using a microorganism. The present invention relates to a novel stevia sweetener having excellent taste and containing dulcoside A, a component obtained by selectively degrading steviol biocide with a microorganism, (2) rebaudioside A, and (3) stevioside, and a method for producing the same.

[Conventional technology]

In recent years, artificial sweeteners such as sodium cyclamate, sodium saccharinate, and zultin have come to be banned or restricted in general foods from the viewpoint of safety, while on the other hand, excessive intake of sugar may lead to health problems. Due to the fact that adverse effects have become a problem, the development of alternative natural sweeteners has been eagerly awaited. Under such circumstances, stevioside and rebaudioside A are low-calorie sweeteners unlike sugar, and the sweetness ratio is about 300 times and 360 times higher than sugar,
The demand is increasing rapidly. Stevioside and rebaudioside A are Stevia rebaudiana BERTON belonging to the Asteraceae family.
It is a sweet component extracted from I) and is a β-glucosyl glycoside having steviol as an aglycone. A mixture of stevioside and rebaudioside A is currently used for sweetening foods and drinks. Of these, rebaudioside A is superior in sweetness to stevioside, but its content is low. The sweetness of stevioside appears later than that of sugar, and it has the drawbacks of bitterness and dislike, in addition to the drawback that it remains as a residual taste for a long time. It is said that there is a limit to the amount and use, and it is necessary to improve the sweetness quality.

[Means for solving problems]

The present inventors have conducted extensive studies to solve the above-mentioned drawbacks in stevia sweeteners, and as a result, stevioside, rebaudioside C, rebaudioside D, rebaudioside E, zulcoside A, steviol biocide ( Hereinafter, stevia sweeteners other than rebaudioside A are referred to as “steabia sweeteners”, and among them, the taste quality is obtained by selectively decomposing stevioside, which has a particularly large amount of bitterness, dislike, residual taste, etc., using a biological method. It was found that the above-mentioned excellent sweetener containing rebaudioside A as a main component was obtained, and the present invention was completed. That is, the present invention is (1) a component obtained by selectively degrading stevioside, rebaudioside C, rebaudioside D, rebaudioside E, zulcocide A, steviol biocide with a microorganism,
(2) Rebaudioside A, and (3) Stevia sweetener containing Stevioside, and an aqueous solution containing a Stevia sweetener component, without decomposing Rebaudioside A, Stevioside, Rebaudioside C, It is intended to provide a method for producing a stevia sweetener, which comprises reacting a microorganism having an ability to selectively decompose rebaudioside D, rebaudioside E, dulcoside A, and steviol biocide.

In the prior art, the sweeteners of the present invention, such as the sweeteners extracted from stevia, other than rebaudioside A, are not known at all for the sweeteners obtained by degrading stevioside. However, a method for decomposing stevioside with an enzyme (hesperidinase) and quantifying it is known (Pharmaceutical Journal 1975, Vol95, 1507-1511).
This hesperidinase differs from the present invention in that it decomposes all stevia sweetening components such as stevioside and rebaudioside A.

〔Constitution〕

The aqueous solution containing the stevia sweetener component used in the production of the sweetener of the present invention, that is, the raw material is limited to an aqueous solution of a highly refined or roughly refined stevia sweetener (mainly consisting of rebaudioside A and stevioside) product. Alternatively, any solution may be used during or after extraction from Stevia leaves, and any solution containing rebaudioside A and other Stevia sweetening components may be used.
A raw material having a high content of rebaudioside A is preferable, and a solution during extraction is more preferable in terms of production efficiency.

Therefore, the aqueous solution containing the stevia sweetening ingredient of the present invention is an aqueous solution or extract containing rebaudioside A, rebaudioside C (dulcoside B), D, E, zulcoside A, steviol biocide, stevioside. is there. If necessary, the aqueous solution may contain an organic solution such as ethanol.

The microorganism used in the present invention may be any microorganism other than rebaudioside A as long as it has the ability to selectively decompose stevia sweetening ingredients, but preferably in an aqueous solution containing stevia sweetening ingredients. Any substance can be used as long as it selectively decomposes only stevioside and does not use rebaudioside A as a reaction substrate. Furthermore, these microorganisms have the ability to cleave one or two or three glucoses bound to stevia sweetening ingredients other than rebaudioside A, such as stevioside, and especially sophorose of stevioside. Those having an ability (glucosidase activity) of degrading terminal glucose of a part or releasing all or one part of glucose bound to aglycone to produce steviol or a steviol-based degradation product are preferable. Stevia sweetness ingredients other than rebaudioside A as used herein include stevioside, rebaudioside C, D, E,
Zulcoside A, Steviol Bioside. Among these sweet components, it is preferable that rebaudioside D is not decomposed from the viewpoint of taste.

Further, as the microorganism used in the present invention, for example, mold, actinomycetes, bacteria, yeast, basidiomycetes, Trichoderma spp., Rhizopus spp., Caetomium spp., Myriococcum spp., Thermomyces spp., Aspergillus spp., Penicillium spp., Milo spp. Various fungi and Streptomyces, Micropolyspora, and various actinomycetes belonging to the genus Microosporia, such as genus Cescium, genus Mucor, genus Sborotricuum, genus Sclerotinia, genus Rhizoctonia, genus Sclerotium, and genus Pelicularia And various bacteria belonging to the genus Bacillus, the genus Previbacterium, the genus Arthrobacter, the genus Flavobacterium, and the yeast of the genus Schizosaccharomyces, and various basidiomycetes belonging to the genus Trametes, the genus Ilpex, the genus Cobrinas, etc. Is mentioned. Particularly, those belonging to the genus Flavobacterium, the genus Bacillus, the genus Streptomyces, the genus Aspergillus, etc. are excellent. The broad distribution of microorganisms that can be used in the present invention is shown in the examples table.
This is as seen in the results shown in 1.

Particularly preferable examples of the Aspergillus strain include Aspergillus nigar ATCC-6275 and Aspergillus niger IFO-6341.

In the reaction of the present invention, the microorganism may be allowed to act in an aqueous solution containing a stevia sweetening ingredient to select conditions for degrading the stevia sweetening ingredient other than rebaudioside A, but the reaction temperature is 15 ° C to 60 ° C. The temperature is preferably 25 ° C. to 50 ° C., the pH is pH 2.0 to 9.0, preferably 4.0 to 7.0, and the reaction time is about 30 minutes to 3 days, preferably 1 to 12 hours.

The concentration of stevia sweetener used in the reaction is about 0.1 to about 1
0 wt% is suitable.

The stevia sweetener containing rebaudioside A as a main component obtained by this reaction is a sweetener containing more than 5.5 parts by weight of rebaudioside A with respect to 1 part by weight of stevioside. Therefore, when the solid content ratio of each component in the sweetener of the present invention is expressed in parts by weight, 1 part by weight of stevioside is rebaudioside A5.5 (excluding 5.5) to 70 parts by weight.
Parts by weight, decomposed products of sweet components other than rebaudioside A
Those having a ratio of 0.01 to 30 parts by weight are preferable.

The sweetener of the present invention may be derived from Stevia sweetener components such as rebaudioside C, D, in addition to the above-mentioned components.
Other sweetening ingredients such as E and Zulcoside A may be contained. The amount is 0.001 to 10 parts by weight in terms of solid content.

Decomposition products of stevia sweetening components other than rebaudioside A which is the first component of the present invention include, for example, stevioside,
Rebaudioside C, D, E, Zulcoside A,
From the same B, steviol biocide etc. glucose 1
5 or Rhamnose is eliminated, and 1 to 4 glucose or Rhamnose-added steviol glycosides such as bubsoside, steviol monoside, steviol biocide, and elimination of all of these sugars. Called Steviol. In addition, many of these decomposition products are derived from stevioside.

The composition of these decomposition products varies depending on the reaction conditions and the microorganisms used, but steviol may be produced when the decomposition reaction is carried out for a sufficient time. Furthermore, these decomposed products are often substances with low sweetness per se or with no taste and odor, and not only do not become causative substances such as disagreeable taste, residual taste, and astringency among the obtained sweeteners,
There is a feeling that the taste quality of rebaudioside A is improved by the presence of these substances. However, when it is required to increase the purity of the main component, these decomposed products can be removed after the reaction by a column purification method or a crystallization method using an ion exchange resin, an adsorption resin or the like described later.

The sweetener of the present invention can be used as it is as a sweetener. In addition, if necessary, after inactivating the microbial cells by heating, purification, for example, a polymerization adsorption resin of styrene and divinylbenzene, such as Diaion HP-20 (trade name, manufactured by Mitsubishi Kasei Co.), Amberlite XAD-2 (commodity) Name, a product of Organo Co., Ltd.) or an ion exchange resin (for example, an H-type strongly acidic ion exchange resin and an OH-type weakly basic ion exchange resin) is used for desalting and then concentrated to obtain a sweetener on syrup. Or
Alternatively, it can be dried and powdered to obtain a sweetener on the powder.

Further, the desalted reaction solution was purified to obtain rebaudioside A.
Can also be separated and collected as a sweetener. At this time, known methods such as concentration under reduced pressure, membrane concentration, vacuum drying, and spray drying can be freely used for concentration, drying, and pulverization. The sweetener of the present invention containing rebaudioside A as a main component thus obtained has a lower sweetness than that of the raw material before the reaction, but the quality of the sweetness is bitterness derived from stevioside. It has no harshness such as astringency or astringency, has a mellow sweetness closer to that of pure Rebaudioside A, is similar to sugar, and has a good residual taste.

The sweetener of the present invention is an odorless, white powder that has no bitterness, odor, taste and taste, and is soluble in water. Therefore, the coexistence ratio of stevioside and glycyrrhizin is arbitrary, and the coexistence is arbitrary under liquid or powdery conditions. Can be made. Further, the sweetener of the present invention can be effectively used in its taste characteristics by sharing it with well-known synthetic sweeteners such as saccharin and salts thereof, sodium cyclamate, dihydrochalcone, and asparatame. One or two synthetic sweeteners
By adding and using the sweetener of the present invention in an amount of more than one species, it is possible to improve the unpleasant taste such as bitterness and dislike of the synthetic sweetener.

Further, the sweetener of the present invention can be added to known sugar sweeteners such as sugar, fructose, glucose, lactose, starch syrup, dextrin and starch which are used as excipients, diluents and adsorbents, and can be used conventionally. It is possible to significantly reduce the amount of use than the amount of use of. Furthermore, when the sweetener of the present invention is used by adding it to a low-calorie sweetening substance having a sweetness lower than that of sugar such as sorbitol, maltitol, mannitol, xylitol, etc., it is possible to enhance the sweetness without impairing the advantages of the sweetening substance, and it is of high quality. A low calorie sweetener is obtained.

It goes without saying that the sweetener of the present invention can be used as a sweetening source for general foods and diet foods, medicines, quasi drugs, cigarettes, feeds and the like.

For example, soy sauce, powdered soy sauce, miso, powdered miso, moromi, mayonnaise, dressing, vinegar, three tablespoons vinegar, powdered sushi vinegar, Chinese cabbage, tempura soup, noodle soup, sauce, ketchup, grilled meat sauce, curry roux, stew sauce, soup base , Dashi, various seasonings, mirin, new mirin, various kinds of seasonings such as table syrup, rice crackers, hail, rice cake, steamed buns, manju, uiro, bean paste, sheep can,
A variety of Japanese sweets such as water sheep, jelly, castella, candy, bread, biscuits, crackers, cookies, pies, pudding, butter cream, custard cream, cream puffs, waffles, sponge cakes, donuts, chocolate, chewing gum, caramel, candy, etc. Western confectionery, ice cream, sorbet, ice candy such as popsicle, syrup pickling of fruits, syrup such as watertight, flower paste, peanut paste, paste such as fruit paste, jam, marmalade, syrup pickling, fruits and vegetables such as confectionery Processed foods, pickles such as Fukujin pickles, Senmai pickles, and Rakkyo pickles, meat products such as ham and sausages, meat hams, fish meat products such as meat hams, fish sausages, kamaboko, chikuwa, and tempura, sea urchin, salted squid, sardines, Blowfish only Boiled vegetables such as dried bonito, seaweed, wild vegetables, sardines, small fish, shellfish, etc., boiled beans, potato salad, kelp rolls, etc., fish meat, meat, fruits, vegetables bottled, Alcoholic beverages such as canned foods, synthetic liquor, fruit liquor, western liquor, coffee, cocoa, juice, carbonated drink, lactic acid beverage, soft drink such as lactic acid bacterium beverage, pudding mix, hot cake mix, instant juice, instant coffee, instant sirko, etc. It can be used for sweetening various foods and drinks such as instant foods and drinks and tastes. Other pharmaceuticals and quasi-drugs include toothpaste, lipstick, lip balm, internal medicine, troches, liver oil drops, mouthwash, mouthwash,
It can also be used freely as a sweetener for mouthwashes and the like.

〔effect〕

Since the sweetener of the present invention contains a large amount of rebaudioside A, it has the same excellent sweetness as rebaudioside A pure product. Its sweetness is more rebaudioside in terms of astringency, residualness, bitterness, and aversion even when compared with the conventional sweetener containing less than 550 parts of revidioside A against 100 parts of stevioside. It is an excellent sweetener having a taste quality close to A.

Hereinafter, the method of the present invention and the sweetener obtained by the method will be specifically described with reference to Examples.
Is based on weight.

Example 1 The microorganisms shown in Table 1 were examined for the presence or absence of stevioside degrading activity and its selectivity. The results are shown in Table-1. The microorganisms shown in Table 1 were grown in a medium containing a stevia sweetening ingredient, or a culture solution of the microorganism was added to an aqueous solution containing a stevia sweetening ingredient and reacted to determine by liquid chromatography.

Example 2 Phosphate-potassium 0.4%, ammonium sulfate 0.5%, magnesium sulfate 0.06%, yeast extract 0.1%, zinc sulfate 0.001
%, Ferrous sulfate 0.005%, (hereinafter referred to as Medium A) basal medium (pH 6.0) containing 1% glucose 100
ml to a 500 ml Sakaguchi flask, and then add 1 g of purified stevia sweetener (composition: stevioside 20%, rebaudioside A41.6%, rebaudioside C8.5%) to dissolve and add to autoclave. And sterilized at 120 ° C for 15 minutes.
After cooling, one platinum loop of Aspergillus niger ATCC6275 was inoculated and shake-cultured at 34 ° C. for 44 hours to carry out a decomposition reaction.

After the reaction, after filtering through Buchner, the obtained supernatant was passed through adsorption resin Diaion HP-20 (manufactured by Mitsubishi Kasei) at SV = 2 to adsorb steviosides. After that, desorption with 95% ethanol, the desorbed ethanol was distilled off under reduced pressure, and then the strongly acidic ion exchange resin Amberlite IR-120B (H
Type, trade name, Rohm and Haas Company), weak basic ion exchange resin Amberlite IRA-93 (OH type, trade name, Rohm and Haas Company) at SV = 2 for desalting. Then, this was concentrated under reduced pressure at 70 ° C or less and dried in vacuum. As a result, HPLC analysis revealed that rebaudioside was found to be a sweet component.
A stevia sweetener having a composition of A40.0%, rebaudioside C 7.2%, stevioside 0.5% or less, and a decomposed product of sweet components other than rebaudioside A 20.2% was obtained.

Example 3 The same basal medium composition (Medium A) as in Example 2 plus 1% glucose was placed in a 3.510-volume jar fermenter and sterilized at 110 ° C. for 30 minutes. After cooling, 0.2 seed culture of Aspergillus niger IFO6341 cultivated in the same medium composition was added and cultivated at 32 ° C. for 30 hours. At the start of the stationary phase, another sterilized refined stevia sweetener (composition: stevioside 21.2%, rebaudioside A55.1
%, Rebaudioside C 6.3%) solution (40g / 0.3)
And aeration rate 4 / min, stirring 400 rpm, temperature 14 ℃ 14
A time-resolved reaction was performed. The reaction solution was purified by the same method as in Example 2. The obtained stevia sweetener was analyzed by HPLC to find that rebaudioside A was 53.3%, rebaudioside C was 5.8%, stevioside was 1.2%, and decomposed products of sweetening components other than rebaudioside A were 22.2%. It was a sweetener of the composition.

Example 4 3.5 volumes of medium containing 1% lactose added to the same basic medium composition (Medium A) as in Example 2 were placed in a 10 jar fermentor and sterilized at 110 ° C. for 30 minutes. After cooling, 0.2 seed culture of Aspergillus niger ATCC6275 cultivated in the same medium composition was added and cultivated at 32 ° C. for 40 hours. Then, 0.3 of Stevia leaf extract (composition: stevioside 9.9%, rebaudioside A 3.8%, rebaudioside C 1.6%) was added in the same manner as in Example 3, and the same as Example 3. The decomposition reaction was carried out for 30 hours under the conditions. After the reaction, Example 2
It refine | purified by the method similar to and dried powder was obtained. The resulting sweeteners are rebaudioside A 44.4%, rebaudioside C 14.3%, stevioside 5.2%, steviol 1.8%.
The composition was

Example 5 (Sweetness Test of Sweetener) The stevia sweetener obtained in Example 2 was used as Sample No. 1 (Stevioside (Stv) 20%, Revasdioside A (RebA) 4).
Purified stevia sweetener before reaction with 1.6% aqueous solution), No.2
(RebA 40%, Stv 0.5% or less of the present invention sweetener) 0.02% and 0.05% aqueous solution was prepared, and a 1-7% aqueous solution of sugar was adjusted to 0.
13 kinds of standard solutions were prepared at the 5% concentration stage, and the sweetness test was performed on these. The test is a two-point comparison method of the sample solution and the standard solution, and is carried out by 20 panel members at room temperature of 20 ° C., and the results are shown in Table 2.

From the results of (a) and (b) in Table-2, the sweetness of sample No. 1 corresponds to a sugar concentration of 4.5% (sweetness 225 times) in a 0.02% aqueous solution, and a sugar concentration of 9.0% in a 0.05% aqueous solution ( The sweetness is 180 times). Similarly, the sweetness of sample No. 2 is 4.
Since the sweetener of the present invention corresponds to 0% and 8.5%, the sweetness of the present invention is judged to be slightly lower than the sweetness corresponding to the raw material used.

Example 6 (Taste test of sweetener) In the same manner as in Example 5, the difference in sweetness quality was compared between the control product of Sample No. 1 and the sweetener of the present invention of Sample No. 2.
Calculated from the sweetness obtained in the sweetness test, each sample was adjusted to an aqueous solution having a sweetness corresponding to a 3%, 6%, or 10% sugar aqueous solution. Then, the quality of the taste of the sample solutions of sample No. 1 and sample No. 2 was compared at each sweetness level.

The test was conducted at room temperature of 20 ° C. by 20 panel members. The results are shown in Table-3.

From the results in Table 3, it is clear that the sweetness of the sweetener of the present invention of Sample No. 2 is superior to that of the control product of Sample No. 1 at any sweetness.

Application Example 1 A powdered juice was prototyped with the following formulation.

Granulated sugar 950 g Citric acid 30 g Sodium citrate 20 g Malic acid 14 g Flavor 10 g β-carotene (1.5%) 3 g The sweetener of the present invention 2.0 g As a comparative example, instead of the sweetener of the present invention in the above-mentioned formulation, A sample was prepared by adding 2.0 g of sample No. 1. As a result of a taste test conducted by 20 panel members, the product of the present invention had no bitterness or dislike, and the comparative product had a bitter aftertaste.

Application Example 2 A 20% fruit juice orange juice was prototyped with the following formulation.

Orange 100% Natural fruit juice 430g White sugar 10g High fructose isomerized sugar 210g Citric acid 3.5g Malic acid 2g Sodium citrate 0.5g Flavor 2g β-carotene (1.5%) 0.5g Sweetener of the present invention 0.5g Total amount with water 2 In addition, as a comparative example, a sample was prepared by adding 0.5 g of sample No. 1 of Example 5 in place of the sweetener of the present invention in the above formulation.
As a result of a taste test by 20 panel members, the applied product of the present invention had a mild sweetness and flavor, and the comparative product had an unpleasant aftertaste.

Application Example 3 A cider was prototyped with the following composition.

Granulated sugar 30g Isomerized sugar 170g Citric acid 3g Na citrate 0.2g Cider essence 0.2g Sweetener of the present invention 0.22g Make whole 2 with carbonated water Also, as a comparative example, it was carried out in place of the sweetener of the present invention in the above formulation. A sample was prepared by adding 0.22 g of the sample No. 1 of Example 5. As a result of a taste test conducted by a panel of 20 people, the product of the present invention applied had a light sweetness and had a good residual taste, and the comparative product had a bitterness and astringency.

Claims (3)

[Claims] (1) Stevioside, rebaudioside C, rebaudioside D, rebaudioside E,
Stevia sweetener containing dulcoside A, a component obtained by selectively degrading steviol biocide with a microorganism, (2) rebaudioside A, and (3) stevioside. 2. The stevia sweetener according to claim 1, which comprises more than 5.5 parts by weight of rebaudioside A per 1 part by weight of stevioside. 3. Rebaudioside A is not decomposed in an aqueous solution containing a stevia sweetening ingredient, and Stevioside, Rebaudioside C, Rebaudioside D, Rebaudioside E, Zulcoside A, Steviol Bioside. A method for producing a stevia sweetener, which comprises allowing a microorganism having an ability of selectively decomposing bacterium to act on the stevia sweetener.