JP2898688B2 - Highly sweetened sugar-added stevia sweetener and process for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a stevia natural sweetener, and particularly to a technique for obtaining a sweetener having a better taste by improving the production method of an α-glucosyl stevia extract. It is a thing.

[Conventional technology]

To improve the taste of sweets consisting of stevia extract, a method of increasing the content ratio of rebaudioside A, which is a good sweetness component (Japanese Patent Publication No. 58-16863), and stevia extract with α-glucosyl glycosyltransferase (cyclohexyl). A method of adding glucose by using destringed lucanotransferase) has been proposed and implemented. The purpose of α-glucosylation by cyclodextrin ganotransferase is to improve the sweetness of stevioside, which has poor sweetness. Therefore, in the actual manufacturing process, in order to reduce the remaining stevioside as much as possible, a heat-producing enzyme that produces Bacillus stearothermotherm with high glycosyl transfer efficiency,
The reaction is carried out at a reaction temperature of 60°C or higher and a reaction time of 24 hours or longer. Also, in Japanese Examined Patent Publication No. 57-18779, the reaction temperature is 60° C. and the reaction time is 4
The α-glucosyl stevia extract is produced in 0 hours.

[Problems to be Solved by the Invention and Means for Solving the Problems]

As a result of examining the reaction conditions for α-glucosylation, the present inventors have found that the reaction temperature is 47°C to 53°C, the reaction time is 6 to 8 hours,
It was found that α-glucose was preferentially transferred to the 13th position of stevioside, although the residual stevioside was large when the amount of added enzyme was 5 to 8 unit/g (stevia extract). Therefore, it was found that the sweetener obtained by separating the remaining stevioside by subjecting it to resin treatment under specific conditions and then treating it with β-amylase has a much better taste, and completed the present invention. It was

The present invention suppresses the production of low-sweetness α-glucosylated stevia extract, increases the content ratio of good-sweetness α-glucosylated stevia extract, and suppresses the residual of poor sweetness stevioside, It is intended to provide a novel sweetener having no bitterness and astringency, having a sharp, mild, sweet aftertaste and having a high degree of sweetness.

According to a recent report on α-glucosylated stevia extract, among α-glucosyl stevioside, α-
Monoglucosyl stevioside and α-diglucosyl stevioside have the best sweetness and sweetness multiple.
In addition, stevioside structurally serves as a cyclodextrin glucanotransferase acceptor at the 13th and 19th positions.
Since it has glucose, α-mono and diglucosyl stevioside are a mixture of two kinds in the case of α-monoglucosyl stevioside and three kinds in the case of α-diglucosyl stevioside as shown below.

Furthermore, of these, only 13-position was glycosylated 13-α-
The sweetness of monoglucosyl stevioside (G1-a) and 13-α-diglucosyl stevioside (G2-a) is the highest among the related compounds. Also, the sweetness multiple is 180 times or more, which is extremely high. On the contrary, the sweetness qualities of G1-b, G2-b, and G2-c are not so good, and the sweetness multiple is also low. (Reference Y.Fkunaga, et a
l., Agric.Biol.Chem.,53(2),1603,1989) Therefore, in order to produce a good-sweetness α-glucosylated stevia extract, α of stevioside, which is the main component, is used.
-It is effective to increase the content rate of 13-α-mono or diglucosyl stevioside among the glucosylated compounds.

First, paying attention to the reaction time, the reaction time of the α-glucosylation reaction and 2 of the α-glucosylated stevia extract produced
The relationship up to the molecular transition was investigated.

Figure 1-1 shows residual (unreacted) stevioside (ST), α
FIG. 2 shows changes in the content rates of monoglucosyl stevioside (G1) and α-diglucosyl stevioside (G2) at each reaction time.

The reaction conditions are 1 g of Stevia extract (75% stevioside,
Rebaudioside A 25%) and dextrin (Sandic #70, manufactured by Sanwa Starch Co., Ltd.) were added at an enzyme amount of 5 unit, a reaction time of 0 to 20 hours, and a reaction temperature of 50°C.

Similarly, FIG. 1-2 shows G1-a, G1-b in G1 and G2- in G2.
It shows changes in the reaction time of a, G2-b and G2-c. As shown in FIG. 1-2, the content of each α-glucosyl stevioside changes with the reaction time. The production of G1-a and G2-a with good sweetness is maximum when the reaction time is 4 to 8 hours, and gradually decreases thereafter. On the contrary, the components such as G1-b, G2-b and G2-c, which have poor sweetness, increase with the reaction time. The content of residual stevioside (ST) is 20%
If it is below, it can be removed by the resin generation described later. As shown in FIG. 1-1, the reaction time is required to be 6 hours or longer because the residual stevioside becomes 20% or less when the reaction time is 6 hours or longer. Also, the production of α-monoglucosyl stevioside (G1) increases rapidly up to 6 hours and then gradually decreases. G2 increases rapidly up to 8 hours and then gradually increases. Therefore, the residual stevioside content is 20% or less, and the content of G1 and G2 is high, and G1-a and G2-a
It was found that the reaction time with a high content ratio of is 6 to 8 hours.

Next, the relationship between the amount of added enzyme and α-glucosyl stevioside was investigated. The reaction conditions were such that the reaction time was 6 hours, the amount of added enzyme was changed in the range of 1.25 to 20 units, and the other conditions were the same as in FIG. As a result, as shown in Fig. 2-1, the production ratio of G1-a and G2-a with good sweetness was 5 to 8 units of added enzyme amount.
It becomes the maximum at, and gradually decreases with the amount of added enzyme beyond that. On the other hand, the contents of G1-b, G2-b, and c with poor sweetness also increase as the amount of added enzyme increases. Also, the G1 and G2 content ratios reached a maximum near 5 units, and no change was observed thereafter. Stevioside sharply decreased until the amount of added enzyme was 10 units, and gradually decreased thereafter. 20% remaining Stevioside
Since the following is desirable, the amount of added enzyme must be 5 units or more. Therefore, it is appropriate to add 5 to 8 units of enzyme.

Furthermore, the relationship between the reaction temperature and the composition ratio of α-glucosyl stevioside was examined. The reaction conditions were such that the reaction time was 6 hours, the other conditions were the same as in FIG. 1, and the reaction temperature was changed in the range of 40 to 70°C. As shown in Fig. 3-1, the residual stevioside decreases with increasing temperature, and G1 and G2 slightly increase with increasing temperature. Further, as shown in Fig. 3-2, the production ratio of G1-a and G2-a with good sweetness sharply decreases with increasing temperature. On the other hand, a reaction temperature of 50°C or higher is required to reduce the residual stevioside to 20% or less. Therefore, a suitable reaction temperature is around 50°C.

From the above results, 13-α-mono, containing a high content of diglucosyl stevioside, and remaining stevioside 20%
It was found that the following reaction conditions were a reaction temperature of 47°C to 53°C, a reaction time of 6 to 8 hours, and an added enzyme amount of 5 to 8 units.

The product thus obtained is 13-α-monoglucosyl stevioside is 70% or more in α-monoglucosyl stevioside, and 13-α-diglucosyl stevioside is 60 in α-diglucosyl stevioside.
It was found that the sugar addition product of stevia extract, which is more than 80%, is more than 80%, and the remaining stevia extract is less than 20%.

This finding was further confirmed by experiments. That is, the reaction temperature
The reaction liquid 1 was prepared by carrying out the reaction under the conditions of 50° C., reaction time 6 hours, and amount of added enzyme 5 unit, and the composition of contained components was measured.

As a result of the measurement, the content rate of G1-a and G2-a with good sweetness is G1-
It was higher than that of b, G2-b and G2-c, and favorable results were obtained for improving the sweetness of the composition ratio of G1 and G2. However, at this stage, the residual stevioside content was 18.8%, 3
It is not preferable for improving sweetness that the content of α-glucosyl stevioside in which glucose is transferred more than a molecule is 38.9% and a large amount of these components with poor sweetness remain. Therefore, the present inventors examined a method of removing residual stevioside by resin treatment and treating 3 or more molecules of α-glucosyl stevioside by amylase treatment.

Usually, separation of stevioside and α-glucosyl stevioside can be performed by a method such as liquid chromatography or column chromatography, but it cannot be industrially applied. Therefore, at present, there is no method for separating stevioside and α-glucosyl stevioside on an industrial level. Therefore, the present inventors have tried separation with a resin.
As a result of investigating various resins and elution conditions, it is easy to produce residual stevioside and α-glucosyl stevioside by elution at a specified methanol concentration of 34%-36% using XAD-7 (Organo Co., Ltd.) resin. I found that it can be separated into. After adsorbing the reaction mixture after α-glucosylation on XAD-7 resin as shown in FIG. 4, elution was carried out in the order of water, 35% methanol, and 50% methanol. As a result of analyzing each elution fraction by TLC (thin layer chromatography) (Fig. 6), unreacted dextrin was eluted by water elution,
Α-Glucosyl stevioside was eluted with 35% methanol, and residual stevioside was eluted with 50% methanol. Similar results were obtained with methanol in the range of 34%-36%, but the separation of residual stevioside and α-glucosylstevioside became unclear in methanol with a concentration of 33% or less and 37% or more.

Next, a method for treating three or more molecules of α-glucosyl stevioside was examined. To treat three or more molecules of α-glucosyl stevioside, it is effective to cleave the glucose chain with α-1,4-glucosidase. α-
1,4-Glucosidase includes α-amylase that randomly cleaves sugar chains, β-amylase that cleaves maltose units from the non-reducing end, and glucoamylase that cleaves glucose units from the non-reducing end. The inventors used β-amylase for the purpose of preferentially obtaining α-mono and diglucosyl stevioside.

Dissolve the XAD-7 resin 35% methanol elution fraction in water,
β-amylase treatment was repeated to obtain a reaction solution 2. As shown in Table 1, in the reaction solution 2, the residual stevioside was significantly reduced as compared with the reaction solution 1, and at the same time, 3 or more molecules of α-
The content of glucosyl stevioside has also decreased significantly. On the other hand, the content of α-mono and diglucosyl stevioside is increased, and the ratio of 13-α-monoglucosyl stevioside in α-monoglucosyl stevioside and 13-α-diglucosyl stevioside in α-diglucosyl stevioside is 76.3 respectively. %, 62.4%, which was a high value.

As shown in Table 1 above, the content of residual stevioside, α-mono, and diglucosyl stevioside, 13-α-mono, by control of reaction conditions and resin treatment under specific conditions, and further by cleavage reaction of sugar chain by β-amylase. , The best sugar adduct was obtained in which the problems in the diglucosyl stevioside (G1-a, G2-a) ratio were solved.

Since the content of stevioside in the total amount of steviol glycosides in Stevia extract (Sanvia Kokusaku Pulp Co., Ltd. Stevia extract) is approximately 75% (rebaudioside A 25%), the sweetness of α-glucosyl stevioside is improved. Is thought to improve the sweetness of stevia extract. In addition, rebaudioside A 25%, other trace glycoside components α
-Glucosylation is expected to behave similarly to α-glucosyl stevioside.

A sweetener was prepared under the reaction liquid 2 (product of the present invention) production conditions,
Sensory tests were conducted by panelists. As a result, both the sweetness quality and the sweetness multiple were improved as compared with the comparative product (manufactured by SK Sweet Z Sanyo Kokusaku Pulp Co., Ltd.). In terms of taste quality, sharpness increased, sweetness started, and post-trendability tended to improve, and favorable results were obtained.

The Stevia extract of the present invention is obtained by extracting non-sweetening components from a Stevia leaf portion by a conventional method using water or alcohol. The α-glucosylated stevia extract is a stevia extract glucosylated by allowing, for example, cyclodextrin glucanotransferase to act on an aqueous solution containing the stevia extract and the α-glucosyl sugar compound (eg, dextrin). In addition, α-glucosyl stevioside is an aqueous solution containing stevioside and an α-glucosyl sugar compound (eg, dextrin), which is glycosylated by reacting, for example, cyclodextrin glucanotransferase.

Further, α-monoglucosyl stevioside is one in which D-glucose is transferred to stevioside with one molecule α-1,4 bond, and α-diglucosyl stevioside is two molecules with D-glucose transferred to stevioside with α-1,4 bond. It was done. 13-α-monoglucosyl stevioside is D-glucose transferred by α-1,4 bond to the terminal glucose of Sophoreth at position 13 of stevioside. 13-α-diglucosyl stevioside is a compound in which D-glucose is transferred by two molecules α-1,4 bond to the terminal glucose of Sophoreth at the 13th position of stevioside.

〔Example〕

Hereinafter, the present invention will be described in detail by experiments and examples, but the present invention is not limited thereto.

Experiment 1 Production of reaction liquids 1 and 2 1 g of Stevia extract (purified by crystallization of Steviafin H manufactured by Sanyo Kokusaku Pulp Co., Ltd.) and dextrin of DE:7 as an α-glucosyl sugar compound (Sanwa Starch Co., Ltd.) (Sandic #70), 2 g, dissolved in 10 ml of water, added with 1 mol of acetate buffer-200μ and 5 units of cyclodextrin glucanotransferase (EC24,1,19) and incubated at 50°C for 6 hours to react. went. This reaction solution
The mixture was kept at 95°C for 30 minutes to inactivate the enzyme by heating to obtain a reaction solution 1. Reaction liquid 1 is an adsorption resin (XAD-7 manufactured by Organo Corporation)
Was adsorbed from the aqueous solution and eluted with water, 35% methanol, and 50% methanol in this order. The 35% methanol fraction containing α-glucosyl stevioside was evaporated to dryness. 600 mg of this solid content was melted in 10 ml of water, 12.5 mg of β-amylase (manufactured by Nagase Co., Ltd.) was added, and the reaction was carried out by incubating at 50° C. for 6 hours. This reaction solution was kept at 95°C for 30 minutes to inactivate the enzyme by heating to obtain a reaction solution 2.

Quantitative method of sweetness ingredient The ingredient composition of the product of the present invention was quantified using an external standard method. The measurement was performed using high performance liquid chromatography under the following conditions.

Column TSK-gel Amide80 4 mm×25 cm Eluent CH ₃ CN:H ₂ O 80:20-60:40 Linear gradient Flow rate 1 ml/min Injection amount 5 μ Detection UV 210 nm The chromatogram of reaction solution 1 is shown in FIG. It was confirmed by the respective standards that peaks 1, 2, 3, and 4 in FIG. 4 correspond to stevioside, α-monoglucosyl stevioside, α-diglucosyl stevioside, and α-triglucosyl stevioside, respectively. Peaks 5 and above are also considered to be compounds in which four or more molecules were similarly transferred. Next, the peaks 2 and 3 of the reaction solution 1 are separately collected and analyzed by high performance liquid chromatography under the following conditions.
The quantitative value was calculated by the peak area ratio.

Column TSK-gel ODS-120T 4mm×25cm Eluent 60% Methanol Flow rate 1ml/min Injection amount 5μ Detection UV 210nm Peaks 1, 2, 3, 4 in Figure 5 are G1-a, G1-
b, G2-a, G2-b, G2-c are shown.

Example 100 g of Stevia extract (produced by crystallization and purification of Steviafin H manufactured by Sanyo Kokusaku Pulp Co., Ltd.) and DE:7 dextrin as an α-glucosyl sugar compound (Sandic #70 manufactured by Sanwa Starch Co., Ltd.) Dissolve 200 g in 100 ml of water, add 20 ml of 1 mol acetate buffer, and add 500 units of cyclodextrin glucanotransferase (EC24,1,19) at 50°C.
The reaction was carried out by incubating for 6 hours. This reaction solution was kept at 95°C for 30 minutes to inactivate the enzyme by heating, and then adsorbed from an aqueous solution onto an adsorption resin (XAD-7 manufactured by Organo Corporation), followed by water, 35% methanol, and 50% methanol in this order. It was eluted with. The 35% methanol fraction containing α-glucosyl stevioside was evaporated to dryness. This solid content 75
%-Dissolved in 1000 ml of water, β-amylase (Nagase Co., Ltd.)
125 mg) was added and the reaction was carried out by incubating at 50° C. for 6 hours. The reaction solution was kept at 95° C. for 30 minutes to inactivate the enzyme by heating, and then the reaction solution was filtered. The filtrate is 2400 ml of synthetic adsorbent Diaion HP-20 (manufactured by Mitsubishi Kasei Co., Ltd.).
Adsorbed to a column packed with, first pass water to elute the dextrins, then 90% methanol is passed to elute the α-glucosyl stevia eluate, and the 90% methanol eluate is diluted to 60%. Concentrate and dry under reduced pressure below ℃, pulverize
65 g of powdered sweetener was obtained. This sweetener was analyzed by the method of Experiment 1, and as a result, the transglycosylation purification ratio was 1G-a (73.8%), 1G
-B (26.2%), G2-a (62.0%), G2-b (20.4%), G
It was 2-c (17.6%).

A sensory test was conducted by 20 panel members on the degree of sweetness and sweetness of this sweetener. For comparison, the conventional sugar-added Stevia sweetener SK Suite Z (Sanyo Kokusaku Pulp Co., Ltd.)
Manufactured) was used. The sweetness test was performed using the sweetener of the present invention of 0.05.
% Aqueous solution and 8% to 1% of 0.07% aqueous solution of SK Sweet Z adjusted to have almost the same sweetness by preliminary tests
The strength of sweetness was examined by adjusting the sucrose aqueous solution up to 13% at intervals. The evaluation was made on the basis of the same three grades: strong sweetness and weak sweetness. The results are shown in Table 2 by the number of panelists for each evaluation of the concentration. From the results in Table 2, 0.05% of the product of the present invention
The sweetness of the aqueous solution is about 200 times that of sucrose, which is between 10% and 11%. Similarly, the sweetness of SK Sweet Z is about 150 times. The degree of sweetness per sweetening ingredient is about 1.5 times that of the conventional type SK Sweet Z. In the sweetness test, a 0.05% aqueous solution of the product of the present invention and a 0.07% aqueous solution of SK sweet Z were compared in a three-stage evaluation for bitterness, lack of sweetness, sweetness onset, sweetness sharpness, and overall sweetness,
The results are shown in Table 3 by the number of panelists for each evaluation. Table 3
From the results of the present invention, the product of the present invention has a sweetness rising, sweetness cutting,
Many panelists rank the products of the present invention in a high rank for their comprehensive evaluation of sweetness, which is highly evaluated in terms of sharpness of sweetness.

〔The invention's effect〕

The α-glucosylated stevia extract having good sweetness according to the present invention can be produced by a simple production process such as control of reaction conditions, resin treatment under specific conditions, β-amylase treatment, and also in terms of cost. It has become possible to produce by a method that has no problem in terms of equipment.

[Brief description of drawings]

All of the drawings show examples of the present invention, FIG. 1-1 is a chart showing changes in the composition ratio of ST, G1 and G2 depending on the reaction time, and FIG. 1-2 is G1-a, b and G2- 6 is a chart showing changes in the composition ratio of a, b, and c. Figure 2-1 is a chart showing changes in the composition ratio of ST, G1 and G2 depending on the amount of added enzyme, and Figure 2-2 is G1-a,b according to the amount of added enzyme.
2 is a chart showing changes in the composition ratio of G2-a, b, and c. Figure 3-1 is a chart showing changes in the composition ratio of ST, G1 and G2 depending on reaction temperature, and Figure 3-2 shows the changes in composition ratio of G1-a,b and G2-a,b,c depending on reaction temperature. It is a chart. 4 and 5 are charts showing chromatograms of the reaction solution 1 and the reaction solution 2, respectively. FIG. 6 shows the result of TLC analysis of the porous resin elution fraction. 1 to 3 ST... Remaining stevioside G1 ... α-monoglucosyl stevioside G2 ... α-diglucosyl stevioside 1 to 9 in Fig. 4 and Fig. 5 refer to the respective peaks in the chromatogram analysis.

Claims (2)

(57) [Claims] 1. When a stevia extract sugar adduct is produced by reacting Stevia extract and dextrin with a cyclodextrin glucanotransferase produced by Bacillus stearothermofilm, the amount of added enzyme is 5 to 8 unit/g ( Stevia extract) The reaction time is 6 to 8 hours. The reaction temperature is set to 47°C to 53°C, and the resulting stevia extract sugar adduct is adsorbed on the porous resin and then treated with 34% to 36% methanol to obtain α.
-Eluting the glucosylstevia extract and adding β to the eluate
A method for producing a high-sweetness sugar-added stevia sweetener having a good taste, which comprises adding amylase. 2. An α-glucosylated stevia extract as a main component, wherein the α-glucosylated stevia extract is
A high-sweetness sugar-added stevia sweetener that satisfies all of the above. 1 13-α-Monoglucosyl stevioside 70% or more in α-monoglucosyl stevioside (weight ratio) 2 13-α-Diglucosyl stevioside 60% or more in α-diglucosyl stevioside (weight ratio) 3 Steviol total dividends On the other hand, the remaining stevioside is 5% or less (weight ratio)