Sweetener containing enzymatically modified stevia composition having improved sweetness quality

Abstract

This invention relates to a sweetener containing an enzymatically modified stevia composition having improved sweetness quality, including 90 wt % or more of steviol glycoside, wherein only glycosylation is carried out using cyclodextrin as a glycosylation material of a stevia extract (steviol glycoside), without a purification process using an existing porous adsorbent resin (aromatic, styrene type), to thus produce enzymatically modified stevia, which can be utilized as an ingredient and a reagent for sweeteners, flavor enhancers and flavor modifiers for a variety of confections, drinks (including alcoholic beverages), foods and food products, thereby providing an enzymatically modified stevia sweetener and products thereof.

Claims

1. A sweetener, comprising steviol glycoside having rebaudioside A including non-glycosylated rebaudioside A and glycosylated rebaudioside A, wherein the sweetener satisfies Relation (1) below:
15≥(RAG1+RAG2)/RA≥3.87   (1) wherein RA represents a proportion of the non-glycosylated rebaudioside A in total wt % of the rebaudioside A, RAG1 represents a proportion of the rebaudioside A having one glycosyl group attached thereto, and RAG2 represents a proportion of the rebaudioside A having two glycosyl groups attached thereto; and a proportion of the total rebaudioside A in the total steviol glycoside is 80 wt % or more, wherein the sweetener satisfies Relation (3) below:
32.8≥RAG1/RAG2≥7.2   (3).

2. The sweetener of claim 1, satisfying Relation (2) below:
1.17≥(RA+RAG1+RAG2)/TRA≥0.8   (2) wherein TRA represents the proportion of the total rebaudioside A in total wt % of the steviol glycoside.

3. The sweetener of claim 1, wherein the sweetener contains 90 wt % or more of the steviol glycoside.

4. A food composition, comprising the sweetener of claim 1.

5. A sweetener, comprising the sweetener of claim 1, in combination with starch, dextrin, and additional sugar.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a flowchart showing the process of preparing a sweetener containing an enzymatically modified stevia composition according to the present invention; and

(2) FIG. 2 shows the chemical formula of rebaudioside A, in which a glycosyl group may be attached to the hydroxyl group thereof.

BEST MODE

(3) A better understanding of the present invention will be given through the following examples. However, the present invention is not limited to the examples, and may be embodied in other forms, and is provided in order to sufficiently transfer the spirit of the present invention to those skilled in the art such that the contents presented therein will be thorough and complete.

EXAMPLE 1

Preparation of Enzymatically Modified Stevia Composition i

(4) An enzymatically modified stevia composition was prepared as follows. Here, intermediate products (numerical indication) and a final product were measured for glycosylation rate.

(5) Specifically, a stevia extract containing 90 wt % of RA (non-glycosylated rebaudioside A) and beta-cyclodextrin were mixed at a weight ratio of 1:2, dissolved in water in an amount three times the total raw material weight thereof, and adjusted to pH 5.5, 70° C., and 30 Brix. For primary enzyme reaction, CGTase (cyclodextrin glucanotransferase) was added such that the amount thereof was 1.6 g based on 100 g of the stevia extract, and was allowed to react.sup.1) for 40 hr.

(6) Next, for secondary enzyme reaction, glucoamylase was added such that the amount thereof was 0.8 g based on 100 g of the stevia extract under conditions of pH 4.5 and 58° C. and was allowed to react.sup.2) for 1 hr 40 min, followed by enzyme deactivation at 90° C. The reaction product, in which the enzyme was deactivated, was filtered with diatomite, subjected to membrane separation.sup.3) through ultrafiltration, followed by concentration under reduced pressure and then spray drying.sup.4), after which the resulting dry product was dissolved at a maximum of 60° C. in a 92% (v/v) ethanol aqueous solution in a volume three times the weight thereof, cooled to 45° C., and crystallized with stirring for 2˜3 hr. Thereafter, centrifugal filtration, washing.sup.5) with a 92% (v/v) ethanol aqueous solution, concentration under reduced pressure, UHT sterilization (ultrahigh-temperature sterilization) and then spray drying.sup.6) were performed. In these processes, the compositions obtained in respective steps.sup.(superscript indication) were measured for RA glycosylation rate, total rebaudioside A proportion in total steviol glycoside, and the proportion of each RA ingredient in the total rebaudioside A. The results are shown in Table 2 below.

(7) In Table 2 below, RAG1 to RAG5 represent glycosylated rebaudioside A, individual numerals showing the number of attached glycosyl groups. Specifically, RAG1 means that one glycosyl group is attached to rebaudioside A, and RAG5 means that five glycosyl groups are attached to rebaudioside A.

(8) TABLE-US-00002 TABLE 2 Total steviol Total RA glycoside proportion content RA in reaction Each RA proportion in reaction product (%) in Glycosylation product RA RAG1 RAG2 RAG3 RAG4 RAG5 RAG6 Yield reaction Process rate (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) product 1) 88.6 94.23 11.4 15.6 16.2 16.3 15.9 13.8 10.8 — — 2) 73.2 87.86 26.8 65.7 7.5 — — — — — — 3) 73.7 87.8 26.3 65.6 8.1 — — — — — — 4) 73.9 86.9 26.1 65.8 8.1 — — — — 135.1 5) 82.2 92.0 17.8 75.1 7.1 — — — — 74.7 — 6) 81.8 92.0 18.5 73.7 7.8 — — — — 66.0 95.7

(9) Here, rebaudioside A content analysis (RA and RAG1˜G6 proportion analysis) was performed through HPLC (high performance liquid chromatography) under the following conditions.

(10) TABLE-US-00003 TABLE 3 Instrument WATERS 2695 Separations Module Detection WATERS Detector (210 nm), Waters 2487 Dual λ Absorbance Detector Lamp Mobile Phase Acetonitrile:Water = 70:30 Column Amino group-bonded silica gel - TOSOH TSKgel Amide-80 or equivalents thereto (5 μm, 4.6 mm × 25 cm) Flow rate 0.8 ml/min Temperature 30° C. Injection 20 μL Volume

(11) $\mathrm{Total}\mathrm{RA}\mathrm{Proportion}=\mathrm{RA}+\mathrm{RA}G1\sim G6\%\mathrm{Area}\mathrm{Sum}$$\mathrm{RA}\mathrm{Glycosylation}\mathrm{Rate}\left(\%\right)=\frac{\begin{array}{c}\mathrm{Total}\mathrm{RA}\mathrm{Proportion}-\\ \mathrm{Unreacted}\mathrm{RA}\mathrm{Proportion}\left(\mathrm{Area}\%\right)\end{array}}{\mathrm{Total}\mathrm{RA}\mathrm{Proportion}}\times 100$$\mathrm{Each}\mathrm{Ingredient}\mathrm{Proportion}\left(\%\right)=\frac{\mathrm{Proportion}\mathrm{of}\mathrm{Each}\mathrm{Ingredient}\left(\mathrm{Area}\%\right)}{\mathrm{Total}\mathrm{RA}\mathrm{Proportion}}\times 100$

(12) In the above equations, Area % is determined by measuring the area of an HPLC peak of each of the RA ingredients.

(13) The total steviol glycoside content was determined by combining steviol glycoside content (%) after glucoamylase treatment of the adsorbed enzymatically modified stevia in accordance with ┌steviol glycoside┘ assay of enzymatically modified stevia assay with the amount (%) of α-glucosyl residue isolated after glucoamylase treatment. The operating conditions of an analyzer for confirming the amount of steviol glycoside are as follows.

(14) TABLE-US-00004 TABLE 4 Detector UV absorption spectrometer (measurement wavelength 210 nm) Column For liquid chromatography, octadecylsilylated silica gel filler 5 μm, inner diameter 4.6 mm, length 250 mm, stainless steel column Column 40° C. Temperature Mobile phase Phosphate buffer (0.01 mol/L, pH 2.6)/acetonitrile mixed solution (17:8) Flow rate 1.0 ml/min

EXAMPLE 2

Preparation of Enzymatically Modified Stevia Composition ii

(15) An enzymatically modified stevia composition was prepared in the same manner as in Example 1, with the exception that ethanol crystallization was performed (at 25° C., without warming) using a 90% (v/v) methanol aqueous solution at room temperature, and washing with a 90% (v/v) methanol aqueous solution was performed after centrifugal filtration. Here, in the same steps as in Example 1, RA glycosylation rate, total rebaudioside A proportion in total steviol glycoside, and the proportion of each RA ingredient in the total rebaudioside A were measured. The results are shown in Table 5 below.

(16) TABLE-US-00005 TABLE 5 Total steviol Total RA glycoside proportion content RA in reaction Each RA proportion in reaction product (%) in Glycosylation product RA RAG1 RAG2 RAG3 RAG4 RAG5 RAG6 Yield reaction Process rate (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) product 1) 90.4 79.97 9.6 15.5 16.6 16.1 15.9 14.4 11.9 — — 2) 73.7 79.18 26.3 66.9 6.8 — — — — — 3) 73.9 80.14 26.1 64.6 8.8 0.5 — — — 117.5 — 4) 74.2 79.79 25.8 60.8 11.9 1.5 — — — — — 5) 82.1 87.9 17.9 73.4 7.9 0.8 — — — 65.2 — 6) 82.0 87.8 18.0 73.6 7.7 0.7 — — — 62.0 95.5

EXAMPLE 3

Preparation of Enzymatically Modified Stevia Composition iii

(17) The primary enzyme reaction.sup.1), secondary enzyme reaction.sup.2), diatomite filtration, membrane separation, concentration and spray drying.sup.3) of the stevia extract were performed in the same manner as in Example 1, after which the powder obtained through spray drying was treated with a 85% (v/v) methanol aqueous solution, a 90% (v/v) methanol aqueous solution or a 95% (v/v) methanol aqueous solution in a volume three times the weight thereof, and crystallized with stirring for 2˜3 hr. Thereafter, centrifugal filtration, washing.sup.5) with the same alcohol as in the crystallization, concentration, UHT sterilization (ultrahigh-temperature sterilization) and then spray drying.sup.6) were performed. In such processes, RA glycosylation rate, total rebaudioside A proportion in total steviol glycoside, and the proportion of each RA ingredient in the total rebaudioside A were measured. The results are shown in Table 6 below.

(18) TABLE-US-00006 TABLE 6 Total Total steviol RA glycoside proportion content RA in reaction Each RA proportion in reaction product (%) in Glycosylation product RA RAG1 RAG2 RAG3 RAG4 RAG5 RAG6 Yield reaction Process rate (%) (%) % % % % % % % (%) product 1) 86.1 67.78 13.9 18.6 19.9 14.2 13.2 10.9 9.3 — — 2) 70.3 66.31 29.7 64.6 5.7 — — — — — — 3) 72.2 70.64 27.8 65.2 7.0 — — — — — — 6) 85% (v/v) 84.5 92.8 15.5 80.7 3.8 — — — — 27.3 96.2 methanol crystallization 90% (v/v) 82.1 87.9 17.9 73.4 7.9 — — — — 60.0 95.9 methanol crystallization 95% (v/v) 80.4 87.46 19.6 76.0 4.4 — — — — 51.2 95.3 methanol crystallization

EXAMPLE 4

Preparation of Enzymatically Modified Stevia Composition iv

(19) An enzymatically modified stevia composition was prepared in the same manner as in Example 1, with the exception that ethanol crystallization was performed using a 90% (v/v) ethanol aqueous solution or a 95% (v/v) ethanol aqueous solution, and washing after centrifugal filtration was performed using the same ethanol aqueous solution as in the crystallization. In such processes, RA glycosylation rate, total rebaudioside A proportion in total steviol glycoside, and the proportion of each RA ingredient in the total rebaudioside A were measured. The results are shown in Table 7 below.

(20) TABLE-US-00007 TABLE 7 Total Total steviol RA glycoside proportion content RA in reaction Each RA proportion in reaction product (%) in Glycosylation product RA RAG1 RAG2 RAG3 RAG4 RAG5 RAG6 Yield reaction Process rate (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) product 1) 90.5 89.22 9.5 14.9 17.1 16.6 16.0 14.2 11.7 — — 2) 73.7 86.86 26.3 65.8 7.9 — — — — — — 3) 70.7 89.56 29.3 65.2 5.5 — — — — — — 6) 90% (v/v) 79.5 86.24 20.5 76.2 3.3 — — — — 53.0 95.1 ethanol crystallization 95% (v/v) 80.1 86.09 19.7 75.1 2.5 — — — — 48.7 95.5 ethanol crystallization

COMPARATIVE EXAMPLE 1

Preparation of Column-Purified Enzymatically Modified Stevia Composition

(21) The primary enzyme reaction.sup.1), secondary enzyme reaction.sup.2), and diatomite filtration of the stevia extract were performed in the same manner as in Example 1, after which an adsorption reaction was carried out using two columns. Specifically, the filtrate obtained through diatomite filtration was passed through a first column packed with an adsorbent resin, and the passed liquid was adsorbed through a second column packed with the same adsorbent resin. After the adsorption procedure, the two columns were eluted.sup.3) with a 50% (v/v) ethanol aqueous solution, and the elution solution thus obtained was subjected to concentration, UHT sterilization (ultrahigh-temperature sterilization) and then spray drying.sup.4). In such processes, RA glycosylation rate, total rebaudioside A proportion in total steviol glycoside, and the proportion of each RA ingredient in the total rebaudioside A were measured. The results are shown in Table 8 below.

(22) TABLE-US-00008 TABLE 8 Total steviol Total RA glycoside proportion content RA in reaction Each RA proportion in reaction product (%) in Glycosylation product RA RAG1 RAG2 RAG3 RAG4 RAG5 RAG6 Yield reaction Process rate (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) product 1) 89.4 79.31 10.6 16.1 17.9 15.5 15.1 13.8 11.0 — — 2) 75.4 75.88 24.6 65.7 9.7 — — — — — — 4) 79.4 81.92 20.6 73.2 6.2 — — — — 65 95.3

COMPARATIVE EXAMPLE 2

Existing Preparation of Enzymatically Modified Stevia Composition

(23) An enzymatically modified stevia composition was prepared by the method of Example 1 disclosed in Korean Patent No. 10-1535427 (Applicant: Daepyung Co. Ltd.). Specifically, a stevia extract and beta-cyclodextrin were mixed, adjusted to pH 5.5, 70° C., and 30 Brix, and subjected to primary enzyme reaction, as in the present invention, after which the reaction solution was warmed to 90° C. for 1 hr to thus deactivate the enzyme, after which the deactivated solution was spray dried without change.

COMPARATIVE EXAMPLE 3

Preparation of Stevia Composition Using Alpha-Amylase in Lieu of Glucoamylase

(24) A stevia composition was prepared in the same manner as in Example 1, with the exception that alpha-amylase was used in lieu of glucoamylase upon secondary enzyme reaction.

(25) The powder thus obtained was measured for RA glycosylation rate, total rebaudioside A proportion in total steviol glycoside, and the proportion of each RA ingredient in the total rebaudioside A. The final RA proportion was less than 60%, and the rate of increase of G1 was judged to be unsuitable for obtaining an enzymatically modified stevia composition.

TEST EXAMPLE 1

Sweetness Quality Sensory Test

Test Example 1-1

Sugar Content Comparison

(26) The sensory test of sweetness and sweetness quality of an aqueous solution obtained by dissolving the sweetener of Example 1 in a concentration of 0.05% was performed through comparison of sweetness quality at different concentrations of the sweetener of Comparative Example 1. The sweetness was tested using a 0.05% aqueous solution of the sweetener of Example 1, prepared by the preparation method of the present invention, and 0.04˜0.07% aqueous solutions of the typical enzymatically modified stevia sweetener of Comparative Example 1, adjusted so as to have almost the same sweetness by the pre-test. The sweetness intensity of the 0.05% aqueous solution of the sweetener of Example 1 according to the present invention based on the aqueous solution at different concentrations of Comparative Example 1 was evaluated by a total of 50 persons. Table 9 shows the number of persons for sweetness evaluation for respective concentrations in three levels of strength, consistency and weakness.

(27) TABLE-US-00009 TABLE 9 0.05% Aqueous solution of Example 1 Strength Consistency Weakness Aqueous solution concentration (No. of (No. of (No. of of Comparative Example 1 (%) persons) persons) persons) 0.04 45 3 2 0.05 37 10 3 0.06 25 14 11 0.07 5 15 30

Test Example 1-2

Quality Analysis

(28) The aqueous solution obtained by dissolving the sweetener of Example 1 in a concentration of 0.05% and the aqueous solution obtained by dissolving the sweetener of Comparative Example 1 in a concentration of 0.06% were evaluated for bitter taste, the quality of sweet taste, and the total taste quality by a panel of 50 persons. The results are shown in Table 9 below.

(29) Based on the above results, it can be confirmed that the stevia composition of the present invention exhibited high proportions of total rebaudioside A and rebaudioside A-G1, and high steviol glycoside content, compared to the stevia composition prepared by the existing method, and was thus suitable for use as a sweetener.

(30) TABLE-US-00010 TABLE 10 0.05% Aqueous solution of Example 1 compared to 0.06% aqueous solution of Comparative Example 1 Strength Weakness (No. of Consistency (No. of Evaluation item persons) (No. of persons) persons) Bitter taste 0 5 45 Remaining sweet 3 8 39 taste (remaining aftertaste) Softness of sweet 40 10 0 taste Total taste quality 41 8 1

(31) Although not shown in Table 10, the aqueous solution of Comparative Example 2 was evaluated as above, and the number of persons who felt the remaining sweet taste was slightly larger, 1.5 times, for the aqueous solution of Comparative Example 2 than for the aqueous solution of Example 1. It is known that an artificial taste or unpleasant and oily taste of a synthetic sweetener increase as the sweet taste remains intense. Thus, the taste quality of the enzymatically modified stevia composition can be judged to be improved through the method of the present invention.

(32) The sweeteners of Examples 1 to 4 satisfy all of Relations (1)˜(3) according to the present invention and are very efficiently used as high-quality sweetener because the proportion of rebaudioside A having one glycosyl group attached thereto is significantly high. However, the sweetener of Comparative Example 1 does not satisfy both of Relations (1) and (2), and the composition of step 4, which is the intermediate product upon preparation of the sweetener of Example 1 or 2, regarded as very similar to the sweetener of Comparative Example 2, does not satisfy Relation (1) or (2). Therefore, it is possible to prepare the enzymatically modified stevia composition having an increased proportion of rebaudioside A having one glycosyl group attached thereto according to the present invention compared to the sweetener of Comparative Example 2.