KR102203086B1 - Sweetening composition comprising transglycosylated neohesperidin dihydrochalcone and manufacturing method thereof - Google Patents

Abstract

The present invention provides a sweetener composition containing transglycosylated neohesperidin dihydrochalcone (NHDC), and a manufacturing method thereof. The sweetener composition manufactured by the manufacturing method provided by the present invention has excellent water solubility compared to NHDC, and has improved sweetness compared to sugar, thereby being able to be used as a sugar substitute sweetener.

Description

[Sweetening composition comprising transglycosylated neohesperidin dihydrochalcone and manufacturing method thereof]

The present invention relates to a sweetener composition comprising an NHDC sugar transfer and a method for producing the same, and provides a sweetener composition having an excellent sugar content and improving water solubility of NHDC.

Among the sweeteners, sugar is the best natural sweetener that can be obtained from nature and has been widely used in the food industry.However, as the interest in health increased with the recent improvement in income level, side effects of excessive intake of sugar, such as dental caries. , Obesity, diabetes, and heart disease patients are increasing, there is a need to develop a sweetener that can replace sugar.

These sugar substitute sweeteners include sugars such as glucose, maltose, and xylose, or sugar alcohols such as sorbitol, xylitol, and erythritol containing alcohol groups in the sugar. (sugar alcohol), or as oligosaccharides, fructo-oligosaccharide, xylo-oligosaccharide, and galacto-oligosaccharide have been suggested. Other non-glycosylated sweeteners include glycyrrhizin, stevioside in the form of glycosides, taumartin, monelin, etc. in the form of proteins.

Neohesperidin dihydrochalcone (NHDC) is a highly sensitive sweetener found in research to minimize the bitter taste of citrus juice.It has an excellent relative sugar content of 1000 to 2000 times that of sugar, but similar to stevioside, the sweet taste is delayed. It has a sweetness that is significantly different from sugar, such as being expressed and has long persistence, and has been used as a sweetener and/or flavor enhancing treatment for feed, cosmetics, etc., not food, due to low water solubility. In the EU, it was approved as a sweetener in 1994, and in order to be used in food, it is necessary to improve the sweetness and water solubility.

It is an object of the present invention to provide a sweetener composition and a method for producing the same, including the NHDC sugar transfer.

The present invention provides a method for producing a sweetener composition. The method for preparing a sweetener composition provided by the present invention includes the steps of (1) preparing a mixture by mixing a substrate of NHDC and a sugar transfer enzyme (2) preparing a reaction product by adding a sugar transfer enzyme to the mixture, and (3) And reacting by adding amylase to the reactant.

In the present specification, "NHDC" refers to'neohesperidine dihydrochalcone' or '1-(4-((2-O-[6-dioxy-α-L-mannopyranosyl]-β-D-glucopyranosyl) Oxy)-2,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone (1-(4-((2-O-[6-Deoxy-α -L-mannopyranosyl]-β-D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone)', represented by the formula of Formula 1 below It can be broken.

[Formula 1]

In the present specification, "relative sugar content" refers to a sugar content measured based on a sugar content of 1, and the sugar content may be measured using a method known in the art without limitation. In an example, the relative sugar content may be measured through regression analysis and/or electronic tongue based on sensory evaluation, but is not limited thereto.

Hereinafter, the present invention will be described in more detail.

The method for preparing a sweetener composition provided by the present invention relates to a method for preparing a sweetener composition comprising an NHDC sugar transfer agent in which glucose is transferred to 10 or less.

In one example, the NHDC glycotransfer may be an NHDC glycotransfer in which glucose is within 10, 8, 5, 3, or 2 or less, but is not limited thereto. The NHDC glycotransfers include, for example, NHDC and 1 glucose alpha-1, NHDC-G 4 binding, maltosyl NHDC, NHDC and maltose (maltose), and NHDC and glucose 1 alpha-1, 3 NHDC+ One or more selected from the group consisting of G, two or more, or all three types may be included, but the present invention is not limited thereto.

In one embodiment, the NHDC glycotransferr may include NHDC-G including glucose in which NHDC and one glucose are alpha-1,4 linked. The NHDC-G may be represented by Formula 2 below.

[Formula 2]

In one embodiment, the content of NHDC-G in the sweetener composition is 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 30 to 90%, 30 to 80%, 30 to 70%, 30 to 60%, 30 to 50%, 33 to 90%, 33 to 80%, 33 to 70%, 33 to 60%, 33 to 50%, 35 to 90%, 35 to 80%, 35 to It may be 70%, 35 to 60% or 35 to 50%, but is not limited thereto.

In one embodiment, as a result of performing the component analysis of the sweetener composition prepared by the method of the present invention by HPLC analysis, when the amylase treatment was performed after the glycotransferase reaction, the NHDC-G ratio was compared to the control group not subjected to the amylase treatment. It was confirmed that it increased. Specifically, in the control group, the NHDC-G ratio was at the level of 28%, but the NHDC-G ratio in the sweetener composition treated with amylase was 35%.

The method for preparing a sweetener composition provided by the present invention includes the steps of (1) preparing a mixture by mixing a substrate of NHDC and a sugar transfer enzyme.

The substrate of the glycotransferase is, for example, selected from the group consisting of alpha-cyclodextrin (a-CD), beta-cyclodextrin (b-CD), gamma-cyclodextrin (r-CD), and starch (Starch). One or more, two or more, three or more, or may be all of the above four types, but is not limited to the exemplified substrate. The starch may be extracted in nature or synthesized chemically, and when extracted in nature, it may be extracted from one or more selected from the group consisting of potato, wheat, corn, rice, cassava, sweet potato, and tapioca, but this Not limited.

The weight ratio of the substrate of the NHDC and the sugar transfer enzyme in the mixture (NHDC: substrate of the sugar transfer enzyme, hereinafter the same) is 1:10 to 100:1, 1:10 to 50:1, 1:10 to 20:1, 1 :10 to 10:1, 1:10 to 5:1, 1:10 to 3:1, 1:5 to 100:1, 1:5 to 50:1, 1:5 to 20:1, 1:5 To 10:1, 1:5 to 5:1, 1:5 to 3:1, 1:2 to 100:1, 1:2 to 50:1, 1:2 to 20:1, 1:2 to 10 :1, 1:2 to 5:1, 1:2 to 3:1, 1:1 to 100:1, 1:1 to 50:1, 1:1 to 20:1, 1:1 to 10:1 , 1:1 to 5:1, or 1:1 to 3:1, but is not limited thereto, and a person skilled in the art may select and use an appropriate mixing ratio according to experimental conditions. In one embodiment, the NHDC and the sugar transfer enzyme were mixed in a weight ratio of 3:1, 3:2, 5:1 or 5:2.

If too much of the glycotransferase substrate is mixed, there is a possibility that the amount of glucose transferred to NHDC will be too high.If the substrate of the glycotransferase is incorporated at a lower ratio than the above ratio, the NHDC glycotransfer is not sufficiently formed. May not.

The method for producing a sweetener composition provided by the present invention includes the step of (2) preparing a reactant by adding a sugar transfer enzyme to the mixture.

The glycotransferase may be used without limitation in the range of the purpose of preparation of the NHDC glycotransferase, for example, cyclomalto dextrin glucantransferase, transglucosidase, maltosyl transferase ( maltosyl transferase), amylosucrase, glycogen synthase, starch synthase, and glucanotransferase.It may be one or more enzymes selected from the group consisting of.

The reaction temperature of the sugar transfer enzyme may be appropriately changed according to the activity temperature of the sugar transfer enzyme, for example, 40 to 110 degrees Celsius, 40 to 105 degrees Celsius, 40 to 100 degrees Celsius, 45 to 110 degrees Celsius, It can be performed at 45 to 105 degrees Celsius, 45 to 100 degrees Celsius, 50 to 110 degrees Celsius, 50 to 105 degrees Celsius, or 50 to 100 degrees Celsius.

The added amount of the sugar transfer enzyme may be appropriately adjusted according to the enzyme activity, for example, 50 to 500 ul, 50 to 400 ul, 50 to 350 ul, 50 to 300 ul, 55 to 40 ml of a substrate mixture of NHDC and sugar transfer enzyme. 500ul, 55 to 400ul, 55 to 350ul, 55 to 300ul, 60 to 500ul, 60 to 400ul, 60 to 350ul, 60 to 300ul, 65 to 500ul, 65 to 400ul, 65 to 350ul, or 65 to 300ul may be added .

The method for producing a sweetener composition provided by the present invention includes the step of (3) reacting by adding amylase to the reactant.

The amylase is also referred to as amylase and serves to hydrolyze starch. The amylase may be used without limitation within a range of purposes for increasing the content of NHDC-G (Formula 2), for example, at least one selected from the group consisting of alpha amylase, beta amylase, and gamma amylase, or It can be used in combination of the above three types. In another example, the amylase may be one or more enzymes selected from the group consisting of exo amylase, for example, amyloglucosidase and beta amylase.

By further treatment with the amylase, the content of NHDC-G (Formula 2) in which one glucose is alpha-1,4 bound in the NHDC glycotransfer may be increased. The content of NHDC-G in the sweetener composition prepared by further including the above step is 30% (w/w) or more, 31% (w/w) or more, 32% (w/w) or more, 33% (w/ w) or more, 34% (w/w) or more, 35% (w/w) or more, 30 to 90% (w/w), 30 to 80% (w/w), 30 to 70% (w/w) ), 30 to 60% (w/w), 30 to 50% (w/w), 33 to 90% (w/w), 33 to 80% (w/w), 33 to 70% (w/w) ), 33 to 60% (w/w), 33 to 50% (w/w), 35 to 90% (w/w), 35 to 80% (w/w), 35 to 70% (w/w) ), 35 to 60% (w/w) or 35 to 50% (w/w), but is not limited thereto.

The sweetener composition prepared by additionally treating the amylase has a content of NHDC-G represented by the structure of Formula 2 above 1.1 times, 1.2 times or more, 1.25 times or more, 1.1 to 5 times as compared to the case where amylase is not treated. , 1.1 to 4 times, 1.1 to 3 times, 1.1 to 2 times, 1.2 to 5 times, 1.2 to 4 times, 1.2 to 3 times, 1.2 to 2 times, 1.25 to 5 times, 1.25 to 4 times, 1.25 to 3 times , Or 1.25 to 2 times, but is not limited thereto.

The sweetener composition prepared by additionally treating the amylase may further contain maltosyl-NHDC, the content of which is 30% (w/w) or less, 25% (w/w) or less, 20% (w/w) ) Or less, 5 to 30% (w/w), 5 to 25% (w/w), 5 to 20% (w/w), 10 to 130% (w/w), 10 to 25% (w/ w), or 10 to 20% (w/w), but is not limited thereto. In the case of maltosyl NHDC, the relative sugar content is lower than that of NHDC-G, but the persistence of sweet taste is reduced, which is predicted to be able to alleviate the delayed expression of sweet taste, which is indicated as a disadvantage of NHDC.

The step of reacting by adding the amylase, 15 to 100 degrees Celsius, 15 to 95 degrees, 15 to 90 degrees, 15 to 80 degrees, 15 to 60 degrees, 20 to 100 degrees, 20 to 95 degrees, 20 to 80 degrees Celsius , Or may be performed at 20 to 60 degrees, but is not limited thereto. In one example, the amylase reaction step is pH 1 to 13, pH 1 to 10, pH 1 to 8, pH 1 to 6, pH 2 to 13, pH 2 to 10, pH 2 to 8, pH 2 to 6, pH 4 To 13, pH 4 to 10, pH 4 to 8, or pH 4 to 6, but may be performed under conditions, but is not limited thereto. In one example, the amylase is 10 to 300 ul, 10 to 200 ul, 10 to 150 ul, 10 to 100 ul, 30 to 300 ul, 30 to 200 ul, 30 to 150 ul, 30 to 100 ul, 50 to 300 ul, 50 to 200ul, 50 to 150ul, or 50 to 100ul may be added, but is not limited thereto. In the step of reacting by adding the amylase, the reaction may be performed for 1 to 24 hours, 1 to 12 hours, 1 to 9 hours, 2 to 24 hours, 2 to 12 hours, or 2 to 9 hours after the amylase addition. , Is not limited thereto.

The present invention also provides a sweetener composition prepared by the above method. The sweetener composition provided by the present invention may include an NHDC sugar transfer agent in which glucose is transferred within 10, 5, 3, or 2 or less. The contents of the NHDC and NHDC sugar transfer are as described above.

The sweetener composition is characterized in that the content of glycosyl NHDC (NHDC-G) in which one glucose is alpha-1,4 bonded to NHDC is increased. The NHDC-G may be represented by the following formula (2).

[Formula 2]

The NHDC-G content in the sweetener composition provided by the present invention is 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 30 to 90%, 30 to 80%, 30 to 70 %, 30 to 60%, 30 to 50%, 33 to 90%, 33 to 80%, 33 to 70%, 33 to 60%, 33 to 50%, 35 to 90%, 35 to 80%, 35 to 70 %, 35 to 60% or 35 to 50%, but is not limited thereto.

When the NHDC-G content is increased, the water solubility of the sweetener increases, and the sweetness is excellent, so that the utilization as a sweetener replacing sugar is excellent.

The sweetener composition provided by the present invention has excellent aqueous solubility compared to NHDCs by including the NHDC glycotransfers in which glucose is transferred within 10, 5, 3 or 2 or less. In one example, the sweetener composition provided by the present invention is 100 times or more, 200 times or more, 250 times or more, 100 to 10000 times, 100 to 1000 times, 100 to 500 times, 100 to 300 times, 200 to 10000 times compared to NHDC , 200 to 1000 times, 200 to 500 times, 200 to 300 times, 250 to 10000 times, 250 to 1000 times, 250 to 500 times, or may have an aqueous solubility of 250 to 300 times, but is not limited thereto.

The sweetener composition provided by the present invention has an excellent relative sugar content compared to sugar, and in one example, the relative sugar content of the sweetener composition is 10 or more, 30 or more, 50 or more, 80 or more, 10 to 1000, 10 to 500, 10 To 300, 10 to 100, 30 to 1000, 30 to 500, 30 to 300, 30 to 100, 50 to 1000, 50 to 500, 50 to 300, 50 to 100, 80 to 1000, 80 to 500, 80 to 300 , Or 30 to 100, but is not limited thereto.

The sweetener composition provided by the present invention may have an improved bitter taste (gourmet) compared to NHDC. In one example, the sweetener composition provided by the present invention may have a bitter taste similar to that of sugar at a concentration of 0.01% (w/v) or more and/or 0.02% (w/v) or more. In one embodiment, as a result of performing a bitter taste sensory evaluation on the sweetener prepared by the method of the present invention, it was confirmed that for a 0.02% or more NHDC sugar transfer (NHDC-G) solution did not show a significant difference from sugar in bitter taste, 0.01 It was found to have a bitter taste similar to that of sugar at concentrations greater than% (w/v) or greater than 0.02% (w/v).

The sweetener composition provided by the present invention may be used alone and/or in the form of a mixture. In one example, the sweetener composition provided by the present invention based on 100 parts by weight of the mixture is 1 part by weight or more, 5 parts by weight or more, 10 parts by weight or more, 20 parts by weight or more, 30 parts by weight or more, 50 parts by weight or more, 70 It may be included in parts by weight or more, 80 parts by weight or more, 90 parts by weight or more, 95 parts by weight or more, or 99 parts by weight or more. The mixture may be one or more, two or more, or all three selected from the group consisting of fructose, glucose, and other reducing sugars in addition to the sweetener composition provided by the present invention, but is not limited thereto.

In one example, the sweetener composition provided by the present invention may be provided in an appropriate formulation according to the purpose, for example, in tablets, capsules, pills, granules, liquids, flakes, pastes, syrups, gels, jelly, bars, and films. It can be provided in one formulation of choice.

In one example, the sweetener composition provided by the present invention may be used without being limited to the type of food in the range of the purpose of enhancing sweetness, and the type of the food may be confectionery, bread, rice cake, frozen fruit, processed cocoa products, chocolates, Sugars, jams, tofu, jelly, edible oils and fats, noodles, beverages, pastes, seasoned foods, pickles, stewed foods, alcoholic beverages, processed agricultural foods, processed meat products, packaged meats, processed egg products, dairy products, processed seafood products, processed animal products, It may be added to one or more, two or more, three or more, five or more selected from the group consisting of honey and pollen processed products, instant foods, other foods, and health functional foods, but is not limited thereto. The sweetener composition provided by the present invention is 0.1% (w/w) or less, 0.3% (w/w) or less, 0.5% (w/w) or less, 1% (w/w) or less, 3% ( w/w) or less or 5% (w/w) or less, but is not limited thereto.

With the manufacturing method provided by the present invention, the sweetener composition contains NHDCs in which glucose is transferred to less than 10, and in particular, the ratio of NHDCs in which glucose is transferred is high, so the water solubility is high, its utilization in food is excellent, and it is superior to sugar. It has a sugar content.

The sweetener composition provided by the present invention can be used as a substitute for sugar and/or mixed with sugar, has high water solubility, and is excellent in food use, and has excellent sweetness compared to sugar, so it can be used for the purpose of reducing the intake of sugar. have.

1 is a flowchart schematically illustrating an example of a method for preparing an NHDC sugar transfer agent.
2 is a graph showing the results of HPLC analysis according to the reaction time of the NHDC sugar transfer enzyme prepared by the method of Example 2.
3 is a graph showing the results of HPLC analysis when the substrate of the sugar transfer enzyme is a-CD (A), b-CD (B) or rice starch (C). In each panel, the mixing weight ratio of NHDC and substrate was 3:1, 3:2, 5:1 and 5:2, respectively, from top to bottom.
4 is a graph showing the results of HPLC analysis before (top) and after (bottom) an amylogglucosidase reaction after the reaction of NHDC and glycotransferase.
5 is a recycling preparative HPLC chromatogram for the separation and purification of NHDC glycotransfers of the present application, and is a chromatogram of the first cycle (top) and the second cycle (bottom).
6 shows the results of performing LC-MS analysis on each of the four representative peaks of the HPLC chromatogram of the NHDC glycotransfer. Peak 1 is the starting material, NHDC, peak 2 is NHDC+G in which one glucose binds to NHDC and alpha-1,4, peak 3 is maltosyl NHDC, and in peak 4, one glucose binds NHDC and alpha-1,3 It is NHDC+G.
7 is a graph showing the absorbance measurement result according to the concentration of aqueous solution of NHDC and NHDC sugar transfer (NHDC-G).
8 is a graph showing the number of respondents who responded that they were sweeter than the sugar solution by concentration of the NHDG-G solution as a result of performing a sensory test for measuring the relative sugar content of the NHDC sugar transfer agent (NHDC-G).

Hereinafter, the content of the present invention will be described in more detail by examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited by the following examples.

Example 1. Preparation of NHDC sugar transfer

Neohesperidin dihydrochalcone (NHDC) is mixed with ethanol and 10 mM citrate buffer (pH 6) in a volume ratio of 1:9 to a concentration of 0.5 to 10% (w/v) (specifically , 1, 2, 3, 5 and 10% (w/v)), and 0.5 to 10% of α-cyclodextrin (α-CD), β-CD, γ-CD, starch, etc. as a substrate After addition as a substrate at a concentration of (w/v) (specifically, 1, 2, 3 and 5% (w/v)), Toruzyme® (Novozyme) sugar transfer enzyme was added at a level of 60 to 300 ul/40 ml (specifically, 60ul/ml and 300ul/ml) and reacted at a temperature of 50 to 100 degrees Celsius (specifically, 55 degrees Celsius) for 24 hours, 48 hours, or 72 hours to prepare an NHDC sugar transfer.

Example 2. Detection of NHDC glycotransfers

The glycotransfers prepared by the method of Example 1 were detected using HPLC. Specifically, the HPLC-PAD system (Waters 2695, PDA detector) was used, and the column was J'sphere ODS-H80 (YMD), and detected under UV 280 nm conditions. As a detection solvent, acetonitrile (ACN) and 10 mM H ₃ PO ₄ were used at a rate of 1 mL/min, and the ACN concentration was 20% (w/v) in the first 0 to 2 minutes, and 25% (w /v), by adjusting to be 30% (w/v) at 13 minutes and 100% (w/v) from 14 minutes to establish detection conditions capable of efficiently eluting a large number of NHDC glycotransfers even at high concentrations.

When the reaction was carried out with Toruzyme 60ul/40ml by adding alpha-cyclodextrin (α-CD) 1% (w/v) to NHDC 3% (w/v) solution as a substrate, reaction time (24 hours, 48 hours, Or 72 hours), the graph of the HPLC result of detecting the NHDC glycotransfer is shown in FIG. 2. As a control, NHDC was used that did not undergo the sugar transfer process (Toruzyme reaction).

-CD(B 패널), 또는 쌀전분(C 패널)을 사용한 경우의 HPLC 결과 그래프를 나타내었다. 모든 패널에서 위에서 아래 방향으로 NHDC와 기질의 혼합비(NHDC:기질 중량비, 이하 동일)가 각각 3:1, 3:2, 5:1 및 5:2인 경우이다.3 shows α-CD as a substrate (FIG. 3A panel),

-A graph of the HPLC results when using CD (Panel B) or rice starch (Panel C) is shown. In all panels, the mixing ratio of NHDC and substrate (NHDC: substrate weight ratio, hereinafter the same) is 3:1, 3:2, 5:1 and 5:2, respectively, from top to bottom.

In general, in the case of saccharides, the relative sugar content of monosaccharides and disaccharides is the highest, and it is known that the sweetness decreases as the degree of polymerization increases.

Many of the glucose transfer products identified as a result of HPLC are NHDC glycotransfers in which glucose has been transferred to within 10, and the yield of glycotransfers (NHDC, NHDC-G to which one glucose has been transferred) is predicted to have the highest relative sugar content. In order to increase, an exo form of amylase (eg, amyloglucosidase) was further treated.

Specifically, NHDC 10% (w/v), starch 3% (w/v), Toruzyme was mixed at a concentration of 300 ul/40 ml and reacted for 48 hours, followed by volatilization of ethanol in 10 ml of the above reaction solution, and 50 ul of amiloglucosidase was added. After addition, the reaction was carried out for another 6 hours.

Figure 4 shows a graph of the HPLC results confirming whether the increase of NHDC-G was successfully performed in the glycotransferase prepared by treatment with amyloglucosidase. The proportion of NHDC-G was only 28% before the amylogglucosidase treatment, but the proportion of NHDC-G with a high relative sugar content increased to 35% after the amylogglucosidase treatment.

Example 3. Separation and purification of NHDC glycotransfers

Preparative HPLC was performed for the separation and purification of the NHDC glycotransfer. The enzyme reaction solution was concentrated in vacuo at 80 degrees Celsius and then filtered, using recycling preparative HPLC (LC-Forte, YMC Inc., Japan) and YMC-Actus C ₁₈ column (20 Х 500 mm, YMC Inc., Japan). Then, the separation process was performed. As the mobile phase, 28% (w/v) ACN was used in isocratic mode at a flow rate of 30 mL/min, and UV detection was performed at 280 nm. After lyophilization of each collected fraction, the purity was confirmed using Waters 2695 HPLC and PDA dectector of Example 2.

Fig. 5 shows a chromatogram for separating and purifying NHDC glycotransfers using recycling preparative HPLC. The top of FIG. 5 is the chromatogram of the first cycle, and the graph below is the chromatogram of the second cycle. In FIG. 5, a denotes NHDC-G, b denotes maltosyl NHDC, and a+NHDC denotes a peak in which NHDC-G and NHDC are mixed.

Example 4. LC-MS analysis of glycotransfer

LC-MS analysis was performed on the four main peaks shown as a result of HPLC analysis of the glycotransferase prepared by the method of Example 2 above. Specifically, the analysis was performed using LCsystem (Ultimate 3000 RS system; Thermo fisher scientific Inc.), and 0.2% acetonitrile (ACN) including 0.1% (v/v) formic acid as a solvent. It was used at a flow rate of ml/min. The sample was injected directly without passing through a column, and the molecular weight was analyzed by electrospray ionization using LTQ (Thermo Fisher Scientific Inc.), and the source voltage was 3.5 kV and negative mode. mode) was applied to perform a full scan in the range of 100 to 2000 m/z.

6 shows a graph of the positions of the four main peaks on the chromatogram and the result of LC-MS analysis of the NHDC or glycotransfers corresponding to each peak. Peak 1 is the starting material, NHDC, peak 2 is NHDC-G in which one glucose binds to NHDC and alpha-1,4, peak 3 is maltosyl NHDC, and in the fourth, one glucose binds NHDC and alpha-1,3 It was identified as NHDC+G.

In other words, it can be predicted that the NHDC glycotransferr prepared by the method provided by the present invention mainly binds one or two glucose to NHDC, and a small number of glucose is transferred to increase water solubility, and that the relative sugar content will be maintained relatively high. have. It is also predicted that the duration of the sweet taste will decrease from the increased water solubility.

In the case of maltosyl NHDC, the relative sugar content is lower than that of NHDC-G, but the persistence of sweet taste is reduced, which is expected to alleviate the delayed expression of sweet taste, which is pointed out as a disadvantage of NHDC.

Example 5. Analysis of water solubility of sugar transfer transfer

In order to check whether the sugar transfer prepared by the method of Example 2 has improved water solubility compared to NHDC, the sugar transfer was dissolved in water at a temperature of 20 degrees Celsius for 5 minutes, and the absorbance was measured at 660 nm to check the water solubility. I did.

Table 1 below shows the absorbance measurement results according to the concentration of NHDC and NHDC sugar transfer (denoted as NHDC-G), and the graph is shown in FIG. 7.

In general, when the water solubility is measured using absorbance, it is considered that a true solution is formed at an absorbance of 1 or less, and when the absorbance is more than 1, it can be seen that a suspension is present without dissolving in water.

Therefore, as shown in Table 1 above, NHDC without sugar transfer process has a solubility of 0.02g/10mL level, but NHDC-G has an aqueous solubility of 6g/10mL or more, and the acceptance of the sugar transferr provided by the present invention It can be seen that the chart has improved more than 300 times.

Example 6. Sensory evaluation

Sensitivity tests for sweet taste and bitter taste were performed on 100 men and women in their 20s to 40s to evaluate the sweetness of the glycosyltransfers prepared by the manufacturing method of the present invention.

First of all, for the selection of the evaluation panel among 100 participants, a three-point test was performed using a caffeine solution and a sugar solution, giving two identical samples and one other sample and selecting the other, and finalizing 44 subjects who answered both sweet and bitter tastes. Relative sugar was selected as an evaluation panel.

The evaluation of the relative sugar content was performed using a two-alternative forced choice (2-afc), and specifically, a 3% (w/v) sugar solution was used as the reference solution, and prepared by the method of Example 2. The resulting NHDC-G solution was prepared in 0.01% (w/v, hereinafter the same) 0.02%, 0.03%, and 0.04%, and each paired with a sugar solution to select a solution having a stronger sweet taste among the two solutions. From the response results, the number of responses that responded that the sweetness of the NHDC-G solution was stronger than that of the sugar solution and the concentration of NHDC-G were plotted in a two-dimensional graph to obtain a regression equation, and the NHDC-G having the same sweetness as the 3% sugar solution. The concentration was calculated.

In the case of bitter taste, the 2-afc method was applied and evaluated in substantially the same manner as in the above-described relative sugar content evaluation.

Table 2 shows the number of panels who answered that NHDC-G was sweeter for each concentration of each NHDC glycotransfer (NHDC-G).

Glycotransfer concentration (%) Sweetness responses (out of 44) 0.01 0 0.02 10 0.03 18 0.04 28 0.05

Table 3 below shows the number of panels who answered that NHDC-G was used more for each concentration of each NHDC glycotransfer (NHDC-G).

Glycotransfer concentration (%) Number of responses to bitter taste (out of 30) 0.02 20 0.03 18 0.04 18 0.05 17

In FIG. 8, a trend line was drawn using the number of responses from the panel, and a graph of the result of calculating the relative sugar content therefrom is shown. Since the concentration of the NHDC sugar transfer agent (denoted as NHDC-G), judged to have the same sweetness as the 3% sugar solution, was 0.0337%, it was confirmed that the relative sugar content reached about 89, confirming that it had a very superior sugar content than sugar. In the case of bitter taste, which is an obstacle to the use of NHDC, it was found that there was a significant difference from sugar at a 95% significance level when 21 or more respondents said they were bitter, and at a concentration of 0.02% or more, there was a significant difference in sugar and bitter taste. There was no difference (less than 21 respondents).

Claims (9)

Preparing a mixture by mixing a substrate of neohesperidin dihydrochalcone (NHDC) and a sugar transfer enzyme;
Preparing a reaction product by adding a sugar transfer enzyme to the mixture; And
A method for producing a sweetener composition comprising an NHDC sugar transfer agent in which glucose is transferred to within 10 or less, including; reacting by adding amylase to the reactant. The method of claim 1, wherein the substrate of the glycotransferase is alpha-cyclodextrin (a-CD), beta-cyclodextrin (b-CD), gamma-cyclodextrin (r-CD), and starch (Starch). It is one or more polysaccharides selected from the group, the manufacturing method. The method of claim 1, wherein the NHDC and the substrate of the sugar transfer enzyme are mixed in a weight ratio of 1:10 to 100:1 (NHDC: the substrate of the sugar transfer enzyme). The method of claim 1, wherein the glycotransferase is cyclomalto dextrin glucantransferase, transglucosidase, maltosyl transferase, and amylosucrase. , Glycogen synthase (glycogen synthase), starch synthase (starch synthase), and glucanotransferase (glucanotransferase) that is one or more enzymes selected from the group consisting of. The method of claim 1, wherein the sweetener composition has a weight ratio of glycosyl NHDC (NHDC-G) represented by the following formula (2) at least 30%:
[Formula 2]

A sweetener composition prepared by the method of any one of claims 1 to 5, comprising an NHDC sugar transfer agent in which glucose is transferred to less than 10. The sweetener composition according to claim 6, wherein the weight ratio of glycosyl NHDC (NHDC-G) represented by the following Formula 2 in 100 parts by weight of the sweetener composition is 30% or more.
[Formula 2]

The sweetener composition of claim 6, wherein the sweetener composition has an increase in water solubility of 100 to 10000 times compared to NHDC. The sweetener composition of claim 6, wherein the sweetener composition has a relative sugar content of 10 to 1000.

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