阅读说明：本技术 使用苹果乳杆菌制造转果糖基化甜菊醇配糖物的方法 (Method for producing transfructosylated steviol glycoside using lactobacillus malate ) 是由 杨泰周 李英美 姜仁声 朴晟喜 李英秀 秋善 金成俌 崔殷姃 于 2018-12-14 设计创作，主要内容包括：本申请涉及使用苹果乳杆菌菌株的粗酶液制备转葡糖基化甜菊醇配糖物的方法。(The present application relates to methods of making transglucosylated steviol glycosides using crude enzyme solutions of lactobacillus malate strains.)

1. A method for preparing transglucosylated steviol glycoside by using Lactobacillus Malus (L actobacterium mali) microorganism or its culture.

2. The method of claim 1, wherein the method comprises reacting steviol glycosides with sugar in the presence of the lactobacillus malate microorganism or culture thereof.

3. The method of claim 2, wherein the steviol glycoside is one or more selected from the group consisting of stevioside, rubusoside, dulcoside a, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, and rebaudioside M.

4. The method of claim 2, wherein reacting the steviol glycoside with the sugar is carried out at a temperature of 10 ℃ to 60 ℃ at a pH of 3 to 8.

5. The method of claim 1, wherein the transglucosylated steviol glycoside is a steviol glycoside, wherein glucose is added by virtue of being linked via an α - (1,6) linkage to glucose linked to the 19-OH site of the steviol glycoside.

6. The method of claim 5, wherein the transglucosylated steviol glycoside comprises 1 to 4 glucose molecules.

7. A transglucosylated steviol glycoside prepared by the method of any one of claims 1 to 6.

8. The transglucosylated steviol glycoside of claim 7, wherein the transglucosylated steviol glycoside is one or more selected from the group consisting of transglucosylated stevioside, transglucosylated rubusoside, transglucosylated dulcitoside A, transglucosylated rebaudioside C, transglucosylated rebaudioside D, transglucosylated rebaudioside E, transglucosylated rebaudioside F, and transglucosylated rebaudioside M.

9. A composition for producing the transglucosylated steviol glycoside, which composition comprises a lactobacillus malate microorganism or a culture thereof.

10. A sweetener comprising a transglucosylated steviol glycoside prepared by the method of any one of claims 1-6.

Technical Field

The present disclosure relates to methods of using crude enzyme solutions of lactobacillus malate (L actobacterium mali) strain to produce transglucosylated steviol glycosides.

Background

As the World Health Organization (WHO) recommends lowering the daily sugar intake for concern over diseases caused by sugar consumption (obesity), the governments of developed countries are actively discussing various policies aimed at reducing sugar intake. Therefore, as the demand for development of various alternative sweeteners in the market is increasing, alternative sweeteners are being developed and commercialized. As alternative sweeteners, these are constantly changing in the form: synthetic high intensity sweeteners (e.g., saccharin, aspartame, Sucralose, etc.), synthetic sugar alcohols (e.g., maltitol and xylitol), and high intensity sweeteners (e.g., Rebaudioside a and licorice). However, due to safety concerns with synthetic sweeteners, consumer demand for natural sweeteners has steadily increased; however, natural sweeteners do not completely replace existing low and zero calorie products based on synthetic sweeteners because of the limitations of their characteristic flavor characteristics, i.e., off-taste and off-flavor.

A natural high intensity sweetener that has attracted much attention in recent years is steviol glycoside (Stevia) extracted from the leaves of Stevia rebaudiana Bertoni. Steviol glycosides are natural materials that have a sweetness that is 200 to 300 times that of sugar. Further, steviol glycosides are composed of Stevioside (Stevioside), rebaudioside A, B, C, D, E, M, and the like. In addition, because steviol glycosides are reported to produce no calories, to have a positive effect on blood glucose and insulin levels, and to have no side effects on the human body, steviol glycosides have potential use as alternative sweeteners; however, steviol glycosides have a bitter taste and have certain limitations in their use.

To date, there have been three approaches to improving the sweetness of steviol glycosides: (1) a method of mixing with a saccharide sweetener, an amino acid, or an amino acid salt, (2) a physical method including a substance such as cyclodextrin; and (3) a method of transferring glucose using an enzyme. As a method for transferring glucose using an enzyme, a method of transferring 1 to 12 glucose molecules to steviol glycoside using cyclodextrin glucosyltransferase (CGTase) is widely used in the art (Korean patent application No. 10-1991-0020769). However, this method has the following disadvantages: all glucose transferred to steviol glycosides is degraded by gut microbes, thereby increasing calories.

It is known that lactobacillus generally produces rubusoside when reacted with β -glucosidase (korean patent application No. 10-17676060000.) furthermore, lactobacillus reuteri (L actinobacillus reuteri)180, a specific genome, is known as the only lactobacillus that recognizes steviol glycoside to produce transglucosylated steviol glycoside.

Disclosure of Invention

[ problem ] to

Under such circumstances, the present inventors completed the present disclosure by finding that lactobacillus appli transglucosylates glucose to steviol glycoside through α - (1,6) -linkage to produce a transglucosylated steviol glycoside that is difficult to digest.

[ means of solution ]

It is an object of the present disclosure to provide a method for preparing transglucosylated steviol glycosides using lactobacillus malate microorganism or culture thereof.

It is another object of the present disclosure to provide transglucosylated steviol glycosides prepared via the above-described preparation methods.

It is yet another object of the present disclosure to provide a composition for producing transglucosylated steviol glycosides, which comprises a lactobacillus malate microorganism or a culture thereof.

It is still another object of the present disclosure to provide a sweetener comprising a transglucosylated steviol glycoside prepared by the above preparation method using a lactobacillus malate microorganism or a culture thereof.

[ advantageous effects ]

The disclosed method for producing transglucosylated steviol glycosides can specifically utilize Lactobacillus Malus microorganisms or cultures thereof to produce transglucosylated steviol glycosides. In addition, the Lactobacillus Malus microorganisms or the culture thereof have a high conversion rate from steviol glycoside to transglucosylated steviol glycoside, and thus transglucosylated steviol glycoside can be efficiently produced. Transglucosylated steviol glycosides according to the present disclosure are materials of high intensity sweeteners with improved bitter taste, and the calorie content thereof is not high compared to known transglucosylated steviol glycosides, and thus can be used in various fields.

Drawings

FIGS. 1 to 9 show the HP L C results for transglucosylated steviol glycosides prepared using crude enzyme solutions of Lactobacillus Mali Pumilae.

FIGS. 10 to 16 show HP L C/MS results for transglucosylated steviol glycosides prepared using crude enzyme solutions of Lactobacillus Mali Pumilae strains.

FIG. 17 is a graph showing the conversion of transglucosylated steviol glycosides (stevioside and rebaudioside A) as a function of temperature.

FIG. 18 is a graph showing the conversion of transglucosylated steviol glycosides (stevioside and rebaudioside A) as a function of pH.

FIG. 19 is a graph showing the conversion of transglucosylated steviol glycosides according to the concentration of steviol glycosides (stevioside and rebaudioside A).

[ solution ]

Hereinafter, the present disclosure will be described in detail. Meanwhile, each of the explanations and exemplary embodiments disclosed herein may be applied to other explanations and exemplary embodiments. That is, all combinations of the various elements disclosed herein are within the scope of the present disclosure. Furthermore, the scope of the present disclosure should not be limited by the specific disclosure provided below.

To achieve the object of the present disclosure, one aspect of the present disclosure provides a method for preparing a transglucosylated steviol glycoside using a lactobacillus malate microorganism or a culture thereof.

The term "steviol glycoside" as used herein refers to a natural sweetener having glucose, rhamnose, xylose, etc. linked to 13-OH or 19-OH of chemical formula 1:

[ chemical formula 1]

Generally, in chemical formula 1, in R₁Where hydrogen (H) may be bound, or 1 to 3 glucose molecules may be bound via β -bonds, and at R₂Here, any one of glucose, xylose and rhamnose may be bound via β -bond, and 0 to 2 glucose molecules may be bound thereto via β -bond, but these are not limited thereto.

Steviol glycosides have the advantage that they have fewer calories than sugar and their sweetness is about 200 to 300 times that of sugar; but has the disadvantage that it is accompanied by a unique astringency or bitterness. Therefore, efforts have been made to improve the sweetness of steviol glycosides.

α -/β -glycosidic linkages are distinguished by the relative stereochemistry (R-or S-form) of the anomeric position (anomeric position) and the stereocenter (stereocenter) farthest from the 1-carbon of the monosaccharide (steroenter). generally, when two carbons have the same stereochemistry, a α -glycosidic linkage is formed, whereas when two carbons have different stereochemistry, a β -glycosidic linkage occurs.

The present inventors have found for the first time that Lactobacillus Malus microorganisms and cultures thereof decompose sugars into glucose using sugars and steviol glycosides as substrates, and selectively attach 1 to 4 glucose molecules to the steviol glycosides through α -linkages moreover, the present inventors have found for the first time that enzymes derived from Lactobacillus Malus of the present disclosure have advantages in that they have excellent conversion rates into transglucosylated steviol glycosides, and their odors are reduced and their sweetness is significantly increased compared to existing steviol glycosides.

As used herein, the term "transglucosylated steviol glycoside" may refer to a steviol glycoside having a form in which 1 to 4 glucose molecules are directly added to the 19-OH site of steviol glycoside via α -bond by Lactobacillus malate by using sugar and steviol glycoside as substrates, more specifically, transglucosylated steviol glycoside may be in a form in which 1 to 4 glucose molecules are added to glucose linked to the 19-OH site of steviol glycoside through α - (1,6) bond, but is not limited thereto.

Each step of the process for producing a transglucosylated steviol glycoside will be described in detail. First, in this method, a lactobacillus appli microorganism or a culture thereof may be prepared.

In the next step of the process for the preparation of transglucosylated steviol glycosides, the sugar may be reacted with the steviol glycoside in the presence of a Lactobacillus Mali microorganism or a culture thereof.

For the purposes of this disclosure, a culture may refer to a medium containing cells or a crude enzyme solution without cells the enzyme having glycohydrolytic activity may function to selectively bind 1 to 4 glucose molecules to glucose α -attached to the 19-OH site of a steviol glycoside, but is not limited thereto.

Herein, the steviol glycoside may be one or more selected from the group consisting of stevioside, rubusoside, dulcoside a (dulcoside a), rebaudioside a, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, and rebaudioside M, but is not limited thereto.

Further, the step of reacting the sugar with the steviol glycoside may be performed at a pH of 1 to 10, more specifically at a pH of 2 to 9, or at a pH of 3 to 8, but the pH is not limited thereto.

Further, the step of reacting the sugar with the steviol glycoside may be performed at a temperature of 1 ℃ to 80 ℃, more specifically at a temperature of 5 ℃ to 70 ℃, 10 ℃ to 60 ℃, or 25 ℃ to 50 ℃, but the temperature is not limited thereto.

In addition, Lactobacillus Malus applanatus with transglucosylated steviol glycoside characteristics is characterized by a higher conversion rate from steviol glycoside to transglucosylated steviol glycoside compared to other microorganisms containing known enzymes.

Conversion the conversion of the disclosed transglucosylated steviol glycoside can range from 40% to 90%, but is not limited thereto. More specifically, the conversion may be 40% to 90%, 50% to 80%, 50% to 85%, 60% to 80%, 70% to 85%, or 70% to 80%, but is not limited thereto.

More specifically, the conversion was measured under the following conditions: wherein a culture medium of Lactobacillus Malus having a characteristic of transglycosylated steviol glycoside therein, which is cultured at 30 ℃ for 24 to 48 hours, is centrifuged at 4000 to 8000rpm for 1 to 20 minutes to separate cells and react the crude enzyme solution with a substrate solution containing steviol glycoside and sugar.

Transglucosylated steviol glycoside in a form in which 1 to 4 glucose molecules are directly added to the 19-OH site of steviol glycoside via α -bond, and more particularly, in a form in which 1 to 4 glucose molecules are directly added to the 19-OH site of steviol glycoside via α - (1,6) bond, but not limited thereto.

More specifically, the transglucosylated steviol glycoside prepared according to the above method may be one or more selected from the group consisting of transglucosylated stevioside, transglucosylated rubusoside, transglucosylated dulcitoside A, transglucosylated rebaudioside C, transglucosylated rebaudioside D, transglucosylated rebaudioside E, transglucosylated rebaudioside F, and transglucosylated rebaudioside M, but is not limited thereto.

Yet another aspect of the present disclosure provides a composition for producing a transglucosylated steviol glycoside, which comprises a lactobacillus malate microorganism or a culture thereof.

Yet another aspect of the disclosure provides a sweetener comprising a transglucosylated steviol glycoside prepared by this method. For the purposes of this disclosure, sweeteners are characterized as having reduced off-flavors and enhanced sweetness.

Yet another aspect of the present disclosure provides a method of enhancing the sweetness of a sweetener, comprising producing a transglucosylated steviol glycoside using a lactobacillus malate microorganism or culture thereof.

Detailed Description

Hereinafter, the present disclosure will be described in detail in connection with the accompanying exemplary embodiments. However, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed to limit the scope of the present disclosure.