KR102141864B1 - Method for manufacturing a recrystallized chestnut starch and recrystallized chestnut starch by using thereof - Google Patents

Abstract

The present invention relates to a food and cosmetic composition for improving obesity disease, comprising a method for producing recrystallized chestnut starch through an enzyme and an enzymatic recrystallized chestnut starch obtained by the above method as an active ingredient, more specifically The night starch production method according to the invention is excellent in thermal stability and makes it possible to obtain recrystallized night starch in a high yield, and also, the recrystallized night starch produced by the above method has an anti-obesity effect. It is expected to be useful as a food and cosmetic composition for improving or treating prevention.

Description

Method for manufacturing recrystallized chestnut starch through enzyme and recrystallized chestnut starch prepared using the same {Method for manufacturing a recrystallized chestnut starch and recrystallized chestnut starch by using thereof}

The present invention relates to a method for producing a recrystallized chestnut starch through an enzyme, and more specifically, a method for producing a recrystallized chestnut starch that induces recrystallization of a chestnut starch using amylosecrase and a preparation using the same Recrystallization relates to the use of chestnut starch.

Starch is a glucose polysaccharide present in plants, a primary primary source of energy for humans, and the starch is a rapidly digestible starch (RDS), which is rapidly digested according to the rate at which glucose is released from the gastrointestinal tract and is absorbed. It is divided into (slowly digestible startch; SDS) and resistant starch (RS) that are not digested by digestive enzymes.

At this time, the resistant starch is not absorbed by the small intestine, but it is decomposed by colonic microorganisms to produce short-chain fatty acids similar to dietary fiber, thereby lowering the pH of the colon and improving the intestinal environment, thereby increasing the amount of feces and preventing colon cancer. have.

In the meantime, cases of using physical treatment, chemical treatment, and enzymatic treatment to increase the content of the resistant starch have been reported, and in particular, Neisseria polysacarea ( Neiserria polysaccharea) to be derived from amyl Klein dehydratase (wateuna method to increase the resistant starch content using the enzymatic process that handles the amylosucrase) is mainly used (Korea Patent Registration 10-0956430), poor thermal stability and activity problems Due to this, there was a problem in that industrial availability was poor.

Therefore, the development of a method for producing starch capable of increasing the content of resistant starch through inducing recrystallization of starch through an enzyme having excellent thermal stability and enzyme activity has become a major subject, and research has been conducted on this. It is losing, but it is still incomplete.

The present invention is conceived to solve the above problems, the present inventors have diethoxy no Lactococcus Geo sseoreu Marlies (Deinococcus geothermalis ) was confirmed to have excellent enzymatic activity and thermal stability of amylosucase (DGAS), and based on the results of the above, DGAS was used to induce recrystallization of chestnut starch. As well as confirming that it has an anti-obesity effect, the present invention was completed based on this.

An object of the present invention is to provide an enzymatic recrystallization balm starch production method comprising the following steps.

(a) after the night starch is gelatinized, a step of inducing an enzymatic reaction by treating sucrose and amylosecrase enzyme solution to the gelatinized night starch solution;

(b) adding ethanol to the night starch liquid gelatinized in step (a), followed by precipitating the recrystallized night starch; And

(c) washing the recrystallized chestnut starch precipitated in step (b), followed by drying.

Another object of the present invention is to provide a food composition for improving obesity disease, comprising the enzymatic recrystallized balm starch obtained by the above-described production method as an active ingredient.

At this time, the food composition includes a health functional food composition.

Another object of the present invention is to provide a cosmetic composition for improving obesity disease, comprising the enzymatic recrystallization balm starch obtained by the above production method as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention

A method for preparing enzymatic recrystallization balm starch comprising the following steps:

(a) after the night starch is gelatinized, a step of inducing an enzymatic reaction by treating sucrose and amylosecrase enzyme solution to the gelatinized night starch solution;

(b) adding ethanol to the night starch liquid gelatinized in step (a), followed by precipitating the recrystallized night starch; And

(c) washing the recrystallized chestnut starch precipitated in step (b), followed by drying.

Preferably, in the step (a), the amylosecrase enzyme solution is deenococcus ( Deinococcus) geothermalis ) derived from amylosecrase or Neisseria polysaccharide Polysaccharea ) may be a culture medium or a crushing solution of a transformed strain capable of producing an amylosekra derived.

Preferably, in the step (a), the enzymatic reaction may be performed at 30°C to 50°C for 35 hours to 50 hours.

Preferably, in step (b), ethanol may be 80% to 99% ethanol.

Preferably, the drying in step (c) may be performed through lyophilization.

The present invention provides a food composition for improving obesity disease, comprising the enzymatic recrystallization balm starch obtained by the above production method as an active ingredient.

At this time, the food composition may include a health functional food composition.

In addition, the present invention provides a cosmetic composition for improving obesity disease, comprising the enzymatic recrystallized balm starch obtained by the above-described production method as an active ingredient.

According to the present invention, as a result of inducing recrystallization from chestnut starch using amylosecrase (DGAS) derived from Deinococcus geothermalis , which has excellent enzymatic activity and thermal stability, the specificity of the crystal structure of chestnut starch The change was confirmed. Therefore, since the enzyme has excellent enzymatic activity and thermal stability, it may be possible to obtain recrystallized chestnut starch excellent in thermal stability with high yield when used industrially. In addition, since the anti-obesity effect of the recrystallized chestnut starch was confirmed, it is expected to be useful as a food or cosmetic composition for improving or treating obesity.

Figure 1 shows the results of confirming the crystal structure of the recrystallized chestnut starch obtained according to the present invention by scanning electron microscopy (SEM).
Figure 2 is a result obtained through X-ray diffractometer (M18XHF, Mac Science Co., Yokohama, Japan) to confirm the X-ray diffractogram of the recrystallized chestnut starch obtained according to the present invention It shows.
Figure 3 shows the result of confirming the amylose (amylose) content using the iodine binding index (iodine binding index; IBI) of the recrystallized chestnut starch obtained according to the present invention.
Figure 4 shows the results of confirming the change in the side-chain distribution of amylopectin (amylopectin) by performing a high performance anionic liquid chromatography on the enzymatic recrystallization chestnut starch obtained according to the present invention.
5 is a result showing the resistance starch (RS) content of the enzymatic recrystallization chestnut starch obtained according to the present invention.
Figure 6 shows the results of confirming the weight change of the control group, natural balm starch + high fat diet group and recrystallized balm starch + high fat diet group.
Figure 7 shows the results of checking the weight gain per week of the control group, natural chestnut starch + high fat diet group and recrystallized night starch + high fat diet group.
Figure 8 shows the results of measuring the weight of white fat cells (abdominal fat and epididymal fat) and liver of the control group, natural chestnut starch + high fat diet group and recrystallized night starch + high fat diet group.
9A to 9D show average daily feed, water, and calorie intake during the 8-week experimental period.
Figure 10 shows the results of the glucose load test.
11 shows a result of confirming a change in deep body temperature.
12A and 12B show the results of confirming the expression rates of CPT1 and CPT2, genes related to fatty acid oxidation process (β-oxidation) in liver tissue.

Hereinafter, the present invention will be described in detail.

The present invention is conventional Neisseria polysaccharide ( Neisseria Polysaccharea ) Deinococcus , compared to the original amilosucrase geothermalis ) was confirmed to have excellent enzymatic activity and thermal stability of amyloseucase (DGAS), and thus, excellent industrial availability was confirmed. As a result of inducing recrystallization of starch using DGAS, night starch In addition to confirming the change in the crystal structure in detail, it was confirmed that the anti-obesity effect, and based on this, the present invention was completed.

Accordingly, in one embodiment of the present invention, the Denococcus geothermalis ( Deinococcus geothermalis )-derived recrystallized chestnut starch through treatment with amyloseucase (DGAS) enzyme and Neisseria polysaccharea amyloseurase (NPAS) enzyme-refined chestnut starch with treatment (Example 2) Reference), recrystallized chestnut starch crystal structure, X-ray diffractogram, amylose content, amylopectin branch distribution change and resistance starch content were specifically confirmed (see Examples 3 to 7), and also anti-obesity through the recrystallized chestnut starch The effect was confirmed (see Example 8). In addition, anti-obesity effects of recrystallized chestnut starch using DGAS and recrystallized chestnut starch using NPAS) were compared (see Examples 9 to 11).

Accordingly, the present invention provides an enzymatic recrystallization balm starch production method comprising the following steps.

(a) after the night starch is gelatinized, a step of inducing an enzymatic reaction by treating sucrose and amylosecrase enzyme solution to the gelatinized night starch solution;

(b) adding ethanol to the night starch liquid gelatinized in step (a), followed by precipitating the recrystallized night starch; And

(c) washing the recrystallized chestnut starch precipitated in step (b), followed by drying.

Hereinafter, each step of the method for preparing a recrystallized balm starch according to the present invention will be described in detail.

In the present invention, step (a) is a step of inducing an enzymatic reaction by treating an amylosecrase enzyme solution at night starch. At this time, the chestnut starch was extracted from the domestic chestnut starch using an alkali extraction method, but any extraction method capable of extracting starch through chestnut can be used without limitation. After the extraction of the night starch was completed, gelatinization of the night starch was performed, and the enzymatic reaction proceeded by processing sucrose and amylosecrase enzyme solution on the gelatinized night starch. At this time, the enzyme reaction may be preferably performed at 30°C to 50°C for 35 hours to 50 hours, and more preferably at 40°C for 48 hours, but is not limited thereto.

On the other hand, Neisseria polysaccharia, which is mainly used as a conventional amylosecrase ( Nisseria) Polysaccharea ) derived amylosecrase has excellent synthesis ability of α-1,4 glucan, but has a problem of poor thermal stability and enzyme activity. To this end, in the present invention, in order to solve the above problems, Deenococcus geothermalis ( Deinococcus geothermalis ) It was confirmed that the superior enzymatic activity and thermal stability of the derived amylosecrase was excellent, and thus, the industrial availability was excellent.

Accordingly, the amylosecrase enzyme solution is preferably amylosecrase or Neisseria polysaccharia derived from Deinococcus geothermalis ( Neisseria Polysaccharea ) may be a culture or a lysate of a transformed strain capable of producing amylosekraase, most preferably Dienococcus geothermalis ( Deinococcus geothermalis ) may be a culture medium or a lysate of a transformed strain capable of producing an amylosekra derived.

In the present invention, step (b) is a step of precipitation of the recrystallized night starch by removing the enzyme through ethanol treatment from the night starch gelatinized in step (a). In this case, the ethanol may be 80 to 99% ethanol, preferably 90 to 99% ethanol, and most preferably 95% ethanol.

In the present invention, step (c) is a step of washing and drying the recrystallized chestnut starch precipitated in step (b). The washing is preferably using distilled water, and the drying is preferably performed by lyophilization, but is not limited thereto.

At this time, the term used in the present invention, "freeze drying (freeze drying)" means a method of obtaining a dried product by removing water by sublimating ice by freezing and depressurizing an aqueous solution or a material containing a large amount of moisture. Since the operation is performed at a low temperature, it is useful as a drying method for materials that are weak to heat, and when dried to form a powder, it can be stored at room temperature for a long time, and has an advantage of excellent re-solubility in water.

As another aspect of the present invention, the present invention provides an enzymatic recrystallization balm starch as an active ingredient, provides a food composition for improving obesity disease.

The disease to be improved, prevented, or treated by the composition of the present invention, "obesity", leads to an inadequate condition for health due to an increase in body weight. It means the state that is accumulated as body fat in the form of or has high lipid content in the blood. When the obesity occurs, various metabolic diseases such as diabetes, hyperlipidemia, heart disease, stroke, arteriosclerosis, fatty liver, and adult disease may be caused.

In addition, the composition provided by the present invention can not only treat obesity by inhibiting fat production, but also obesity-related diseases derived from obesity, such as diabetes, hyperlipidemia, heart disease, stroke, arteriosclerosis, fatty liver Disease can be improved, prevented or treated.

As used herein, the term "improvement" means any action that at least reduces the severity of a parameter associated with the condition being treated, such as symptoms. At this time, the health functional food composition may be used before or after the onset stage of the disease in order to improve obesity disease, at the same time as the drug for treatment or separately.

The term "food composition" used in the present invention is a formulation selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, including one or more of carriers, diluents, excipients, and additives. It is characterized by. Foods that can be added to the extract of the present invention include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods. Additives that may be further included in the present invention include natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), colorants, fillers, pactic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, antioxidants, glycerin, alcohol, carbonic acid, and one or more components selected from spheres consisting of flesh can be used. Examples of the natural carbohydrates described above include monosaccharides, such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, etc.; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (taumatine, stevia extract (for example, rebaudioside A, glycyrrhizine, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The composition according to the invention is a variety of nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and neutralizing agents, pactic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, etc. Other compositions in the present invention may contain flesh for the preparation of natural fruit juices and vegetable drinks These components may be used independently or in combination The specific examples of the carrier, excipients, diluents, and additives are not limited thereto, but lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia rubber , Calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylkyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methyl hydroxy benzoate, propylhydroxy benzo It is preferred that at least one selected from the group consisting of eight, talc, magnesium stearate and mineral oil is used.

As another aspect of the present invention, the present invention provides a cosmetic composition for the improvement of obesity disease comprising an enzymatic recrystallization balm starch as an active ingredient.

The cosmetic composition of the present invention may include enzymatic recrystallization balm starch, as well as ingredients commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances Adjuvants, and carriers.

In addition, the composition of the present invention may be used in addition to the enzymatic recrystallization balm starch, organic sunscreens that have been conventionally used to the extent that they do not impair the skin protective effect by reacting with the enzymatic recrystallization balm starch. Examples of the organic sunscreen include glyceryl parva, drometrizolitrisiloxane, drometrizole, digaloyl trioleate, disodium phenyldibenzimidazole tetrasulfonate, diethylhexylbutamidotriazone, diethylamino Hydroxybenzoylhexylbenzoate, die-methoxycinnamate, mixture of Lawson and dihydroxyacetone, methylenebis-benzotriazolyltetramethylbutylphenol, 4-methylbenzylidene camper, menthyl anthranilate, benzophenone -3 (oxybenzone), benzophenone-4, benzophenone-8 (dioxyphenbenzone), butylmethoxydibenzoylmethane, bisethylhexyloxyphenolmethoxyphenyltriazine, cynoxate, ethyldihydroxypropylparva, Octocrylene, ethylhexyldimethylparba, ethylhexylmethoxycinnamate, ethylhexyl salicylate, ethylhexyltrizone, isoamyl-p-methoxycinnamate, polysilicon-15 (dimethycodiethylbenzalmalonate) , Terephthalylidene di camphor sulfonic acid and salts thereof, T- salicylate and one or more selected from the group consisting of aminobenzoic acid (pava) can be used.

As a product to which the cosmetic composition of the present invention can be added, for example, cosmetics and cleansing agents such as convergent makeup, softening makeup, nutrient makeup, various creams, essences, packs, foundations, cleansing agents, soaps, treatments, and cosmetic solutions And so on. Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Contains formulations such as soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, latex, lipstick, makeup base, foundation, press powder, rust powder, and eye shadow.

According to a preferred embodiment of the present invention, the content of the enzymatic recrystallization balm starch of the present invention is 0.00001-30% by weight, preferably 0.5-20%, more preferably 1.0-10% by weight of the total weight of the composition. %to be. If the content of the enzymatic recrystallization balm starch is less than 0.00001% by weight, the ultraviolet absorption effect is greatly reduced, and if it exceeds 30% by weight, skin irritation may occur, and formulation problems may occur.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[ Example ]

Example One. Dienococcus Geothermalis And Nigeria Polysaccharide origin Amylosecrase Production and purification

1-1. Dienococcus Geothermalis ( Deinococcus geothermalis ) Origin Amilosucrase (amylosucrase) ( DGAS ) Production and purification

In this example, amylosucrase was prepared to be used to prepare recrystallized chestnut starch according to the present invention.

More specifically, by using a pHCEXHD expression vector diethoxy no Lactococcus Geo sseoreu Marlies (Deinococcus geothermalis) sucrase azepin recombinant vector pHC-DGAS genes contain as derived from amyl to (His tag including vectors), transformed with pHC-DGAS E. coli MC1061 was prepared. Next, the transformed E. coli was inoculated into 5 ml of LB liquid medium containing ampicillin, cultured at 37° C. for 12 hours, and the culture was completed by centrifugation to obtain cells.

Next, in order to purify His-tag (his-tag)-binding recombinant protein, the transformed E. coli MC1061 was cultured at 7° C. for 15 hours, and then at 4° C. and 4,000 rpm. The cells were collected by centrifugation for 30 minutes. Then, lysis buffer (Lysis buffer) [50 mM NaH ₂ PO ₄ , pH 7.0, 300 mM NaCl, 10 mM imidazole, pH 7.0] was suspended, and ultrasonic grinding was performed to remove the pulverized grinding liquid at 4°C and 12,000. Centrifugation was performed for 10 minutes under the condition of rpm, and after completion of the centrifugation, the supernatant was injected into Ni-NTA affinity chromatography to purify the recombinant protein.

1-2. Nigeria Polysaccharide ( Neisseria polysaccharea ) Origin Amilosucrase ( amylosucrase ) (NPAS) production and purification

In this example, amylosucrase was prepared to be used to prepare recrystallized chestnut starch according to the present invention.

More specifically, using the recombinant vector pET-NPAS containing the Neisseria polysaccharea gene in the pET-21a expression vector (His tag-containing vector), it was transformed into pET-NPAS. E. coli BL21 (DE3) was prepared. Next, the transformed E. coli was inoculated into 5 ml of LB liquid medium containing ampicillin, cultured at 37° C. for 12 hours, and the culture was completed by centrifugation to obtain cells.

Next, in order to purify His-tag (his-tag)-coupled recombinant protein, the transformed E. coli ( E. coli ) BL21 (DE3) was incubated at 37°C for 3 hours, and then 0.1 mM IPTG (Isopropyl) β-D-1-thiogalactopyranoside) was added. Subsequently, the recombinant protein was forcibly expressed by culturing at 18°C for 16 hours.

The cells were collected by centrifugation for 30 minutes at 4°C and 4,000 rpm. Then, lysis buffer (Lysis buffer) [50 mM NaH ₂ PO ₄ , pH 7.0, 300mM NaCl, 10mM imidazole, pH 7.0] was suspended, and ultrasonic grinding was performed to remove the pulverized grinding liquid at 4°C and 12,000 rpm. Centrifugation was performed for 10 minutes under conditions, and after completion of the centrifugation, the supernatant was injected into Ni-NTA affinity chromatography to purify the recombinant protein.

Example 2. Preparation and purification of recrystallized chestnut starch according to the present invention

2-1. Dienococcus Geothermalis origin Amylosecrase Preparation and purification of used recrystallized chestnut starch

Deinococcus geothermalis obtained through Example 1-1 above geothermalis ) Recrystallized chestnut starch was prepared using derived amylosecrase (DGAS). More specifically, first, domestic chestnut starch was extracted using a known alkali extraction method (Isolation and characterization of starches from chestnuts cultivated in three regions of Korea, STARCH, 67(7-8), July 2015, 585-594) , 60 g of the night starch extracted by the above method was gelatinized by adding 50 mM Tris-HCL pH 8.0 buffer. Thereafter, 300 mM sucrose and 0.01 U/L DGAS were added to the luxurious night starch. Next, after adjusting the 2 L volume with 50 mM Tris-HCL pH 8.0 buffer solution, each was reacted at 40°C for 48 hours, and 2 L, 95% ethanol was added to the reaction solution to complete the reaction for 24 hours at 4°C. Allowed to stand, precipitated recrystallized starch. Next, the precipitated recrystallized starch was washed with distilled water, lyophilized, and then pulverized of the dried sample was passed through a 100-mesh sieve to obtain a recrystallized chestnut starch according to the present invention. .

2-2. Nigeria Polysaccharide origin Amylosecrase Preparation and purification of used recrystallized chestnut starch

Neisseria polysaccharide obtained through Example 1-2 above ( Neisseria Polysaccharea ) Recrystallized chestnut starch was prepared using amylosecrase (NPAS). More specifically, first, domestic chestnut starch was extracted using a known alkali extraction method (Isolation and characterization of starches from chestnuts cultivated in three regions of Korea, STARCH, 67(7-8), July 2015, 585-594) , 60 g of the night starch extracted by the above method was gelatinized by adding 50 mM Tris-HCL pH 8.0 buffer. Thereafter, 300 mM sucrose and 0.01 U/L NPAS were added to the luxurious night starch. Next, after adjusting the 2 L volume with 50 mM Tris-HCL pH 8.0 buffer solution, each was reacted at 35°C for 48 hours, and 2 L, 95% ethanol was added to the reaction solution to complete the reaction for 24 hours at 4°C. Allowed to stand, precipitated recrystallized starch. Next, the precipitated recrystallized starch was washed with distilled water, lyophilized, and then pulverized of the dried sample was passed through a 100-mesh sieve to obtain a recrystallized chestnut starch according to the present invention. .

Example 3. Enzymatic recrystallization Crystal structure of chestnut starch

In order to confirm the crystal structure of recrystallized chestnut starch using amenosukraase derived from Dienococcus geothermalis obtained by the method of Example 2-1, using a scanning electron microscopy (SEM, Japan) It was speculative.

More specifically, each starch is frozen at -72°C and dried for 48 hours in a freeze dryer (Freeze Dryer, Ilsin lab Co., Korea), and ion-sputtered with gold-palladium. , C1010 Hitachi, Japan), and microstructures of starch at an acceleration voltage of 20 kV were observed at a magnification of 250 times and 1000 times, respectively.

As a result, as shown in FIG. 1, it was confirmed that the crystal structure was different from the natural chestnut starch structure and that the starch-specific structure was destroyed and recrystallized.

Example 4. Enzymatic recrystallization X-ray diffraction diagram of night starch

In order to confirm the X-ray diffraction pattern of recrystallized starch using amenosukraase derived from Dienococcus geothermalis obtained by the method of Example 2-1, an X-ray diffractometer ( X-ray diffractometer) (M18XHF, Mac Science Co., Yokohama, Japan) using a target: Cu-Kα, voltage: 40 kV, current: 300 mA under the conditions of diffraction angle (2θ) scan speed up to 3.5-40° : X-ray diffraction was observed by diffraction at 3°/min (0.02o Step).

As a result, as shown in FIG. 2, as a result of confirming the X-ray diffraction diagram, it was confirmed that a peak appeared in the vicinity of 2θ = 17°, 19°, and 22°, through which the recrystallized night starch according to the present invention was B It was confirmed that it was recrystallized as a type (B-type).

Example 5. Enzymatic recrystallization Check amylose content of chestnut starch

In order to confirm the amylose content of the recrystallized chestnut starch using amenosukraase derived from Dienococcus geothermalis obtained by the method of Example 2-1, an iodine binding index (IBI) was used. More specifically, 100 mg of the sample was added to 10 ml of a urea-dimethylsulfoxide (Urea-DMSO) solution, and then stirred by boiling the sample well in boiling water for 15 minutes. Then, after standing for 45 minutes in a 100°C dry oven, 1 ml of the sample was mixed with 95 ml of distilled water, and then ₂ ml of an I ₂ -KI solution was added, and distilled water was used as the mixed solution. The dose was adjusted to 100 ml. Next, after reacting the solution for 15 minutes, IBI was measured by measuring the optical density at 660 nm.

As a result, as shown in Figure 3, it was confirmed that the IBI of the recrystallized chestnut starch according to the present invention was significantly increased compared to the natural chestnut starch. Thus, through the above results, it was confirmed that when recrystallization of starch according to the present invention, the amylose content was significantly increased.

Example 6. Enzymatic recrystallization Analysis of changes in amylopectin length of chestnut starch

In order to confirm the change in amylofectin side-chain distribution of recrystallized chestnut starch using amenosukraase from dienococcus geothermalis obtained by the method of Example 2-1, high performance anionic liquid chromatography was performed. Was performed.

More specifically, 50 mg of recrystallized chestnut starch was placed in a container, and 0.5 g of distilled water was added to hydrate, and then 4.5 mL of DMSO was added and heated and stirred for 24 hours. Then, after mixing 2 ml of the sample dissolved in DMSO and 12 ml of 99% ethanol, centrifugation was performed at 1,500 × g for 10 minutes, the supernatant was removed, 12 ml of 99% ethanol was mixed, and 10 at 1,500 × g Centrifugation was performed for a minute. Next, stand in a dry oven at 70° C. for 30 minutes, add 1.8 ml of distilled water to the dried sample, dissolve the sample in boiling water for 30 minutes with stirring, and 20 μl of 40 mM sodium phosphate buffer. Add and mix well. Thereafter, 1 ml was collected and reacted with 2.5 µl of isoamylase at 40° C. for 24 hours, and when the reaction was completed, the reaction product was filtered through a 0.20 µm thin filter paper to inject 50 µl, and the flow rate was analyzed at 0.5 ml/min. In addition, HPAEC analysis was performed using a GS50 gradient pump of EDONEX (Dionex, Sunnyvale, CA, USA) and ED40 detector and PA-200 column, and the solvent was A solution (100 mM NaOH), B solution (10 mM + Analysis was performed using 500 mM Sodium acetate) under the conditions shown in Table 1 below.

Solvent gradient over time Time (min) Gradient (%) A B 0 90 10 10 70 30 16 60 40 27 50 50 44 40 60 60 36 64

As a result, as shown in FIG. 4, it was confirmed that the branch length of amylopectin of the recrystallized chestnut starch increased.

Example 7. Confirmation of the resistance starch content of enzymatic recrystallization chestnut starch

Resistant starch (RS) content of enzymatic recrystallization chestnut starch using amenosukraase from dienococcus geothermalis obtained by the method of Example 2-1 according to the present invention is as follows It was measured.

100 mg of starch was measured in a cap tube, followed by 10 mg/ml of pancreatic α-amylase and 3 U/ml of amyglucosidase (Amyloglucosidase; AMG) 4 ml of the solution was added. Thereafter, to confirm the thermal stability of the reaction product, heat treatment was performed at 100°C for 10 minutes before adding the enzyme, and then mixed well to react for 16 hours in an incubator at 37°C. After this, when the reaction is complete, 4 ml of 99% ethanol is added, mixed and centrifuged at 1,500 × g for 10 minutes to carefully pour out the supernatant, and again, 8 ml of 50% ethanol is added, 1,500 × g Centrifugation was performed for 10 minutes at. This process was repeated twice, the supernatant was decanted, and 2 ml of 2 M KOH was added to each sample, and then a magnetic stirrer bar was added to mix well in a stirrer. Then, 0.1 ml of AMG (Solution 1: 3300 U/ml) was added immediately after 8 ml of 1.2 M sodium acetate buffer (pH 3.8) was added, and the mixture was reacted at 50°C for 30 minutes. At this time, the enzyme-treated sample was diluted with distilled water up to 100 ml, and the enzyme-untreated sample was centrifuged at 1,500 × g for 10 minutes without dilution. Thereafter, 0.1 ml and GOD-POD reagent (Glucose oxidase plus peroxidase and 4-aminoantipyrine) were added to the sample, reacted at 50° C. for 20 minutes, and absorbance was measured at 510 nm. At this time, 0.1 ml of 100 mM sodium acetate buffer (pH 4.5) was added to the blank instead of the sample, and 3 ml of GOD-POD reagent was added and reacted at 50°C for 30 minutes before use.

As a result, as shown in FIG. 5, it was confirmed that the content of the resistant starch of the recrystallized chestnut starch according to the present invention was significantly increased compared to the natural chestnut starch.

Example 8. Confirmation of anti-obesity effect of enzymatic recrystallization chestnut starch

In order to confirm the anti-obesity effect of recrystallized chestnut starch using amenosukraase from dienococcus geothermalis obtained by the method of Example 2-1 according to the present invention, 6-week-old C57BL/6 male rat Was purchased from Hallym Experimental Animal Co., Ltd. and purified for 2 weeks. During this period, litter was mixed with feed at intervals of 2 to 3 days for uniformity of intestinal microbial flora. Subsequently, the group was divided into 4 groups by a randomized complete block design, and 8 animals were placed per experimental group to provide a normal diet to the normal group and 3 groups to provide a high-fat diet of Reserch Diets' Rodent diet with 45% Kcal Fat. . High-fat diets were given orally by administration of natural chestnut starch and enzymatic recrystallized chestnut starch to rats of group 3 except for control group 1, wherein the concentration of resistant starch was 1,500 mg/kgbw and suspended in 200 μl of sterile PBS. After that, it was administered for 10 weeks at a fixed time, 5 times per week. The body weight was measured once a week, and the dietary intake was measured twice a week. The liver, abdominal fat, and epididymal fat were extracted and washed with saline to remove moisture, and then weighed.

As a result, as shown in FIG. 6, it was confirmed that the recrystallized chestnut starch + high fat diet group significantly lowered the weight by 9 weeks compared to the control group and the natural night starch + high fat diet group.

In addition, as shown in FIG. 7, as a result of checking the weight gain per week, it was confirmed that the weight gain in the recrystallized chestnut starch + high fat diet was significantly reduced only in the administration group.

Moreover, as shown in FIG. 8, the content of white fat cells (abdominal fat and epididymal fat) compared to body weight in the recrystallized balm starch + high fat diet group was significantly reduced compared to the control and natural balm starch + high fat diet groups. Was confirmed.

Through this, it was confirmed that when the ingested re-crystallized chestnut starch using a dietary diet derived from Dienococcus geothermalis was consumed together with a high-fat diet, weight loss was confirmed.

Example 9. Preparation and purification of non-enzymatically recrystallized balm starch

For comparison with recrystallized chestnut starch using amylosecrase derived from Dienococcus geothermalis obtained by the method of Example 2-1 of the present invention, non-enzymatically treated recrystallized chestnut starch was prepared.

More specifically, first, domestic chestnut starch was extracted using a known alkali extraction method (Isolation and characterization of starches from chestnuts cultivated in three regions of Korea, STARCH, 67(7-8), July 2015, 585-594) , 60 g of the night starch extracted by the above method was gelatinized by adding 50 mM Tris-HCL pH 8.0 buffer. Next, after adjusting the 2 L volume with 50 mM Tris-HCL pH 8.0 buffer solution, each was reacted at 35°C for 48 hours, and 2 L, 95% ethanol was added to the reaction solution to complete the reaction for 24 hours at 4°C. The precipitated recrystallized starch was precipitated. Next, the precipitated recrystallized starch was washed with distilled water, subjected to lyophilization, and then subjected to pulverization of the dried sample, passed through a 100-mesh sieve to obtain a non-enzymatically treated recrystallized chestnut starch. Obtained.

As a result, as shown in Table 2 below, after the starch was gelatinized, it was left for 48 hours at 40° C. without enzymatic treatment, and when recovered by ethanol precipitation (control, no enzymatic treatment), DGAS was obtained. After 48 hours of treatment at 40° C., the production efficiency when recovering with ethanol precipitation (C-DGAS) was found to be higher when the DGAS treatment was performed.

Production efficiency (%) Control 46.1 ± 1.5 C-DGAS 101.4 ± 2.3

Example 10. Each Dienococcus Geothermalis And Nigeria Polysaccharide origin Amylosecrase Comparison of anti-obesity effects of recrystallized chestnut starch

10-1. Animal experiments and biochemical index analysis

Recrystallized chestnut starch using amylosucrase (DGAS) derived from Deinococcus geothermalis obtained by the method of Example 2-1 of the present invention and obtained by the method of Example 2-2 Neisseria polysaccharea ) To compare the anti-obesity effect of recrystallized chestnut starch using amylosecrase (NPAS), animal experiments were performed as follows.

More specifically, a 6-week-old C57BL/6 male mouse was purchased from the central laboratory animal and purified for 1 week, and litter was mixed at intervals of 2-3 days for uniformity of intestinal microbial flora during the purification period. Subsequently, a total of 7 groups per experiment group were placed in a randomized complete block design, and the normal diet group fed the normal diet and the other 6 groups fed the high-fat diet of Research Diets' Rodent diet with 60% kcal Fat.

As shown in Table 3 below, the high fat diet was supplied to the rats of group 5 except the control group 1 by using natural chestnut starch, recrystallized chestnut starch using each DGAS and NPAS enzyme, and the concentration of starch at this time. Was suspended in 200 μl of sterile PBS at 33 mg/head, and administered 5 times a week for 8 weeks at a constant time. Body weight was measured once a week and dietary intake was measured twice a week.

N. Oral administration concentration Oral administration days Normal diet (ND) 8 200 μl PBS/head 5 times/week High fat diet (HFD) 8 200 μl PBS/head 5 times/week HFD + Control 8 33 mg/200 μl PBS/head 5 times/week HFD + DGAS-modified chestnut powder 8 33 mg/200 μl PBS/head 5 times/week HFD + DGAS-modified chestnut starch 8 33 mg/200 μl PBS/head 5 times/week HFD + NPAS-modified chestnut starch 8 33 mg/200 μl PBS/head 5 times/week HFD + dextrin 8 33 mg/200 μl PBS/head 5 times/week

Then, after fasting for 12 hours at the end of the experiment period, blood was taken through orbital blood collection, liver, abdominal fat, and epididymal fat were extracted and washed with saline to remove the water, and then weighed. The small intestine and large intestine were stored at 4°C immediately after extraction and stored at -70°C until the experiment. Blood was centrifuged at 1,690 x g for 10 minutes to separate serum and used for the experiment. Organs and serum were stored in a -70°C freezer until used in the experiment.

In addition, in order to confirm the tendency of insulin resistance to be improved by DGAS enzyme-treated night starch as an indicator of insulin resistance, a glucose load test (OGTT) was performed to determine insulin resistance and the ability to secrete insulin from pancreatic beta cells. After fasting for more than 12 hours, the body weight is measured and the glucose dose is calculated at a weight of 2 g/kg to prepare glucose by dissolving glucose in drinking water at a concentration of 400 mg/ml. After measurement, glucose was administered orally. Blood glucose was measured in the same manner 30, 60, 90, and 120 minutes after administration. In order to confirm the energy metabolic rate and energy consumption pattern, the deep body temperature of the mouse was measured using a rectal thermometer without fasting on the 10th week of the experiment.

10-2. DGAS And NPAS Using recrystallized night starch Antiobesity Effect comparison

Anti-obesity effect of night starch recrystallized using DGAS and NPAS by calculating daily average feed, water, and caloric intake during the 8-week experiment period through the animal experiment according to the method of Example 10-1. As a result of comparison, as shown in FIGS. 9A to 9D, in the daily feed intake, the high-fat diet had no significant difference compared to the control group, but as a result of converting it into calorie intake, chestnut powder treated with Control and DGAS enzyme (CP- DGAS) group showed fewer calories.

As such, when calculating the total calories consumed during the 8-week experiment period, the fact that the high-fat diet consumed significantly less than the control group may explain some of the effects of suppressing weight gain in the Control and CP-DGAS groups, but CS- In the group ingested with DGAS, CS-NPAS, and dextrin, it was confirmed that the high-fat diet decreased body weight despite ingesting calories similar to the control group.

On the other hand, as a result of the glucose load test, as shown in FIG. 10, it was found that the blood glucose concentration was increased throughout the test process from the fasting state by the high-fat diet, which tended to decrease slightly by ingestion of starch. Looked.

In addition, deep body temperature, such as rectal temperature, is lower than that of a normal body weight when it is obese, and the body's metabolic rate is low, so energy accumulation to fat occurs more efficiently, so when the body temperature is increased by 1 degree, the body metabolic rate is 10~ It is considered to be important for the prevention and treatment of obesity, with an increase in energy consumption of about 100-130 kcal per day in humans in a static state of not exercising and increasing by 13%. Thus, the rectal temperature of the animal was checked to evaluate anti-obesity activity due to increased energy consumption in the body.

As a result, as shown in FIG. 11, the deep body temperature, which means an increase in metabolic rate in the body and an increase in energy consumption through heat generation, was reduced by a high fat diet, which resulted in body fat accumulation and obesity by reduction of energy consumption. I could see that.

On the other hand, it was found that in the group consuming CS-DGAS, the deep body temperature was most similar to that in the normal diet group, which showed that the mechanism for generating heat was higher in the group consuming CS-DGAS than the group consuming other high-fat diets. It may be a direct reason for body fat loss by suggesting that it is actively occurring.

In addition, when comparing the indices such as deep body temperature, calorie intake, and body weight in the CS-DGAS and CS-NPAS groups, similar results were obtained in other indicators, but CS- in deep body temperature. It was confirmed that the CS-DGAS group maintained a significantly higher body temperature than the NPAS intake group.

The anti-obesity effect appears through mechanisms such as body fat reduction through increased energy consumption such as heat generation, absorption of dietary fat or inhibition of body fat accumulation through various mechanisms such as suppression of fat cell differentiation and accumulation. -DGAS intake is estimated to show anti-obesity activity through increased energy consumption, such as heat generation, unlike CS-NPAS.

Example 11. According to the increase in energy consumption in the body Antiobesity Active check

In order to confirm the anti-obesity activity according to the increase in energy consumption in the body through the expression rate of the gene involved in the oxidation reaction of fatty acids, it is a gene related to the fatty acid oxidation process (β-oxidation) in the liver, one of the most active metabolic rates in the body. The gene expression rates of carnitine palmitoyltransferase CPT1 and CPT2 were confirmed.

CPT1 contributes to the formation of acyl-carnitine by promoting the migration of the acyl group from coenzyme A to l-carnitine in the mitochondrial membrane, and the acyl-carnitine thus formed passes through the mitochondrial membrane and passes acyl-CoA to β-oxidation of fatty acids. It is an enzyme that plays an important role in providing. In contrast, CPT2 breaks down acyl-carnitine into acyl-CoA and carnitine.

As a result, as shown in FIGS. 12A and 12B, the expression rate of CPT1 mRNA in liver tissue was decreased by a high fat diet, and it was confirmed that the expression rate was significantly increased when recrystallized chestnut starch was administered. This means that the fatty acid oxidation process in the liver is likely to be accelerated.

From the above, administration of DGAS enzyme-treated night starch in a high-fat diet-induced obesity animal model can be inferred that SCFA was produced according to the content of the resistant starch contained in the DGAS enzyme-treated night starch, which is GPR43-mediated insulin of adipocytes. Signaling is suppressed, and as a result, fat accumulation in adipose tissue is reduced to suppress the increase in body weight and body fat. It can be considered as contributing.

The above description of the present invention is for illustration only, and those skilled in the art to which the present invention pertains can understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (7)

A method for preparing an anti-obesity food composition comprising an enzymatic recrystallization balm starch comprising the following steps:
(a) after the night starch is gelatinized, a step of inducing an enzymatic reaction by treating sucrose and amylosecrase enzyme solution to the gelatinized night starch solution;
(b) adding ethanol to the night starch liquid gelatinized in step (a), followed by precipitating the recrystallized night starch; And
(c) washing the recrystallized chestnut starch precipitated in step (b), followed by drying, wherein the method for producing enzymatic recrystallized chestnut starch,
In the step (a), the enzyme solution of amylose-craase is a culture medium or crushing solution of a transformed strain capable of producing amylo- sucase derived from Deinococcus geothermalis ,
The enzymatic recrystallization balm starch, characterized in that the recrystallization of type B, the production method.
The method of claim 1, wherein the gelatinization of step (a) is gelatinization with a Tris-HCL buffer.
The method of claim 1, wherein the enzymatic reaction in step (a) is performed at 30° C. to 50° C. for 35 hours to 50 hours.
The method of claim 1, wherein in step (b), ethanol is 80% to 99% ethanol.
The method of claim 1, wherein the drying in step (c) is performed through lyophilization.
A food composition for improving obesity disease, comprising the enzymatic recrystallized balm starch obtained by the method of claim 1 as an active ingredient.
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