KR101925096B1 - A manufacturing method of hangover-eliminating enzyme powder and a composition for relieving hangover comprising thereof - Google Patents

Abstract

The present invention relates to a method for producing a hangover-eliminating enzyme powder and a composition for hangover detoxification comprising the same, and more particularly to a method for producing a hangover-releasing enzyme powder, which comprises a) a step of culturing a strain of Acetobacter pasteurianus VA2 in a solid medium containing a yeast extract powder Culturing the yeast extract powder, fermenting the yeast extract powder and fermented juice in a liquid medium to inoculate the strain; b) centrifuging the fermentation broth in step a), and removing the supernatant to obtain acetic acid cells; c) disrupting the acetic acid bacterium obtained in the step b), and then mixing the excipient; And d) drying and pulverizing the mixture of step c), and a pharmaceutical composition and food composition for preventing and eliminating a hangover containing the hangover enzyme powder as an active ingredient. will be.
The method of the present invention is useful for preparing a composition exhibiting an alcohol dehydrogenase (ADH) activity and an acetaldehyde dehydrogenase (ALDH) enzyme activity from acetic acid bacteria having excellent acetic acid production ability and preventing liver damage due to hangover and drinking .

Description

Technical Field [0001] The present invention relates to a method for preparing a hangover enzyme powder and a hangover containing the same,

The present invention relates to a method for producing a hangover-eliminating enzyme powder and a composition for hangover detoxification comprising the same, and more particularly to a method for producing a hangover-releasing enzyme powder, which comprises a) a step of culturing a strain of Acetobacter pasteurianus VA2 in a solid medium containing a yeast extract powder Culturing the yeast extract powder, fermenting the yeast extract powder and fermented juice in a liquid medium to inoculate the strain; b) centrifuging the fermentation broth in step a), and removing the supernatant to obtain acetic acid cells; c) disrupting the acetic acid bacterium obtained in the step b), and then mixing the excipient; And d) drying and pulverizing the mixture of step c), and a pharmaceutical composition and food composition for preventing and eliminating a hangover containing the hangover enzyme powder as an active ingredient. will be.

Alcohol is mainly absorbed in the digestive tract, absorbing 30% in the upper part and absorbing 60% in the small intestine. Absorbed alcohol is metabolized in the liver and the remaining 10% is excreted by breathing, urine, and sweat. Generally, the alcohol absorbed in the body is oxidized to acetaldehyde by the alcohol dehydrogenase (ADH). Acetaldehyde is then oxidized to acetic acid by acetaldehyde dehydrogenase (ALDH), which is used as an energy source in the body (Shumate RP et al, J Forensic Med 14: 83-100, 1967; Lieber CS., Clin Liver Dis 9: 1-35, 2005; Gill, K. et al., Alcohol 13 (4): 347-355, 1996).

In addition, in the process of alcohol decomposition, reactive oxygen species (ROS) is formed during the oxidation of acetaldehyde to acetic acid by ALDH, which causes hangover such as headache, nausea and abdominal pain (Gemma S. et al ., Ann Ist Super Sanita 42: 8-16, 2006)

On the other hand, Acetobacter spp. Is a vinegar producing bacterium which is most important in industry because of its high alcohol tolerance and strong acetic acid resistance. In order to produce acetic acid by acetic acid bacteria, ADH is decomposed into acetaldehyde through oxidation reaction with coenzyme NAD ⁺ ALDH is degraded to produce acetic acid (Kall, L. el al., J. Mol. Biol . 338, 1027-1036, 2004). Currently, the GRAS (generalized as as safe) strain for vinegar of the Korea Food and Drug Administration is the Acetobacter Pasteurianus is permitted (Eun-Jung Yim et al., Korean Journal of Food Preservation , 22 (1), 108-118, 2015).

Recently, it is expected that the effect of continuously ingesting the liver can be enhanced, and when it is consumed before and after drinking, the hangover drink prepared so as to expect hangover relief and liver protection is commercially available, and such a hangover drink The demand for the same activity as the usual one without the hangover phenomenon is increasing, regardless of sex between the ages.

However, existing hangover beverage drinks focus on physiologically active ingredients that inhibit ADH function or promote the activity of ADH, which is an intracerebral enzyme, and many commercial products of medicinal herb extracts are commercialized as a drink, Or temporarily relieving symptoms, not only for the purpose of relieving a hangover or for alcohol, but also for the treatment of headaches such as abdominal pain, headache, nausea, or gastrointestinal problems that occur after drinking.

Therefore, there is a demand for the invention of a hangover relieving product which exhibits a faster hangover resolution effect, can prevent liver damage, and has no adverse effect on the human body.

Therefore, the inventors of the present invention have been studying a hangover remedy which can prevent a hangover effect and liver damage and have no adverse effects on human body, and it has been found that alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) The present inventors have found that the present invention has the effect of rapidly decomposing alcohol and acetaldehyde into acetic acid.

It is therefore an object of the present invention

a) culturing a strain of Acetobacter pasteurianus VA2 in a solid medium containing a yeast extract powder, inoculating the strain into a liquid medium containing a yeast extract powder and fermented alcohol, and fermenting the strain;

b) centrifuging the fermentation broth in step a), and removing the supernatant to obtain acetic acid cells;

c) disrupting the acetic acid bacterium obtained in the step b), and then mixing the excipient; And

and d) drying and pulverizing the mixture of step c).

Another object of the present invention is to provide a pharmaceutical composition for prevention and elimination of hangover which contains the hangover-resolving enzyme powder as an active ingredient.

It is still another object of the present invention to provide a food composition for prevention and elimination of hangover which contains the hangover-resolving enzyme powder as an active ingredient.

To achieve these and other advantages and in accordance with the purpose of the present invention,

a) culturing a strain of Acetobacter pasteurianus VA2 in a solid medium containing a yeast extract powder, inoculating the strain into a liquid medium containing a yeast extract powder and fermented alcohol, and fermenting the strain;

b) centrifuging the fermentation broth in step a), and removing the supernatant to obtain acetic acid cells;

c) disrupting the acetic acid bacterium obtained in the step b), and then mixing the excipient; And

d) drying and pulverizing the mixture of step c).

In order to accomplish still another object of the present invention, the present invention provides a pharmaceutical composition for prevention and elimination of hangover, which contains the hangover-resolving enzyme powder as an active ingredient.

In order to accomplish still another object of the present invention, the present invention provides a food composition for prevention and elimination of hangover which contains the hangover-resolving enzyme powder as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for producing a yeast extract, comprising the steps of: a) culturing an Acetobacter pasteurianus VA2 strain in a solid medium containing a yeast extract powder, inoculating the strain into a liquid medium containing a yeast extract powder and a fermented alcohol, ;

b) centrifuging the fermentation broth in step a), and removing the supernatant to obtain acetic acid cells;

c) disrupting the acetic acid bacterium obtained in the step b), and then mixing the excipient; And

d) drying and pulverizing the mixture of step c).

Each step will be described in detail.

In the method of the present invention, the step a) is carried out in a solid medium containing a yeast extract powder, such as Acetobacter pasteurianus VA2) is cultured, and the strain is inoculated into a liquid medium containing a yeast extract powder and a fermented alcohol to effect fermentation.

The < RTI ID = 0.0 > Acetobacter < / RTI > pasteurianus VA2) is deposited under the deposit number (KCTC13506BP).

In the present invention, the ' Acetobacter pacigianus' strain is acetic acid bacteria corresponding to the genus Acetobacter . Acetobacter spp. Is a gram-negative aerobic bacterium and is also called "acetic acid bacteria". It causes fermentation of acetic acid by changing alcohol to acetic acid by using oxygen, and it is the most important vinegar producing industry in industry because of its high alcohol resistance and strong acetic acid resistance. The optimum temperature for growth is 20 to 30 캜, and when the temperature is below 10 캜 or above 45 캜, the propagation ability is very weak. In addition, if the alcohol concentration is not in the range of 5 to 10%, the bacteria will not develop, and if the concentration of acetic acid produced exceeds 10%, all of them will die. It has been approved as a GRAS (generally recognized as safe) strain for vinegar by the Korean Food and Drug Administration (Eun-Jung Yim et al., Korean Journal of Food Preservation , 22 (1), 108-118, 2015).

In the step a) of the present invention, the liquid medium comprises 0.5 to 1% by weight of the yeast extract powder.

In the production example of the present invention, the strain is cultured in a solid culture medium composed of yeast extract powder, mannitol and peptone for 16 to 24 hours, and then cultured in a liquid culture medium containing yeast extract powder, soytone, glucose, MnSO ₄ After mixing the fermented alcohol, the strain was inoculated and cultured.

In the step (a) of the present invention, 1 to 5% by weight of the fermentation alcohol is mixed.

The 'fermented liquor' of the present invention means a fermented product of rice, barley, sweet potato, tapioca, sugarcane, sugar beet, etc. as a raw material by microorganisms or enzymes and then distilled using a continuous distillation method. do. It is mainly used as a main raw material for alcoholic drinks. In addition, it is widely used in food, medicine, beverage, cosmetics and other industrial use areas, and preferably ethanol.

The term 'fermentation' of the present invention means a process in which a microorganism decomposes an organic substance using an enzyme of its own, and preferably means 'acetic acid fermentation'. The acetic acid fermentation is also called acetic acid fermentation, and acetic acid bacteria (acetic acid bacteria) oxidize alcohol to produce acetic acid through acetaldehyde. The optimum temperature for fermentation may be 20 to 30 캜, which is the optimum temperature for the growth of acetic acid bacteria, and preferably fermented for 24 to 36 hours at a temperature of 25 to 30 캜 in the present invention.

In the method of the present invention, in the step b), the culture broth fermented in the step a) is centrifuged and the supernatant is removed to obtain acetic acid cells.

The 'fermented culture solution' of the present invention means a culture solution obtained by fermentation of acetic acid with acetic acid. After fermentation, the concentration of acetic acid should be less than 10% by weight. Preferably, the pH value of the culture medium is 3.5 to 4.0, and the acetic acid concentration is 2.0 to 4.0 wt%.

In the preparation example of the present invention, the fermented culture broth was centrifuged at 10,000 x g for 20 minutes, the supernatant was removed, and the precipitate was washed with physiological saline two to three times to obtain acetic acid cells.

In the present invention, the step c) is a step of disrupting the acetic acid bacterium obtained in the step b), followed by mixing the excipient.

The 'excipient' of the present invention may be selected from the group consisting of starch, glucose, cellulose, lactose, glycogen, D-mannitol, sorbitol, lactitol, maltodextrin, calcium carbonate, synthetic aluminum silicate, calcium monohydrogenphosphate, calcium sulfate, , Hydroxypropylmethylcellulose, propylene glycol, casein, calcium lactate, pre-mosel, gum arabic, and the like, and may be, for example, sodium lauryl sulfate, sodium lauryl sulfate, Starch, glucose, cellulose, lactose, dextrin, glycogen, D-mannitol, maltodextrin, and most preferably maltodextrin.

In the preparation example of the present invention, the obtained acetic acid bacterium was disrupted by a high-pressure disruption method using a cell crusher, and then maltodextrin was added to the disrupted acetic acid bacterium body solution at a concentration of 50 to 60% by weight of the acetic acid bacterium.

The 'maltodextrin' of the present invention is a kind of dextrin which is formed by decomposing starch into dilute acid or amylase. It means a low molecular dextrin having a lower degree of polymerization than that of acrodextrin and having a maltose content. Maltodextrin is a functional saccharide which is widely used in food such as a protein denaturing effect, a masking effect of food, and a soft texture. Maltodextrins are maltodextrins, which are maltose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltohexaose, maltodextrins, G7, maltoheptaose, maltooctaose, malononaose, and the like.

In the present invention, the step d) is a step of drying and pulverizing the mixture of the step c).

In the production example of the present invention, the acetic acid bacteria solution mixed with the soil dextrin was dried for 2 to 4 days by using a freeze dryer, and the dried product was pulverized and powdered and powdered.

The 'hangover elimination enzyme powder' of the present invention is characterized by containing an alcohol dehydrogenase (ADH) and an aldehyde dehydrogenase (ALDH).

The above-mentioned 'alcohol dehydrogenase (ADH)' is an enzyme that catalyzes the reaction of removing hydrogen from alcohol to generate aldehyde or ketone. It contributes to chemical changes in alcohol that can be poisonous in humans and in many other animals, yeast, higher plants, bacteria, and the like.

The 'aldehyde dehydrogenase (ALDH)' is an enzyme that generates a carboxylic acid or an acyl group by oxidizing acetaldehyde produced by an alcohol, and is present in microorganisms, green plants, animals, and the like. Liver enzymes mainly produce acetic acid in acetaldehyde. In the process of decomposing acetaldehyde in the body, reactive oxygen species (ROS) is formed, which causes hangover such as headache, nausea and abdominal pain.

The present invention provides a pharmaceutical composition and a food composition for preventing and eliminating hangovers containing the hangover-eliminating enzyme powder as an active ingredient.

In the pharmaceutical composition and the food composition of the present invention, the hangover elimination enzyme powder and the grain fermentation enzyme powder may be mixed in a ratio of 1: 0.5 to 5, preferably 1: 0.5 to 3, Most preferably 1: 1 by weight.

The pharmaceutical composition according to the present invention may be formulated into a suitable form together with the hangover elimination enzyme powder alone or together with a pharmaceutically acceptable carrier, and may further contain an excipient or a diluent. &Quot; Pharmaceutically acceptable " as used herein refers to a nontoxic composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery materials used for oral administration to peptide preparations. In addition, the carrier for parenteral administration may contain water, a suitable oil, a saline solution, an aqueous glucose and a glycol, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components. Other pharmaceutically acceptable carriers and preparations can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

The composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration Lt; / RTI >

The pharmaceutical composition of the present invention may be formulated into oral or parenteral dosage forms according to the route of administration as described above.

In the case of a preparation for oral administration, the composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a liquid, a gel, a syrup, a slurry, . For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include, but are not limited to, sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition of the present invention may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In the case of a preparation for parenteral administration, it can be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol and nasal aspirate. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa., 1995, which is a commonly known formulary for all pharmaceutical chemistries.

 The total effective amount of the composition of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses. In the pharmaceutical composition of the present invention, the content of the active ingredient may be varied depending on the degree of the disease. Preferably, the total preferred dose of the pharmaceutical composition of the present invention may be from about 0.01 μg to about 10,000 mg, and most preferably from about 0.1 μg to 500 mg, per kilogram of patient body weight per day. However, the dosage of the pharmaceutical composition may be determined depending on various factors such as the formulation method, administration route and frequency of treatment, as well as the patient's age, body weight, health condition, sex, severity of disease, diet and excretion rate, It will be possible to determine the appropriate effective dose of the composition of the present invention by those of ordinary skill in the art in view of this point. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.

The food composition using the hangover elimination enzyme powder according to the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. These types can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the food composition itself of the present invention may be prepared in the form of tea, juice, and drink and then consumed, granulated, encapsulated, and powdered. In addition, the food composition of the present invention can be prepared in the form of a composition by mixing together with a known substance or active ingredient known to have a hangover resolution effect.

Functional foods also include beverages (including alcoholic beverages), fruits and their processed foods (such as canned fruits, bottled, jam, maalmalade, etc.), fish, meats and processed foods such as ham, (Such as udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, , Vegetable protein, retort food, frozen food, various kinds of seasoning (for example, soybean paste, soy sauce, sauce, etc.).

The preferred content of the food composition according to the present invention is not particularly limited, but is preferably 0.01 to 50% by weight based on the total weight of the final food product. In order to use the food composition of the present invention in the form of a food additive, it may be used in the form of powder or concentrate.

In one embodiment of the present invention, the Acetobacter pasteurianus VA2, ATCC (American Type Culture Collection) and the other Acetobacter sold in KCTC (Korean Collection for Type Cultures) As a result of comparing the enzymatic activities of the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH) of four strains of Pasteurianus and four kinds of lactic acid bacteria (Lb. brevis, Lb. reuteri, Lb. fermentum and Lb. plantarum) (See Example 2, Table 1 and Table 2). In addition, it was confirmed that the enzymatic activity of Acetobacter pacrylanus VA2 used in Example 2 was high.

In another embodiment of the present invention, the hangover elimination enzyme powders of different concentrations of the present invention were each treated with alcohol and reacted for 3 hours. The changes in alcohol content and acetaldehyde content were measured, It was confirmed that both the alcohol concentration and the acetaldehyde content were further reduced when the hangover resolution enzyme powder was treated as compared with the control group (see Example 3, Figs. 2 and 3).

In another embodiment of the present invention, rats were administered hangover elimination enzyme powder, complex, and dairy products, respectively. After 30 minutes, alcohol was administered, and blood was collected over time to measure blood alcohol concentration and acetaldehyde concentration As a result, the alcohol concentration increased rapidly after 30 minutes in the alcohol-treated group, and the blood alcohol concentration and blood acetaldehyde concentration were lower when the hangover enzyme powder was administered compared with the control group, It was confirmed that the dissolving enzyme powder had an effect of rapidly decomposing alcohol in the body (see Example 4, Figs. 4 and 5).

In another embodiment of the present invention, the rats were administered the hangover elimination enzyme powder, complex, and dawning, respectively. After 30 minutes, the alcohol was administered to the rats and the blood was collected to determine the hepatotoxicity index AST (aspartate aminotransferase), ALT alanine transferase in the control group showed that the concentration of AST and ALT in the control group was increased and the concentration of AST and ALT in the blood was decreased when the hangover enzyme powder was treated (see Examples 5 and 6) 6).

In another embodiment of the present invention, the rats were administered the hangover elimination enzyme powder, complex, and date of birth, and after 30 minutes of alcohol administration, liver tissues were extracted and liver tissues were observed by H & E staining. As a result, And the liver damage was increased in the control group compared to the control group. The treatment of hangover enzyme powder showed less liver damage, and when the high concentration hangover enzyme powder was treated, the liver condition was almost similar to that of the normal group (See Example 6 and Fig. 7).

Accordingly, the present invention provides a process for producing a hangover-eliminating enzyme powder and a composition for hangover solubility comprising the same. The method of the present invention is useful for preparing a composition exhibiting an alcohol dehydrogenase (ADH) activity and an acetaldehyde dehydrogenase (ALDH) enzyme activity from acetic acid bacteria having excellent acetic acid production ability and preventing liver damage due to hangover and drinking .

Fig. 1 schematically shows a process for preparing hangover elimination enzyme powder.
FIG. 2 is a graph showing the concentration of alcohol (FIG. 2A) and the rate of alcohol concentration decrease (FIG. 2B) over time when 5% alcohol was administered at different concentrations of hangover enzyme powder.
FIG. 3 is a graph showing changes in acetaldehyde concentration over time when 5% alcohol was administered at different concentrations of hangover enzyme powder. FIG.
FIG. 4 shows the blood alcohol concentration (FIG. 4A) and the blood concentration-time-area under-curve (AUC) (FIG. 4B) over time when the hangover resolution enzyme powder was administered to an animal model.
FIG. 5 shows the blood acetaldehyde concentration (FIG. 5A) and the blood concentration-time-area under-curve (AUC) (FIG. 5B) over time when the hangover resolution enzyme powder was administered to an animal model.
FIG. 6 shows the blood level of aspartate aminotransferase (AST) (FIG. 6A) and the alanine aminotransferase (ALT) blood concentration (FIG. 6B) as hepatotoxicity index when the hangover elimination enzyme powder was administered to an animal model.
Fig. 7 shows the result of observing the liver tissue when the hangover-eliminating enzyme powder was administered to an animal model. Fig.
Statistical significance of the control of the control group in the drawings ^{* p <0.05, ** p <} 0.01, *** p <0.001 , and the test group relative to the control group (L-CA, H-CA , CA + GF) and positive statistical the statistical significance of the control group (YM) is ^{# p <0.05, ## p <} 0.01, ### p <0.001 , and the group (L-CA, H-CA , CA + GF) for the positive control (YM) The significance is ^$ p <0.05.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Manufacturing example  1> hangover elimination enzyme powder

Acetobacter pasteurianus VA2) were fermented with acetic acid to prepare an enzyme complex containing ADH and ALDH expressed therein. The concrete method is as follows.

The cultured bacteria were cultured in a pre-culture medium for 16 to 24 hours to a concentration of 0.5% of yeast extract powder, 2.5% of D-mannitol and 0.3% of peptone. Then, a mixture of 0.5% to 1% of yeast extract powder, 0.5% to 1.5% of soytone, 2.5% to 7.5% of hydrous crystalline glucose, and 0.01% to 0.05% of manganese sulfate were mixed to prepare a fermented alcohol of 1% to 5% After the mixture was inoculated with the pre-cultivated strain, it was cultured at 25 to 30 DEG C at 200 to 300 rpm for 24 to 36 hours. After cultivation, the pH is preferably 3.5 to 4.0, and the acetic acid concentration is preferably 2.0 to 4.0 wt%. Then, the supernatant was removed by centrifugation (10,000 x g, 20 min), and the precipitate was washed with 0.85% physiological saline two to three times. The precipitate was suspended in a potassium phosphate buffer solution (pH 7.0-8.0) at a concentration of 20 to 50% by weight, and acetic acid cells were disrupted with a cell crusher. Then, maltodextrin excipient was added to the crushed acetic acid bacteria solution in an amount of 50 to 60% by weight of acetic acid cell mass and stirred. After drying for 2 to 4 days in a freeze dryer, the dried product was pulverized and powdered to prepare a hangover-eliminating enzyme powder. This process is shown in Fig.

Then, the hangover elimination enzyme powder was administered to an animal model at a concentration of 103 mg / kg, hereinafter referred to as L-CA, administered at a concentration of 206 mg / kg, and expressed as H-CA.

< Manufacturing example  2> Hangover elimination enzyme complex

A hangover elimination enzyme complex was prepared by adding a grain fermentation enzyme powder (Application No. 10-2017-0080405) to the hangover elimination enzyme powder obtained in <Preparation Example 1>. Then, the animal model was administered with 103 mg / kg of hangover enzyme powder and 103 mg / kg of grain fermentation enzyme powder (1: 1 weight ratio).

< Comparative Manufacturing Example  1>

As a control group, a third-party product, Yeast 808, was administered at a concentration of 12 ml / kg, hereinafter referred to as YM.

< Example  1>

Determination of enzymatic activity of hangover enzyme powder

<1-1> Analysis of enzyme activity of alcohol dehydrogenase

First, 200 μl or more of the prepared sample was placed in an E-tube and heated at 100 ° C. for 30 minutes to remove the enzyme activity. 1.3 mL of 50 mM sodium phosphate buffer (pH 8.8) and 1.5 mL of 15 mM β-NAD solution were mixed with 0.1 mL of 95% (v / v) ethanol to prepare a substrate reaction solution such that the total solution was 2.9 mL.

6.67 占 퐇 of the sample and 193.3 占 퐇 of the reaction solution prepared above were mixed and dispensed in a 96-well microplate (blank = enzyme solution heated at 100 占 폚 for 30 minutes). After measuring the absorbance at 340 nm and 25 ° C for 0-10 minutes using a multiple reader, the enzyme activity of the alcohol dehydrogenase was analyzed using the following equation (1).

<1-2> Acetate Aldehyde  Dehydrogenase activity assay

First, 200 μl or more of the prepared sample was placed in an E-tube and heated at 100 ° C. for 30 minutes to remove the enzyme activity. 100 μl of 20 mM β-NAD solution, 100 μl of 3M KCl, 50 μl of 100 mM acetaldehyde solution, and 30 μl of 1 M 3-mercaptoethanol solution were mixed, To prepare a reaction solution. Then, 6.67 μl of the sample and 193.3 μl of the reaction solution were mixed and shaken (blank = enzyme solution heated at 100 ° C. for 30 minutes) in a 96-well microplate. Thereafter, the absorbance was measured at 340 nm and 25 ° C for 0-10 minutes using a multiple reader, and the acetaldehyde dehydrogenase activity was analyzed using the following formula (1).

<Formula 1>

※ 1 unit contains enzyme activity (pH 8.8, 25 ℃) which decomposes 1.0 umol of ethanol with acetaldehyde or 1.0 um of acetaldehyde with acetic acid for 1 minute.

< Example  2>

Comparison of Enzyme Activity of Acetic Acid Bacteria

<2-1> Comparative analysis of enzymatic activities of different kinds of bacteria

In order to compare enzyme titers of A. pasteurianus VA2 and A. pasteurianus strains used in the preparation of the hangover elimination enzyme powder of the present invention, the following experiment was conducted.

A. pasteurianus was distributed from ATCC (American Type Culture Collection) and KCTC (Korean Collection for Type Cultures), A. pasteurianus VA2 (KCTC 13506BP) was obtained from natural fermented persimmon.

Acetobacteria strains (VA2) and A. pasteurianus strains were cultured under the same culture conditions and cultured and then subjected to high pressure disruption. Then, enzyme activity was measured according to the method described in Example 1, and the enzyme activity of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) was compared.

As a result, as shown in Table 1, the acetic acid bacterium (VA2) used for the hangover elimination enzyme powder of the present invention had an ADH activity of 7.53 unit / g and an ALDH activity of 2.77 unit / g. As a result, it was confirmed that the enzyme activity was higher than that of other acetic acid bacteria.

<2-2> Acetic acid bacteria  Comparison of Enzyme Activity of Lactic Acid Bacteria

Lactobacillus, Lb. reuteri, Lb. fermentum, Lb. reuteri, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus reuteri, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus acidophilus, plantarum), the following experiment was carried out.

Lactobacillus brevis, Lb. reuteri, Lb. fermentum and Lb. plantarum were identified by ATCC (American Type Culture Collection), KCTC (Biological Resources Center, Korean Collection for Type Cultures), KFRI (Korea Food Research Institute), KACC (National Institute of Agricultural Science and Technology), and cultured by adding 1 to 3% of ethanol to the optimal medium of each strain.

Then, each of the lactic acid bacteria was subjected to high-pressure disruption, and the activity of the enzyme was measured according to the method described in Example 1, as in the <Example 2-1>, and the alcohol dehydrogenase (ADH) and the acetaldehyde dehydrogenase (ALDH) Enzyme activity was compared.

As a result, as shown in Table 2, Lb. brevis showed the highest ADH activity, and Lb. reuteri showed the highest ALDH activity, but it was confirmed that the activity of ADH and ALDH was lower than that of the acetic acid bacterium (VA2) used in the present invention.

&Lt; Example 3 >

Alcohol degradation effect of hangover elimination enzyme powder

In order to confirm the alcohol decomposing effect of the hangover elimination enzyme powder prepared according to the method described in the above Production Example, the following experiment was conducted.

First, the hangover enzyme powder prepared in Preparation Example 1 was mixed with 5% alcohol at 1% (w / v) and 3% (w / v) ) Were mixed. Then, the reaction was carried out at 25 DEG C at 100 rpm for 3 hours. The mixture was reacted and sampled at intervals of 0, 1, and 3 hours to measure the alcohol contained in the mixture. The content of alcohol was determined by the specific gravity method through the esophagus of the General Test Methods for Ethanol in Food and Drug Administration, Food and Drug Administration, the Republic of Korea.

50 mL of the mixed solution was boiled in boiling water using an alcohol distillation apparatus for 20 minutes to distill the alcohol. After distilled alcohol was collected, it was diluted to 4 times (v / v) with respect to 50 mL of the mixed solution to a final volume of 200 mL, and then cooled to 15 째 C, and the alcohol content was measured by floating the incubation system.

As for the method for confirming the reduction of acetaldehyde, the hangover elimination enzyme powder was mixed with 1% (w / v) and 3% (w / v) in 5% And reacted for 3 hours. The acetaldehyde assay kit (megazyme) was used to measure the amount of acetaldehyde after sampling at 0, 0.5, 1, 3, 5, 7, 10, and 22 hours after mixing.

The results are shown in Fig. 2 and Fig.

As shown in FIG. 2, when the hangover elimination enzyme powder was mixed, the alcohol concentration was decreased compared with the control group, and the alcohol concentration was further decreased according to the enzyme powder content.

As shown in Fig. 3, the acetaldehyde content of the control group showed almost no change, and the hangover enzyme enzyme powder 3% (w / v) / v), the acetaldehyde content decreased more rapidly.

It was confirmed that the hangover elimination enzyme powder had the effect of reducing the alcohol, and the alcohol decomposition rate was faster than the concentration.

< Example  4>

Hangover resolution Enzymatic powder is a blood alcohol and To acetaldehyde  Impact

Hangover elimination To investigate the effect of enzyme powder on alcohol degradation in the body, the following experiment was conducted.

First, Sprague-Dawley (SD) rats were purchased and allowed to circulate for one week under conditions of a temperature of 22 ± 2 ° C, a humidity of 40 to 60%, and a light-dark cycle of 12 hours. Thereafter, the H-CA group receiving 10 mg of each of the normal, non-treated, alcohol-only control, L-CA group receiving 103 mg / kg of hangover dissolution enzyme powder and 206 mg / kg hangover enzyme powder , The CA + FG group to which the hangover elimination enzyme complex of Preparation Example 2 was administered, and the YM group to which 12 mg / kg of YM was administered at the concentration of 12 ml / kg. And fasted for 12 hours and then administered orally to each group.

After 30 minutes of administration, 0.4 g / ml of alcohol (40% alcohol) was orally administered, and blood was collected through the veins after 0, 1, 3, 5 and 8 hours. The blood was centrifuged at 3,000 rpm for 15 minutes to separate the serum. The serum was then measured using a commercial alcohol concentration measurement kit (EnzyChrom ™ Ethanol Assay Kit (BioAssay System, USA)) and an Enzymatic Acetaldehyde Assay Kit , USA) was used to measure the absorbance at 565 nm by ELISA and the blood concentration was measured. Then, the blood concentration-time-area under curve (AUC) was calculated using the following equation (2).

<Formula 2>

AUC = ((0 hour concentration + 1 hour concentration) * 1) / 2) + (((1 hour concentration + 3 hour concentration) * 2) / 2) + Concentration + 5 hours concentration) * 2) / 2) + (((5 hours concentration + 8 hours concentration) * 3) / 2)

As a result, as shown in Fig. 4, in all the groups except the normal group, blood alcohol concentration increased rapidly after 30 minutes of alcohol administration. In comparison with the control group, L-CA group, H- FG group, and YM group showed low alcohol content. In addition, in the H-CA group and the CA + FG group, the blood alcohol concentration was lower than that in the YM group.

As shown in FIG. 5, in all the groups except the normal group, blood acetaldehyde concentration was rapidly increased after 1 hour of alcohol administration. In comparison with the control group, L-CA group, H-CA group, CA + FG group and YM group showed low acetaldehyde content.

&Lt; Example 5 >

Effect of hangover elimination enzyme powder on serum AST and ALT concentration

AST (alanine aminotransferase) and alanine transferase (ALT) were used as a marker of hepatotoxicity. In order to confirm the effect of hangover enzyme powder on blood AST and ALT levels, The following experiment was conducted.

Animal experiments were carried out in the same manner as described in Example 4 above, and the rats were administered hangover elimination enzyme powder, complex, and dairy, and oral administration of 0.4 g / ml of alcohol (40% alcohol) After 0.5, 1, 3, 5, and 8 hours of administration, blood was collected through the vein. The blood was centrifuged at 3,000 rpm for 15 minutes to separate the serum and then the commercial AST, ALT kit (GOT, GPT Assay kit (Asan Pharmaceutical Co., Seoul, Korea)) was used and the absorbance at 490 nm was measured by ELISA And the blood concentration was measured.

As a result, as shown in FIG. 6, the blood levels of AST and ALT in the control group were significantly increased compared to the normal group. In addition, AST and ALT levels in the L-CA, H-CA, CA + FG, and YM groups were decreased compared to the control group. In particular, in the H-CA group and the CA + FG group, the ALT content was significantly decreased as compared with the YM group.

Thus, it was confirmed that the hangover elimination enzyme powder and the hangover elimination enzyme complex of the present invention are excellent in the hangover resolution effect and have the effect of preventing liver damage.

&Lt; Example 6 >

Effect of hangover elimination enzyme powder on liver tissue

Animal experiments were carried out in the same manner as described in Example 4, and the rats were administered hangover elimination enzyme powder, complex, and dairy, and oral administration of 0.4 g / ml (40% alcohol) . One hour after the administration of the alcohol, the rat embryo was removed and liver tissues were extracted. The tissue was then paraffin-embedded by a method commonly used in the art and then sliced to a thickness of 3-4 [mu] m. Tissue sections were stained with H & E (hematoxylin andeosin) and observed under an optical microscope.

As a result, as shown in FIG. 7, liver damage was more intensified in the control group than in the normal group. On the other hand, liver damage was less in the L-CA group (low), H-CA group (high), CA + FG group (grain) and YM group (dawn). In the H-CA group (high) and CA + FG group (grain), the liver status was almost similar to that of the normal group.

Thus, it was confirmed that the hangover elimination enzyme powder and the hangover elimination enzyme complex of the present invention are effective in inhibiting liver damage.

As described above, the method of the present invention can produce a composition exhibiting an alcohol dehydrogenase (ADH) activity and an acetaldehyde dehydrogenase (ALDH) enzyme activity from acetic acid bacteria having excellent acetic acid producing ability to prevent hangover and alcoholic liver damage As shown in FIG.

Korea Biotechnology Research Institute KCTC13506BP 20180328

Claims (13)

a) Acetobacter pasteurianus strain deposited with the deposit number KCTC13506BP in a solid medium containing a yeast extract powder was cultured, and then cultured in the above solid medium in a liquid medium in which yeast extract powder and fermented alcohol were mixed, Inoculating a strain to be fermented;
b) centrifuging the fermentation broth in step a), and removing the supernatant to obtain acetic acid cells;
c) disrupting the acetic acid bacterium obtained in the step b), and then mixing the excipient; And
d) drying and pulverizing the mixture of step c).
The method according to claim 1, wherein the hangover enzyme powder comprises an alcohol dehydrogenase (ADH) and an aldehyde dehydrogenase (ALDH).
delete [2] The method according to claim 1, wherein the liquid medium in step a) comprises 0.5 to 1% by weight of the yeast extract powder.
[2] The method according to claim 1, wherein 1 to 5% by weight of fermentation alcohol is mixed in step a).
The method according to claim 1, wherein the fermentation alcohol in step (a) is ethanol.
The method according to claim 1, wherein the fermentation in step a) is performed at a temperature of 25 to 30 ° C for 24 to 36 hours.
The method according to claim 1, wherein the culture medium in step b) has a pH of 3.5 to 4.0 and a nitric acid concentration of 2.0 to 4.0% by weight.
The method of claim 1, wherein the excipient is selected from the group consisting of starch, glucose, cellulose, lactose, glycogen, D-mannitol, sorbitol, lactitol, maltodextrin, calcium carbonate, synthetic aluminum silicate, calcium monohydrogen phosphate, Which is composed of sodium chloride, sodium hydrogen carbonate, refined lanolin, dextrin, sodium alginate, methylcellulose, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose, propylene glycol, casein, calcium lactate, Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;
The method according to claim 1, wherein mixing the excipient in step c) comprises mixing maltodextrin at 50 to 60% by weight of acetic acid cell mass.
A pharmaceutical composition for prevention and elimination of hangover comprising the hangover-resolving enzyme powder of claim 1 as an active ingredient.
A food composition for prevention and elimination of hangover comprising the hangover-eliminating enzyme powder of claim 1 as an active ingredient.
The composition according to claim 11 or 12, wherein the composition is prepared by mixing the hangover dissolving enzyme powder and the grain fermentation enzyme powder at a weight ratio of 1: 0.5 to 5: 1.

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