KR20220117742A - Hangover Treatment Composition Which Comprises Aldehyde Dehydrogenase and Glutathione - Google Patents

Abstract

The present invention relates to a hangover relief composition containing dry powder, lysate or extract of yeast producing glutathione and acetaldehyde dehydrogenase. More specifically, the present invention relates to a hangover relief composition containing dry powder, lysate or extract of Saccharomyces cerevisiae Kwon P-1 KCTC 13925BP, Saccharomyces cerevisiae Kwon P-2 KCTC14122BP, and Saccharomyces cerevisiae Kwon P-3 KCTC14123BP yeasts which simultaneously produce glutathione and acetaldehyde dehydrogenase.

Description

Hangover Treatment Composition Which Comprises Aldehyde Dehydrogenase and Glutathione

The present invention relates to a hangover reliever containing glutathione (GSH) and aldehyde dehydrogenase (hereinafter, ALDH). More specifically, the present invention Saccharomyces cerevisiae Kwon P-1 KCTC 13925BP and Saccharomyces cerevisiae Kwon P-2 KCTC14122BP or Saccharomyces cerevisiae Kwon P-3 KCTC14123BP It is about a hangover reliever containing glutathione derived from yeast and aldehyde dehydrogenase at the same time.

Alcohol is a favorite food with human history, but excessive drinking leads to a hangover that causes physical and mental discomfort, nausea, vomiting, dizziness, thirst, lethargy, drowsiness, headache, and abnormalities in the brain and nervous system (Alcohol Use Disorder). , AUD) (Shao-Cheng Wang et al, 2020) is emerging as a social problem that induces severe alcohol addiction and even mental panic disorder (Choi Song-sik 2013).

Alcohol is absorbed 5% from the oral cavity, 10-15% from the stomach, 80% from the small intestine and enters the bloodstream, 2-4% from the lungs, 2-4% from the kidneys, 2-6% from sweat, and the liver. 90% is decomposed in

Alcohol dehydrogenase in the liver is oxidized by alcohol dehydrogenase (ADH), converted to acetaldehyde, and again oxidized and detoxified by aldehyde dehydrogenase (ALDH).

However, 15% to 50% of Asians who lack aldehyde dehydrogenase or genetically have an aldehyde dehydrogenase allele (ALDH2*2) are unable to break down acetaldehyde that occurs when drinking alcohol, resulting in blushing alcohol. Syndrome (Alcohol Flushing Syndrome) (Brooks, PJ et al . 2009) appears, and is reported to have a high probability of getting alcoholism or liver disease due to acetaldehyde accumulation (Larson, HN et al , 2007). Acetaldehyde is not decomposed and remains in the human body, causing death and disability due to alcoholic hepatitis or liver cirrhosis (Gilpin, NW. et al , 2008).

On the other hand, excessive residual of aldehydes in the body produced by alcohol intake is associated with cardiovascular diseases, diabetes, neurodegenerative diseases, upper digestive and respiratory tract cancer, radiation dermatitis, Fanconi's anemia, peripheral nerve damage, inflammation, osteoporosis and aging. It has been reported to cause diseases due to the same oxidation (Chen et al . 2014).

In addition, it is reported that the amount of socioeconomic loss due to drinking amounts to about 0.5-2.7% of GDP in most countries. There is a report that the loss of productivity due to illness, accident, and hangover is estimated to be 6.28 trillion won (Woojin Jung et al. 2006).

In order to solve these social problems, research and experiments on many substances that can reduce the toxicity of ethanol or inhibit the expression of toxicity are in progress, and the results are being developed into various health supplement-related products. Alcohol introduced into the body is absorbed in the stomach or small intestine, enters the blood vessels, is transported to the liver, and is decomposed and detoxified.

Alcohol dehydrogenase (ADH) present in hepatocytes first oxidizes alcohol to acetaldehyde, and acetaldehyde is converted back to acetic acid (Acetate) by acetaldehyde dehydrogenase (ALDH, ALdehyde DeHydrogenase) in hepatocytes. It is decomposed and transferred to muscle or adipose tissue throughout the body, where it is finally decomposed into carbon dioxide and water. Acetaldehyde, the first metabolite of ethanol, is highly reactive and more toxic than ethanol, and is the main cause of hangover and alcoholic liver disorders.

19 types of aldehyde dehydrogenase present in the human body have been reported (Marchitti et al . 2007, 2008), and among them, acetaldehyde dehydrogenase2, which is mainly present in mitochondria, was analyzed by enzymatic engineering, When acetaldehyde was analyzed as a substrate for the enzyme, it showed the lowest Km value (~0.2 μM) compared to when other types of aldehydes were used as substrates, so it is evaluated that acetaldehyde derived from alcohol is best oxidized and removed.

It is very important for human health to remove aldehyde by most effectively converting acetaldehyde, which is a hangover-causing substance produced in ethanol metabolism in vivo, into acetic acid (Eriksson et al . 1977). In addition, acetaldehyde dehydrogenase 2 is used not only in acetaldehyde but also in the metabolism of aldehydes such as aliphatic aldehydes, aromatic aldehydes, and polycyclic aldehydes to remove toxic substances from the body (Klyosov et al. 1996).

As a representative example, it removes 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA), which are oxidized aldehyde substances generated during oxidative stress, and acrolein generated from cigarette smoke and automobile exhaust. role (Chen et al . 2010, Yoval-Sanchez et al. 2012). People who have low expression of acetaldehyde dehydrogenase 2 enzyme in the human body or whose amino acid residue 487 of this enzyme is mutated from glutamic acid to lysine show a reddening and flushing phenomenon, and are sensitive to even a small amount of alcohol. However, the concentration of acetaldehyde in the blood is high when drinking alcohol because it cannot be converted (Yoshida et al. 1984).

In particular, if you have ALDH2-2, a homozygous for acetaldehyde dehydrogenase 2, you are known to be vulnerable to drinking, and this genetic mutation is rarely seen in Westerners, but in Koreans, Chinese, and Japanese, it is found in 50% of the total population. . (Brooks et al. 2009)

As for R&D on aldehyde dehydrogenase 2, research on promoters and inhibitors of aldehyde dehydrogenase 2 in the body has been actively studied for medical purposes, highlighting the importance of aldehyde dehydrogenase 2 (Budas et al. 2009, Chen et al. 2014). , M.zel et al. 2018), studies on the development of microbial breeding or mass production technology for overproduction of aldehyde dehydrogenase 2 are still lacking.

The development of a strain that produces an excess of aldehyde dehydrogenase 2 is to express human-type aldehyde dehydrogenase 1 and 2 proteins using a protein expression system using E. coli as a host, of which about 30% are active. It has been reported to produce 2 to 4 mg/L of protein by expression as a soluble form of enzyme (Zheng et al. 1993), and in the case of Rat aldehyde dehydrogenase 2, 95% of the active soluble protein was expressed, but it was reported that very little protein of 1 to 2 mg/L was produced (Jeng et al. 1991).

However, there have been no reports of cases of increasing the production of acetaldehyde dehydrogenase 2 using a mutagenesis method that is easy to use due to small legal restrictions. Therefore, there is an urgent need to develop microorganisms with high activity aldehyde dehydrogenase 2 by mutagenesis to expand the range of use of acetaldehyde dehydrogenase 2.

Ethanol absorbed in the body is oxidized to acetaldehyde by the enzyme ADH (alcohol dehydrogenase), and in the process of decomposition/oxidation of acetaldehyde produced by alcohol oxidation, an enzyme called ALDH (aldehyde dehydrogenase) acts to release carbon dioxide from the body. , it is decomposed into water and discharged.

ALDH not only decomposes Acetaldehyde, but also Nonenal (4-hydroxy-2-nonenal), HNE (4-hydroxy -trans-2-nonenal), Malondialdehyde, DOPAL (3,4-dihydroxy- It also decomposes phenylacetaldehyde), DOPEGAL (3,4-dihydroxy-phenylglycolaldehyde), 5-HIAL (5-hydroxy indole-acetaldehyde), and Retinaldehyde (Arnold SL et al , 2015).

These various types of aldehydes destroy DNA in the human body (Garaycoechea, JI et al , 2018), and cause serious diseases by making mitochondria, an important energy-producing organ of cells, abnormal (Mitochondrial dysfunction) (Gomes, KM et al , 2014). do.

For the decomposition of these various aldehydes, ALDH from Saccharomyces , a yeast, is mainly used. According to the genome genome database of yeast, about six types of ALDH (Datta S. et al , 2017) are known in the genus Saccharomyces .

Among them, ALDH2 is structurally similar to human ALDH in the binding site of the coenzyme NAD (Mukhopadhyay, A. et al , 2013), uses NAD as a coenzyme, and acts in the mitochondria. In yeast, it also acts in the cytoplasm other than the mitochondria. works As for the specific activity of the enzyme, yeast ALDH ( y ALDH) is more than 20 times higher than that of human ALDH ( h ALDH) (M.-F. Wang et al , 2009), so a high effect can be expected when used in the human body. .

Conventional rice-derived yeast ALDH produced by solid culture in rice has the advantage of being easy to purify, but there is a limit to the commercial mass production of hangover relievers due to the low ALDH production yield. In order to improve this problem, a method for mass production of rice-derived yeast ALDH has emerged. A method for producing recombinant yeast by securing the ALDH gene is described in Korean Patent Laid-Open No. 10-2005-0052664 (PCT/EP2003/01049).

A technique for recombination of yeast-derived aldehyde dehydrogenase gene (ALDH gene) is described in Patent No. 10-1664814. A method for producing an ADH enzyme that oxidizes alcohol by genetic recombination is described in Patent Application No. 10-2020-0045978.

On the other hand, various efforts are being made to develop an activator that activates the ALDH enzyme in the human body with various health food materials to prevent hangover caused by alcohol intake, relieve hangover, and prevent liver damage. (US 10,406,126) B2 (2019), US Pub. NO US2020/0237716 A1 (2020)).

In Korea, as an activator, herbal extracts (Korean Patent Application No. 10-2020-0142768) prepared alone or in combination with herbal preparations such as chrysanthemum, licorice, galgeun, dermis, and Heotgae are known.

In addition, Korean Patent Registration No. 10-0696589 discloses a hangover relieving composition containing Hwangtae, Heotgae tree, mistletoe extract and arrowroot ingredients, and Korean Patent Publication No. 10-2012-0123860 discloses turmeric, alder, and Heotgae tree. It discloses the use of glutathione as a reducing agent by providing a hangover relieving composition comprising a gwabyeong, a spiny hornwort concentrate, an unfermented soybean ferment extract, milk thistle and glutathione.

However, most of the patented technologies so far have focused more on relieving hangover than preventing hangover, and in many cases the effect of relieving hangover is insignificant. Therefore, there is a need in the art to develop a hangover relief composition containing ALDH that can directly and quickly detoxify acetaldehyde, which is the root problem of a hangover phenomenon.

The present inventor rapidly decomposes alcohol and aldehyde by acting quickly in the body, and further rapidly decomposes aldehyde products that cause various ROS, which are reactive oxygen species (ROS) generated during human metabolism, and the effect is sustained in the human body. As a result, a hangover relieving composition containing glutathione and ALDH, which can contribute to the protection of human physiological functions as well as a hangover, was developed.

An object of the present invention is to provide a novel hangover relieving composition that contains sufficient amounts of ALDH enzyme and glutathione, so that the effect of removing aldehyde toxins from the body is fast and continuous. The hangover relieving composition according to the present invention maintains a variety of tolerable aldehyde detoxification activity in the human body as well as aldehyde detoxification activity in the digestive system.

On the other hand, Glutathione (γ-L-glutamyl-L-cysteinylglycine, GSH) is a physiologically active substance present in cells and is a tripeptide composed of three amino acids: glutamate, cysteine, and glycine, It is present in the cells of animals, plants and microorganisms at a concentration of 0.1 to 10 mM, and accounts for more than 90% of the total non-proteinaceous activity of the cells.

In vivo, glutathione is known to play an important role as an antiviral agent by causing an increase in immune activity through the generation of white blood cells, and acts as a substrate for GST (glutathione S-transferase) and is harmful to the living body (xenobiotics) It plays an important role in detoxification by combining toxic substances such as conjugation in the form of conjugation.

In addition, glutathione prevents necrosis by damaging cell membranes, nucleic acids and cellular structures through oxidation within the cell, and plays a role in alleviating the toxicity of reactive oxygen species (ROS), the cause of aging. At this time, reactive oxygen species are formed in various biological metabolism, and include superoxide, peroxide, hydroxyl radical, etc., endogenous reactive oxygen species produced as biological metabolites of substances and tobacco , radioactive and other exogenous reactive oxygen species.

Oxidative stress caused by reactive oxygen species can impair cognitive function (Liu et al . 2002) and cause male infertility by destroying sperm DNA (Wright et al . 2014), cellular proteins, lipids And it can cause cancer by damaging nucleic acids, and acts as a causative factor of various diseases and aging by lowering physiological functions. Therefore, antioxidants that play a role in disease prevention, immunity enhancement, and anti-aging are very important in our body, and the function of glutathione as an antioxidant in cells is important in many fields including enzymology, pharmacology, therapy, toxicology, endocrinology and microbiology. It is attracting attention in the medical field.

Glutathione is basically synthesized in the body, but as abnormal conditions such as disease outbreaks, weakened immunity, and aging progress, the absolute content of glutathione in the human body decreases and deteriorates health. Therefore, glutathione supplied from the outside can remove intracellular reactive oxygen species to maintain health and slow aging. Due to the physiologically active factors of glutathione in the human body, glutathione is currently being used for food, cosmetics, feed, and pharmaceuticals, and its usage is gradually increasing.

On the other hand, the production of glutathione is currently produced using edible microorganisms, but the intrinsic content of glutathione that microorganisms can produce is very low. Research on mass production with strains is being actively conducted.

Therefore, the development of a strain with a high glutathione content is to develop a fundamental material that increases economic value, and it enables the market to have competitive power to use glutathione in a wide range of health foods, medicines and feeds.

However, strain development by genetic recombination technology cannot be free from the various problems of GMO, which are currently an issue, so the scope of its use is limited. However, the performance-improved strain by mutation technology has relatively few limitations, so it is relatively easy to develop for various uses. Therefore, the breeding technology for strains producing high content of glutathione using mutagenesis technology is suitable for the production of glutathione used as an active ingredient in food or drugs.

However, as described above, in order to remove chemicals such as reactive oxygen species and various aldehydes among various harmful substances accumulated in the human body, using glutathione and aldehyde dehydrogenase at the same time is effective, but so far, glutathione and aldehyde A strain that simultaneously produces a large amount of dehydrogenase has not been commercialized.

In order to prepare the hangover cure composition of the present invention, a strain capable of producing aldehyde dehydrogenase and glutathione at the same time with high efficiency is made a mutant strain using a primary chemical mutation method, and a secondary selection factor is adapted (Adaptation). ) mutants were selected and developed.

Mutant strains that simultaneously produce excessive amounts of glutathione and acetaldehyde dehydrogenase 2 are used in food, health food, and feed. Wild Saccharomyces cerevisiae is reported as GRAS (Generally Recognized As Safe) with no problems for cosmetic and medicinal use, and is known as a strain that produces both glutathione and aldehyde dehydrogenase although production efficiency is low. ) was selected and used.

Through this mutation, the production capacity of glutathione is increased, and at the same time, the genus Saccharomyces cerevisiae sp., which is a new improved strain with increased aldehyde dehydrogenase production capacity, was prepared, and the dry powder, lysate of this strain (lysate) or ALDH-containing extract was prepared to complete the hangover reliever of the present invention.

The active ingredient of the hangover remedy composition of the present invention is described in detail in Korean Patent Application No. 10-2020-0019858, and Saccharomyces cerevisiae Kwon P-1 (KCTC13925BP), Saccharomyces cerevisiae , which have been deposited with the International Depositary Organization (KCTC) Dry powder, lysate, or ALDH-containing extract of Kwon P-2 (KCTC14122BP) or Saccharomyces cerevisiae Kwon P-3 (KCTC14123BP).

The present inventors have a high ALDH production ability by mutation and a high glutathione production ability Saccharomyces cerevisiae Kwon P-1 ( Saccharomyces cerevisiae Kwon P-1, accession number: KCTC13925BP), such as three strains alone or mixed A new fermentation process that dramatically increases the production yield of ALDH in a two-step process that includes performing the first liquid fermentation step using invented.

In addition, in the new fermentation process of the present invention, one of three strains such as Saccharomyces cerevisiae Kwon P-1 (Accession No.: KCTC13925BP) or a mixture thereof is used in the first liquid fermentation step and the second By proceeding with the solid-state fermentation step, it was possible to complete a fermentation process capable of simultaneously producing glutathione and ALDH enzymes, which are powerful reducing agents, in high yield.

1. Korean Patent Application No. 10-2020-0019858 “Saccharomyces cerevisiae winding 1,2,3 producing glutathione and aldehyde dehydrogenase” 2. Korean Patent Application Publication No. 10-2005-0052664 3. Korean Patent No. 10-1664814 4. Korean Patent Publication No. 10-2020-0045978 5. United States Patent 10,406,126 B2 ALDH2 ACTIVATOR 6. United States Patent Application US2020/0237716 A1 7. Korean Patent Publication No. 10-2020-0142768 8. Korean Patent Registration No. 10-0696589 9. Korean Patent Application Publication No. 10-2012-0123860

1. Gilpin, N.W.; Koob, G. F. Neurobiology of alcohol dependence: Focus on motivational mechanisms. AlcoholRes. Health, 2008, 31, 185. 2. Shao-Cheng Wang et al, Alcohol Addiction, Gut Microbiota, and Alcoholism Treatment: A Review. Int. J. Mol. Sci. 2020, 21, 6413 3. Song-Sik Choi, A Study on the Relapse Prevention Strategies of Alcoholics in Korean Society, Korean National Culture No. 48, 2013, 307-348 4. Arnold SL, Kent T, Hogarth CA, Schlatt S, Prasad B, Haenisch M, Walsh T, Muller CH, Griswold MD, Amory JK, et al. Importance of ALDH1A enzymes in determining human testicular retinoic acid concentrations. J Lipid Res. 2015, 56: 342-357. 5. Garaycoechea J.I. , G.P. Crossan, F. Langevin., Alcohol and endogenous aldehydes damage chromosomes and mutate stem cells, Nature, 553 ,2018, 171e177 6. Gomes K.M. , J.C. Campos, L. R. Bechara, et al., Aldehyde dehydrogenase 2 activation in heart failure restores mitochondrial function and improves ventricular function and remodeling, Cardiovasc. Res. 103,2014, 498e508. 7. Datta, S., Annapure, U.S. and Timson, D. J., Different specificities of two aldehyde dehydrogenases from Saccharomyces cerevisiae var. boulardii, Bioscience Reports, 2017, 37 BSR20160529 8. Brooks, P. J., Enoch, M. A., Goldman, D., Li, T. K., & Yokoyama, A., The alcohol flushing response: an unrecognized risk factor for esophageal cancer from alcohol consumption. PLoS medicine, 2009, 6(3), e1000050. 9. Larson, H.N., Zhou, J., Chen, Z. et al., Structural and functional consequences of coenzyme binding to the inactive Asian variant of mitochondrial aldehyde dehydrogenase: Roles of residues 475 and 487, J. Biol. Chem. Vol. 282,2007 pp. 12940-12950. 10. M.-F Wang et al., Chemico-Biological Interrations 178, 2009, 36-39 11. Budas, G. R., Disatnik, M. H., & Mochly-Rosen, D. (2009). Aldehyde dehydrogenase 2 in cardiac protection: a new therapeutic target. Trends in cardiovascular medicine, 19(5), 158-164. 12. Chen, C. H., Ferreira, J. C. B., Gross, E. R., & Mochly-Rosen, D. (2014). Targeting aldehyde dehydrogenase 2: new therapeutic opportunities. Physiological reviews, 94(1), 1-34. Eriksson, C. J. (1977). Acetaldehyde metabolism in vivo during ethanol oxidation. Advances in experimental medicine and biology, 85, 319-341. Hamad, G. M., Taha, T. H., Alshehri, A. M. & Hafez, E.E., (2018). Enhancement of the Glutathione Production by Mutated Yeast Strains and its Potential as Food Supplement and Preservative. Res. J. Microbiol., 13: 28-36. Jeng, J., & Weiner, H. (1991). Purification and characterization of catalytically active precursor of rat liver mitochondrial aldehyde dehydrogenase expressed in Escherichia coli. Archives of biochemistry and biophysics, 289(1), 214-222. 16. Klyosov, A. A., Rashkovetsky, L. G., Tahir, M. K., & Keung, W. M. (1996-1). Possible role of liver cytosolic and mitochondrial aldehyde dehydrogenases in acetaldehyde metabolism. Biochemistry, 35(14), 4445-4456. Klyosov, A. A. (1996-2). Kinetics and specificity of human liver aldehyde dehydrogenases toward aliphatic, aromatic, and fused polycyclic aldehydes. Biochemistry, 35(14), 4457-4467. Liu, J., Head, E., Gharib, A. M., Yuan, W., Ingersoll, R. T., Hagen, T. M., & Ames, B. N. (2002). Memory loss in old rats is associated with brain mitochondrial decay and RNA/DNA oxidation: Partial reversal by feeding acetyl-L-carnitine and/or R-α-lipoic acid. Proc. Natl. Acad. Sci., 99(4), 2356-2361. Marchitti, S. A., Deitrich, R. A., & Vasiliou, V. (2007). Neurotoxicity and metabolism of the catecholamine-derived 3,4-dihydroxyphenyla cetaldehyde and 3,4-dihydroxyphenylglycolaldehyde: the role of aldehyde dehydrogenase. Pharmacological reviews, 59(2), 125-150. Marchitti, S. A., Brocker, C., Stagos, D., & Vasiliou, V. (2008). Non-P450 aldehyde oxidizing enzymes: the aldehyde dehydrogenase superfamily. Expert opinion on drug metabolism & toxicology, 4(6), 697-720. Najafi, MBH, & Pezechki, P. (2013) Bacterial mutation; types, mechanisms and mutant detection methods: a review. European Scientific Journal, 4 Ohtake, Y., Satou, A., & Yabuuchi, S. (1990). Isolation and Characterization of Glutathione Biosynthesis-deficient Mutants in Saccharomyces cerevisiae. Agric. Biol. Chem., 54(12), 3145-3150. Wright, C., Milne, S., & Leeson, H. (2014). Sperm DNA damage caused by oxidative stress: modifiable clinical, lifestyle and nutritional factors in male infertility. Reprod. BioMed. Online, 28(6), 684-703. Yoshida, A., Huang, I. Y., & Ikawa, M. (1984). Molecular abnormality of an inactive aldehyde dehydrogenase variant commonly found in Orientals. Proceedings of the National Academy of Sciences, 81(1), 258-261.

It is an object of the present invention to mutate yeast in wild Saccharomyces cerevisiae to increase the simultaneous production capacity of glutathione and aldehyde dehydrogenase 2 To provide a hangover relief composition containing as an active ingredient.

Another object of the present invention is Saccharomyces cerevisiae KwonP-1 ( Saccharomyces cerevisiae KwonP-1: KCTC13925BP), KCTC14122BP ( Saccharomyces cerevisiae KwonP-2) or KCTC14123BP ( Saccharomyces cerevisiae KwonP-2) that simultaneously produces glutathione and aldehyde dehydrogenase 2 -2) To provide a hangover relieving composition containing the dry powder, lysate, or ALDH-containing extract of mutant yeast as an active ingredient.

Another object of the present invention is Saccharomyces cerevisiae KwonP-1 ( Saccharomyces cerevisiae KwonP-1: KCTC13925BP), KCTC14122BP ( Saccharomyces cerevisiae KwonP-2) or KCTC14123BP ( Saccharomyces cerevisiae KwonP-2) that simultaneously produces glutathione and aldehyde dehydrogenase 2 -2) It is to provide a method for producing in a two-step process of culturing yeast in a liquid phase, adding the liquid culture to rice and culturing in a solid phase.

Another object of the present invention is Saccharomyces cerevisiae KwonP-1 ( Saccharomyces cerevisiae KwonP-1: KCTC13925BP), KCTC14122BP ( Saccharomyces cerevisiae KwonP-2) or KCTC14123BP ( Saccharomyces cerevisiae KwonP-2) that simultaneously produces glutathione and aldehyde dehydrogenase 2 -2) A hangover containing dry powder, lysate, or ALDH-containing extract of yeast produced in a two-step process of culturing the yeast in liquid phase, adding the liquid culture to rice and culturing the solid phase as an active ingredient It is to provide a dissolving composition.

In order to achieve the object of the present invention as described above, after mutating wild yeast, the strain producing an excess of glutathione is a methylglyoxal-adapted mutant, and the selection of an aldehyde dehydrogenase excess producing strain is a lysine-adapted mutant selection method, Finally, Saccharomyces cerevisiae, which simultaneously produces excessive amounts of glutathione and aldehyde dehydrogenase, was selected.

Saccharomyces cerevisiae strain that produces excess glutathione and acetaldehyde dehydrogenase by inoculating rice with Saccharomyces cerevisiae KwonP-1 (accession number KCTC13925BP) cultured in large quantities again and proceeding with solid-phase fermentation 2 After culturing on a larger scale in the step process, the hangover relieving composition of the present invention containing the dry powder, lysate or extract powder of the strain was prepared.

1 is a graph showing changes in blood acetaldehyde content of experimental animals according to administration of a composition of the present invention.
2 is a graph showing changes in blood acetaldehyde content of experimental animals according to administration of the composition of the present invention.

Hereinafter, the configuration and effects of the present invention will be described in more detail through the following examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples. Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. The following examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

glutathione and Preparation of Yeast Lysates of the Invention Containing ALDH

Example 1-1: Saccharomyces cerevisiae yeast fermentation process containing glutathione and ALDH

The ALDH-containing Saccharomyces cerevisiae yeast seed was fermented and cultured for 24 hours in an incubator at 160 rpm and 30° C. using YPD medium (yeast extract, peptone, and glucose containing medium) in a 200 mL flask. The culture was carried out for 72 hours through a 5 L fermenter (Marado-05D-PS, CNS, Korea). After completion of the culture, the yeast was centrifuged using a high-speed centrifuge (Supra R22, Hanil, Korea).

Example 1-2: Preparation of yeast lysate containing glutathione and ALDH

The centrifuged ALDH-containing yeast was frozen in a cryogenic freezer (CLN-52U, Nihon freezer, Japan) for 2 days, and then freeze-dried for 2 days in a freeze dryer (FDU-7006, Operon, Korea). After dissolving 3 g of lyophilized yeast powder in 50 mL phosphate-buffered saline (PBS) containing a protease inhibitor (A32955, Thermo fisher, USA), 0.5 mm glass beads for cell disruption (11079105, Biospec) 10 g was put into the bead homogenizer (Mixer Mill MM400, Retsch, Germany) for 2 minutes a total of 3 times to disrupt the yeast. After centrifugation using a high-speed centrifuge (Supra R22, Hanil, Korea), only the supernatant was separated and freeze-dried for 2 days with a freeze dryer (FDU-7006, Operon, Korea).

Mass production of Saccharomyces cerevisiae strain by two-step fermentation process

Saccharomyces cerevisiae KwonP-1 ( Saccharomyces cerevisiae KwonP-1) strain (KCTC13925BP) was inoculated in YPD medium containing 2% peptone, 1% yeast extract, and 2% glucose and cultured at 30 ° C to increase the OD value of _600nm . It was fermented at 200 rpm, 1 vvm in a fermentor (Fermentor, Cobiotech) until it reached 50. Cells were recovered from the culture solution using a membrane filter.

The recovered cells are mixed with the already sterilized rice fermented powder at a ratio of 10%, the moisture content is adjusted to 60%, and the solid phase is cultured for 2 days at 30°C and then dried at 50°C to adjust the final moisture content to 7%. Yeast fermented rice fermented powder was prepared.

The fermentation composition of the present invention thus prepared contained a maximum of 600 units/g of ALDH. Considering that the rice fermented powder by the wild-type Saccharomyces cerevisiae yeast strain known to date generally contains about 2unit/g of ALDH, it was confirmed that the ALDH content of the fermented composition of the present invention was increased by about 300 times. did.

Table 1 shows the results of evaluating the acetaldehyde decomposition ability of the compositions (1 to 4) of the present invention prepared by the two-stage fermentation process of the present invention for 5 minutes.

Reaction time (m) NADPH (mM) Suspension vol. (ml) Unit/g powder Average One 5 1.732 0.20 693.0 681.4 One 5 1.675 0.20 669.8 2 5 1.722 0.20 688.9 682.2 2 5 1.689 0.20 675.6 3 5 1.770 0.20 707.9 702.1 3 5 1.741 0.20 696.3 4 5 1.639 0.20 655.8 665.7 4 5 1.689 0.20 675.6

NAD, an ALDH coenzyme, was added as an enzyme activator to the dry pulverized powder of the fermentation composition of the present invention prepared in this way, and citric acid, magnesium stearate, DL methionine, vitamin C and lactic acid bacteria ( Lactobacillus plantium 10 ⁷ /g), zinc oxide, and silicon dioxide were added. Added to prepare a hangover reliever of the present invention.

Through animal testing of the hangover reliever of the present invention, the concentration of acetaldehyde in the blood was measured after alcohol intake to confirm the result that the hangover reliever of the present invention significantly and rapidly reduced the concentration of acetaldehyde in the blood compared to the conventional hangover reliever did.

In addition, for human clinical trials of the hangover reliever of the present invention, voluntary clinical trial volunteers were subjected to a genomic test to the experimental group, which is a group with the ALDH2 gene that degrades aldehyde, and the group with the ALDH2*2 mutant gene, which is genetically deficient in the ability to degrade aldehydes. The experiment was divided into experimental groups.

As a result of the experiment to confirm the effect of human hangover relieving for 15 hours, significant differences in aldehyde degradation ability were confirmed in both the ALDH2 retention test group and the ALDH2*2 gene mutation test group. The hangover reliever of the present invention was able to efficiently remove acetaldehyde in both experimental groups. In particular, the aldehyde decomposition and hangover relieving effect of the hangover reliever of the present invention due to the reinforcement of the contents of ALDH and Glutathione was confirmed by efficiently removing the aldehyde even in the ALDH 2*2 gene mutation test group, which is difficult to degrade aldehyde.

Measurement of hangover relief effect of the composition of the present invention

Example 3-1: Animal test for changes over time of acetaldehyde in blood

Table 2 shows the animal test results for the temporal change of acetaldehyde in blood after ethanol administration.

Blood Acetaldehyde (mg/L) 0hr 1hr 3hr 5hr 8hr control 0 0.03 0.02 0.03 0.02 Ethanol administration group 0 0.52 0.49 0.3 0.19 Ethanol and the composition administered at 73 mg/kg 0 0.48 0.33 0.23 0.18 Ethanol and the composition 220mg/kg administered group 0 0.46 0.24 0.22 0.12

Table 3 shows the results (mg/L·hr) of the cumulative amount of aldehydes in the blood.

Accumulated amount of acetaldehyde in the blood decrease rate control 0.26 Ethanol administration group 2.57 Ethanol and the composition administered at 73 mg/kg 2 -22% Ethanol and the composition 220mg/kg administered group 1.27 -51%

Example 3-2: Analysis of ethanol and acetaldehyde in blood of human clinical trial volunteers

Clinical trial volunteers were selected from 43 healthy adult males in their 20s and 40s who can drink soju based on an average alcohol content of 20 degrees at a time of drinking, and the clinical trial was conducted once a week on Friday evenings at 17:00 for a total of 4 weeks. The clinical trial was carried out through a camp for 15 hours the day after entering the hospital at 8 am, and a total of 23 patients finally completed the clinical trial due to personal circumstances during the clinical process.

On the first day of the camp, after taking 10 glasses of soju, blood alcohol metabolism, that is, changes in alcohol concentration and acetaldehyde concentration by time period, were measured, and on the second day of the camp, the amount of change in blood alcohol metabolism after taking 73 mg/kg of the composition of the present invention 30 minutes after taking 10 glasses of soju On the second day of the camp, after taking 220 mg/kg of the composition of the present invention, 30 minutes after taking 10 glasses of soju, the amount of change in blood alcohol metabolism was measured.

In the group taking the composition containing 500 mg/day of the double-fermented dry powder of the present invention and 1500 mg of fermented rice powder, the blood concentration of Acetaldehyde, which is a causative agent of hangover and a strong carcinogen in the body, was significantly reduced in a dose-dependent manner compared to the group administered with alcohol alone. . In addition, the residual amount of blood alcohol was also significantly reduced in a dose-dependent manner with the composition of the present invention.

Table 4 shows the decrease in blood alcohol concentration of volunteers in the human impression test.

alcohol accumulation
(g hr/dL) alcohol reduction
(%) decrease rate peak Cmax
(g/L) Alcohol alone group 30.852 100% 7.583 Ethanol and composition 73mg/kg administration group 29.693 96.2% -3.8% 5.548 Ethanol and composition 220mg/kg administration group 25.271 85.7% -14.9% 4.18

Table 5 shows the decrease in the residual amount of acetaldehyde in the blood of human impression test volunteers.

Residual amount of acetaldehyde in blood
(mg hr/dL) Residue reduction
(%) decrease rate high
Cmax
(mg/dL) Ethanol administration group 13.02 100% 1.65 Ethanol and composition 73mg/kg administration group 9.39 72.1% -27.9% 1.2 Ethanol and composition 220mg/kg administration group 5.22 68.0% -32.0% 1.3

Example 3-4: Confirmation of changes in ethanol and acetaldehyde according to ALDH gene mutation

For the recruitment of clinical trial volunteers, 43 male and female adults in their 20s and 40s who can drink soju with an average alcohol content of 20 degrees per drink were selected. For a total of 4 weeks, the clinical trial was conducted once a week from 17:00 on Friday evening to 8:00 am on the next day after admission to the hospital. During the clinical trial, only 23 patients completed the clinical trial for a total of 15 hours due to personal circumstances.

Of these, about 22 of them participated in the genetic test related to alcohol metabolism, and with the consent of the experiment and information use, a total of 22 people involved in alcohol metabolism in vivo, including ADH1B (Alcohol dehydrogenase 1B), ALDH2 (Aldehyde dehydrogenase 2), and CPY2E1 P450, 3 types of genome tests were performed. Through this, it was confirmed that the blood concentration of Acetaldehyde, which is a causative agent of hangover and a strong carcinogen in the body, decreased in a dose-dependent manner in both the ALDH2 non-mutant group and the ALDH2*2 mutant group compared to the alcohol-only group.

The ALDH2*2 gene mutation group is known to exhibit a very high blood acetaldehyde concentration even with a small amount of alcohol. never had However, in the case of administration of the hangover relieving composition of the present invention, the effect of reducing blood Acetaldehyde in the ALDH2*2 gene mutant group is very remarkable.

Table 6 shows the alcohol content (g hr/L) of the normal ALDH gene-bearing group and the ALDH gene mutant group.

ALDH gene variants group with normal ALDH gene Ethanol administration group 42.55 28.13 Ethanol and composition 73mg/kg administration group 36.13 29.86 Ethanol and composition 220mg/kg administration group 40.87 24.94

Table 7 shows the average blood acetaldehyde content (g hr/L) of the normal ALDH gene-bearing group and the ALDH gene mutant group.

ALDH gene variants group with normal ALDH gene Ethanol administration group 10.56 13.47 Ethanol and composition 73mg/kg administration group 8.78 7.57 Ethanol and composition 220mg/kg administration group 8.43 3.92

Toxicity test of the hangover relieving composition of the present invention

Example 4-1. Preparation of laboratory animals

As experimental animals, female and male ICR mice (7 weeks old) were received and acclimatized for 7 days, and only healthy animals were used for the test by observing general symptoms during the acclimatization period. Feed and water were ingested ad libitum, and group separation was performed so that there were 5 males and 5 females in each group based on an average body weight of about 20 g the day before oral administration.

Example 4-2 Administration of the hangover relieving composition of the present invention

The test substance was prepared by dissolving in physiological saline so that the doses of the test animals were 0, 750, 3000, and 5000 mg/Kg, respectively, based on the content of the yeast lysate containing GSH and ALDH of the present invention. The standard of administration dose complied with the Korea National Toxicology Program (KNTP) toxicity test manual of the Ministry of Food and Drug Safety, and the maximum applied dose 5000mg/Kg guided by the KNTP manual was applied as the maximum concentration of this experiment. Samples prepared for each group were orally administered once to each test animal, and physiological saline was administered to the normal group (G1).

Example 4-3. Observation and autopsy

For all the animals in the test group, symptoms were observed at least once a day from the date of acquisition to the day of autopsy, and symptoms were observed for 7 days after oral administration. After the type of symptom observation, an autopsy was performed, and changes in each organ were visually observed at the time of autopsy.

As a result of a single-dose toxicity test using the yeast lysate containing GSH and ALDH of the present invention in mice, no deaths were observed for 7 days at a concentration of up to 5000 mg/kg, weight gain, feed intake, etc. No singularity could be found. Also, no unusual findings were found in the autopsy results after the observation was completed.

Korea Institute of Bioscience and Biotechnology KCTC13925BP 20190822 Korea Institute of Bioscience and Biotechnology KCTC14122BP 20200130 Korea Institute of Bioscience and Biotechnology KCTC14123BP 20200130

Claims (5)

A hangover relieving composition containing glutathione and aldehyde dehydrogenase. The method according to claim 1, wherein the glutathione and aldehyde dehydrogenase are Saccharomyces cerevisiae yeast, Saccharomyces cerevisiae Kwon P-1 KCTC13925BP, Saccharomyces cerevisiae Kwon P-2 KCTC14122BP and Saccharomyces. Myces cerevisiae Kwon P-3 KCTC14123BP Any one selected from the group consisting of, or a hangover relieving composition, characterized in that derived from a mixture thereof. A first step of culturing the Saccharomyces cerevisiae strain in a liquid medium, and a second step of further culturing the Saccharomyces cerevisiae strain cultured in the first step in a solid medium again, Saccharomyces cerevisiae strain mass culture method. The method of claim 3, wherein the solid medium is any one selected from the group consisting of rice, barley, wheat, corn, and soybeans, or a mixture thereof, Saccharomyces cerevisiae strain mass culture method. The method of claim 3 or 4, wherein the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae Kwon P-1 (KCTC13925BP), Saccharomyces cerevisiae Kwon P-2 (KCTC14122BP) And Saccharomyces cerevisiae Kwon P-3 KCTC14123BP, characterized in that selected from the group consisting of, mass culture method.

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