Hangover reliever containing glutathione and aldehyde dehydrogenase

Abstract

To provide a method for mass-cultivating Saccharomyces cerevisiae strains.SOLUTION: Provided is a method for mass-cultivating a Saccharomyces cerevisiae strain, comprising: a first step of culturing the Saccharomyces cerevisiae strain in a liquid-phase medium; and a second step of further culturing the Saccharomyces cerevisiae strain cultured in the first step in a solid-phase medium. The solid-phase medium is preferably any one selected from the group consisting of rice, barley, wheat, corn, and beans, or a mixture thereof. The mass-cultivation method is characterized in that the Saccharomyces cerevisiae strain is selected from the group consisting of Saccharomyces cerevisiae Kwon P-1 (KCTC13925BP), Saccharomyces cerevisiae Kwon P-2 (KCTC14122BP), and Saccharomyces cerevisiae Kwon P-3 KCTC14123BP.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a hangover reliever containing glutathione (GSH) and aldehyde dehydrogenase (hereinafter referred to as ALDH). More specifically, the present invention relates to a hangover reliever containing glutathione and aldehyde dehydrogenase derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae Kwon P-1 KCTC 13925BP), Saccharomyces cerevisiae Kwon P-2 KCTC14122BP, or Saccharomyces cerevisiae Kwon P-3 KCTC14123BP yeast. [Background technology]

[0002] Alcohol is a recreational food that has existed throughout human history, but excessive drinking can lead to hangovers that cause physical and mental discomfort, as well as nausea, vomiting, dizziness, thirst, lethargy, drowsiness, and headaches. It can also cause abnormalities in the nervous system (Alcohol Use Disorder, AUD) (Shao-Cheng Wang et al., 2020), leading to serious alcohol addiction symptoms and even panic disorder, making it a social problem (Choi, Seong-sik, 2013).

[0003] When you drink alcohol, 5% of the alcohol is absorbed in the mouth, 10-15% in the stomach, and 80% in the small intestine and flows into the bloodstream. 2-4% is broken down in the lungs, 2-4% in the kidneys, 2-6% in sweat, and 90% in the liver.

[0004] In the liver, alcohol is broken down by being oxidized by alcohol dehydrogenase (ADH) to be converted into acetaldehyde, which is then further oxidized and detoxified by aldehyde dehydrogenase (ALDH).

[0005] However, 15% to 50% of Asians who lack aldehyde dehydrogenase or who inherit the aldehyde dehydrogenase allele (ALDH2*2) are unable to break down the acetaldehyde produced when drinking alcohol, resulting in Alcohol Flushing Syndrome (ALF) (Brooks, PJ et al. 2009), a phenomenon in which the face turns red. It has also been reported that the accumulation of acetaldehyde increases the risk of alcoholism and liver disease (Larson, HN et al. 2007). When acetaldehyde is not broken down, it remains in the body, causing alcohol hepatitis and liver cirrhosis, leading to death and disability (Gilpin, NW et al. 2008).

[0006] On the other hand, excessive aldehydes produced by alcohol consumption and remaining in the body have been reported to cause oxidative diseases such as cardiovascular disease, diabetes, neurodegenerative diseases, upper digestive and respiratory tract cancer, radiation dermatitis, Fanconi anemia, peripheral nerve damage, inflammation, osteoporosis, and aging (Chen et al. 2014).

[0007] It has also been reported that the socioeconomic losses caused by drinking amount to approximately 0.5-2.7% of GDP in most countries. In South Korea, the socioeconomic costs caused by drinking in 2000 were estimated at 14,935.2 billion won, of which 6,284.5 billion won was lost in productivity and losses due to illness, accidents, and hangovers (Jung Woo-jin et al. 2006).

[0008] To solve this social problem, research and experiments are being conducted on many substances that can reduce the toxicity of ethanol or inhibit its manifestation, and the results are being developed into various health supplement products. Alcohol that enters the body is absorbed in the gastrointestinal tract or small intestine, enters the bloodstream, and is transported to the liver where it is broken down and detoxified.

[0009] Alcohol dehydrogenase (ADH) in liver cells first oxidizes alcohol to acetaldehyde, which is then broken down into acetate by acetaldehyde dehydrogenase (ALDH) in liver cells, and transported to muscle and fat tissue throughout the body, where it is ultimately broken down into carbon dioxide and water. Acetaldehyde, the first metabolic product of ethanol, is much more reactive and toxic than ethanol, making it the main cause of hangovers and alcoholic liver damage.

[0010] Nineteen types of aldehyde dehydrogenases have been reported to exist in the human body (Marchitti et al. 2007, 2008). Among these, acetaldehyde dehydrogenase 2, which is primarily present in mitochondria, was analyzed using enzyme engineering techniques and found to have the lowest Km value (~0.2 μM) when acetaldehyde was used as the enzyme substrate compared to when other types of aldehydes were used as substrates. This indicates that acetaldehyde derived from alcohol is most effectively oxidized and removed.

[0011] The removal of aldehydes, which is produced during ethanol metabolism in the body and causes hangovers, by converting acetaldehyde to acetic acid most efficiently is crucial for human health (Eriksson et al. 1977). Furthermore, acetaldehyde dehydrogenase 2 is used in the metabolic processes of not only acetaldehyde but also other aldehydes, such as aliphatic aldehydes, aromatic aldehydes, and polycyclic aldehydes, to remove toxic substances from the body (Klyosov et al. 1996).

[0012] Representative examples include the removal of oxidized aldehydes 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) produced during oxidative stress, and the removal of acrolein produced in cigarette smoke and automobile soot (Chen et al. 2010, Yoval-Sanchez et al. 2012). Individuals with low expression of acetaldehyde dehydrogenase 2 or a mutation in the 487th amino acid residue of this enzyme from glutamic acid to lysine exhibit sensitive reactions to even small amounts of alcohol, including facial flushing, and also have high blood acetaldehyde levels after drinking alcohol due to the lack of conversion (Yoshida et al. 1984).

[0013] In particular, people who are homozygous for acetaldehyde dehydrogenase 2, ALDH2-2, are known to be more susceptible to alcohol. This genetic mutation is rarely found in Westerners, but has been found in 50% of the Korean, Chinese, and Japanese populations (Brooks et al. 2009).

[0014] Research and development into aldehyde dehydrogenase 2 has been actively conducted, with studies into stimulators and inhibitors of aldehyde dehydrogenase 2 in the body for medical purposes highlighting its importance (Budas et al. 2009, Chen et al. 2014, M.zel et al. 2018). However, research into breeding microorganisms that overproduce aldehyde dehydrogenase 2 and developing mass production techniques is still lacking.

[0015] To develop a strain that overproduces aldehyde dehydrogenase 2, a protein expression system using E. coli as a host was used to express human aldehyde dehydrogenase 1 and 2 proteins, and it was reported that approximately 30% of the proteins were expressed as active soluble enzymes, producing 2-4 mg / L of protein (Zheng et al. 1993).In the case of rat aldehyde dehydrogenase 2, it was reported that 95% was expressed as active soluble protein, but very little protein was produced, only 1-2 mg / L (Jeng et al. 1991).

[0016] However, there have been no reported cases of increasing the production of acetaldehyde dehydrogenase 2 using mutation methods, which are easy to use and have few legal restrictions. Therefore, in order to expand the range of uses of acetaldehyde dehydrogenase 2, there is an urgent need to develop microorganisms that have highly active aldehyde dehydrogenase 2 using mutation methods.

[0017] Ethanol absorbed into the body is oxidized to acetaldehyde by the enzyme ADH (alcohol dehydrogenase), and the enzyme ALDH (aldehyde dehydrogenase) acts to decompose / oxidize the acetaldehyde produced by alcohol oxidation, and it is broken down into carbon dioxide and water and excreted from the body.

[0018] ALDH not only degrades acetaldehyde, but also other compounds that cause oxidative stress in the human body, such as nonenal (4-hydroxy-2-nonenal), 4-hydroxy-trans-2-nonenal (HNE), malondialdehyde, 3,4-dihydroxy-phenylacetaldehyde (DOPAL), 3,4-dihydroxy-phenylglycolaldehyde (DOPEGAL), 5-hydroxy-1H-indole-3-acetaldehyde (5-HIAL), and retinaldehyde (Arnold SL et al., 2015).

[0019] These various types of aldehydes damage human DNA (Garaycoechea, JI et al., 2018) and cause mitochondrial dysfunction (Gomes, KM et al., 2014), which are important energy-generating organelles in cells, and can lead to serious diseases.

[0020] To decompose these diverse aldehydes, many ALDHs derived from the yeast Saccharomyces are used. According to the yeast Genome Database, approximately six types of ALDHs are known to exist in Saccharomyces (Datta S. et al., 2017).

[0021] Of these, ALDH2 has a structurally similar binding site for crude NAD to human ALDH (Mukhopadhyay, A. et al., 2013), and it uses NAD as a crude enzyme to function not only in mitochondria but also in the cytoplasm in yeast. The specific activity of yeast ALDH (yALDH) is more than 20 times higher than that of human ALDH (hALDH) (M.-F.Wang et al., 2009), suggesting that it may be highly effective when used in humans.

[0022] While conventional rice-derived yeast ALDH, produced by solid-state fermentation in rice, has the advantage of being easy to purify, the low production yield of ALDH limits its commercial mass production as a hangover reliever. To address this issue, a method for mass-producing rice-derived yeast ALDH has emerged. A method for isolating the ALDH gene and creating recombinant yeast is disclosed in Korean Patent Publication No. 10-2005-0052664 (PCT / EP2003 / 01049).

[0023] The technology for recombinant aldehyde dehydrogenase (ALDH) gene from yeast is disclosed in Korean Patent No. 10-1664814. A method for recombinantly producing ADH enzyme, which oxidizes alcohol, is disclosed in Korean Patent Application No. 10-2020-0045978.

[0024] Meanwhile, various efforts are being made to develop activators that activate ALDH enzymes in the human body using various health food ingredients to prevent and alleviate hangovers and protect against liver damage caused by alcohol consumption (US 10,406,126 B2 (2019), US Pub. No. US2020 / 0237716 A1 (2020)).

[0025] In Korea, herbal extracts (Korean patent application 10-2020-0142768) are known as activators, and are made by using herbal preparations such as szechuan cucumber, licorice root, kudzu root, dried orange peel, and Chinese quince, either alone or in combination.

[0026] Furthermore, Korean Patent Registration No. 10-0696589 discloses a hangover relief composition containing Alaska pollack, Kenponashi, mistletoe extract, and kudzu ingredients, and Korean Patent Publication No. 10-2012-0123860 provides a hangover relief composition containing turmeric, alder, Kenponashi fruit stalk, Eleuthero concentrated solution, fermented ungerminated soybean extract, milk thistle, and glutathione, and discloses the use of glutathione as a reducing agent.

[0027] However, most of the patented technologies to date have focused on relieving hangovers rather than preventing them, and the hangover relief effect has often been insignificant. Therefore, there is a need in the industry to develop a hangover relief composition containing ALDH that can directly and quickly detoxify acetaldehyde, the fundamental problem behind hangovers.

[0028] The inventors have developed a hangover relief composition containing glutathione and ALDH, which acts quickly in the body to quickly decompose alcohol and aldehydes, and in turn, quickly decomposes aldehyde products that induce various ROS, which are reactive oxygen species (ROS) generated during the human metabolic process, and whose effects last in the body, contributing to the protection of not only hangovers but also the physiological functions of the human body.

[0029] The present invention aims to provide a novel hangover relief composition that contains sufficient amounts of ALDH enzyme and glutathione, ensuring rapid and sustained detoxification of aldehydes from the body. The hangover relief composition of the present invention maintains the activity of not only detoxifying aldehydes in the digestive tract but also various endogenous aldehyde detoxification activities within the human body.

[0030] Meanwhile, glutathione (γ-L-glutamyl-L-cysteinylglycine, GSH) is a physiologically active substance present within cells. It is a tripeptide composed of three amino acids, glutamate, cysteine, and glycine, and is present in animal, plant, and microbial cells at concentrations of 0.1 to 10 mM, accounting for more than 90% of the total non-protein active components of the cell.

[0031] Glutathione in the body is known to play an important role as an antiviral agent by increasing immune activity through the production of white blood cells. It also plays an important role in detoxification by acting as a substrate for GST (glutathione S-transferase) and binding toxic substances such as xenobiotics that are harmful to the body in a conjugated form.

[0032] Glutathione also plays a role in preventing damage to cell membranes, nucleic acids, and cell structures through intracellular oxidation, leading to necrosis, and mitigating the toxicity of reactive oxygen species (ROS), which are the cause of aging. ROS are formed through various metabolic processes in the body and include superoxide, peroxide, and hydroxyl radicals. They can be divided into endogenous ROS, which are produced as metabolic products of substances, and exogenous ROS, such as tobacco and radioactivity.

[0033] Oxidative stress caused by reactive oxygen species can damage cognitive function (Liu et al. 2002), damage sperm DNA, causing male infertility (Wright et al. 2014), damage cellular proteins, lipids, and nucleic acids, leading to cancer, and impair physiological functions, acting as a causative factor for various diseases and aging. Therefore, antioxidants, which play roles in disease prevention, immune enhancement, and anti-aging, are extremely important in our bodies. The function of glutathione, which plays an antioxidant role within cells, has attracted attention in many medical fields, including enzymology, pharmacology, therapy, toxicology, endocrinology, and microbiology.

[0034] Although glutathione is essentially synthesized in the body, the absolute content in the human body decreases as abnormal conditions such as disease onset, weakened immunity, and aging progress, leading to deterioration of health. Therefore, glutathione supplied from the outside can remove reactive oxygen species from cells, thereby maintaining health and delaying aging. Due to the physiological activity of glutathione in the human body, glutathione is currently being used in foods, cosmetics, feed, and pharmaceuticals, and its usage is on the rise.

[0035] Meanwhile, glutathione is currently produced using edible microorganisms, but the inherent glutathione content that can be produced by microorganisms is very low. Therefore, research is being actively conducted to increase the glutathione content of microorganisms using mutation and recombinant technology, and then mass-produce high-content glutathione-producing strains using fermentation techniques.

[0036] Therefore, the development of a strain with high glutathione content will develop a fundamental material that will increase economic value and give glutathione market competitiveness that will enable it to be widely used in health foods, pharmaceuticals, feed, etc.

[0037] However, the development of strains using genetic engineering technology is limited in its scope of use due to the various issues surrounding GMOs. However, performance-improved strains using mutation technology have fewer limitations and are easier to develop for various uses. Therefore, breeding techniques for high-content glutathione-producing strains using mutation technology are suitable for producing glutathione, which is used as an active ingredient in foods and pharmaceuticals.

[0038] However, as explained above, the simultaneous use of glutathione and aldehyde dehydrogenase is highly effective in removing various harmful substances that accumulate in the human body, particularly chemical substances such as reactive oxygen species and various aldehydes. However, to date, no strain capable of simultaneously mass-producing glutathione and aldehyde dehydrogenase has been commercialized.

[0039]

[0040]

[0041] [Prior art documents] [Patent documents]

[0042] [Patent Document 1] 1. Korean Patent Application No. 10-2020-0019858 "Saccharomyces cerevisiae Kwon P1, 2, 3 producing glutathione and aldehyde dehydrogenase"

[0043] [Patent Document 2] 2. Korean Patent Application Publication No. 10-2005-0052664

[0044] [Patent Document 3] 3. Korean Patent No. 10-1664814

[0045] [Patent Document 4] 4. Korean Patent Publication No. 10-2020-0045978

[0046] [Patent Document 5] 5. US Patent 10,406,126 B2 ALDH2 ACTIVATOR

[0047] [Patent Document 6] 6. U.S. Patent Application US2020 / 0237716 A1

[0048] [Patent Document 7] 7. Korean Patent Publication No. 10-2020-0142768

[0049] [Patent Document 8] 8. Korean Patent No. 10-0696589

[0050] [Patent Document 9] 9. Korean Patent Application Publication No. 10-2012-0123860

[0051]

[0052] [Non-patent literature]

[0053] [Non-Patent Document 1] 1.Gilpin, NW; Koob, GFNeurobiology of alcohol dependence:Focus on motivational mechanisms.AlcoholRes.Health,2008,31,185.

[0054] [Non-patent document 2] 2.Shao-Cheng Wang et al,Alcohol Addiction,Gut Microbiota,and Alcoholism Treatment:A Review.Int.J.Mol.Sci.2020,21,6413

[0055] [Non-patent document 3] 3. Choi Seong-sik, Study on Relapse Prevention Strategies for Alcoholics in Korean Society, Korean Folk Culture No. 48, 2013, 307-348

[0056] [Non-patent document 4] 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.

[0057] [Non-Patent Document 5] 5.Garaycoechea JI, GPCrossan, F. Langevin., Alcohol and endogenous aldehydes damage chromosomes and mutate stem cells, Nature, 553, 2018, 171e177

[0058] [Non-patent document 6] 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.

[0059]

Non-Patent Document 7

[0060]

Non-Patent Document 8

[0061]

Non-Patent Document 9

[0062] [Non-Patent Document 10] 10.M.-F Wang et al.,Chemico-Biological Interrations 178,2009,36-39

[0063] [Non-Patent Document 11] 11. Budas, GR, Disatnik, MH, & Mochly-Rosen, D. (2009). Aldehyde dehydrogenase 2 in cardiac protection: a new therapeutic target. Trends in cardiovascular medicine, 19(5), 158-164.

[0064] [Non-Patent Document 12] 12. Chen, CH, Ferreira, JCB, Gross, ER, & Mochly-Rosen, D. (2014). Targeting aldehyde dehydrogenase 2: new therapeutic opportunities. Physiological reviews, 94(1), 1-34.

[0065] [Non-Patent Document 13] 13. Eriksson, C. J. (1977). Acetaldehyde metabolism in vivo during ethanol oxidation. Advances in experimental medicine and biology, 85, 319 - 341.

[0066]

Non - Patent Document 14

[0067]

Non - Patent Document 15

[0068]

Non - Patent Document 16

[0069]

Non-Patent Document 17

[0070]

Non-Patent Document 18

[0073] [Non-Patent Document 21] 21. Najafi, M.B.H., & Pezechki, P. (2013) Bacterial mutation; types, mechanisms and mutant detection methods: a review. European Scientific Journal, 4

[0074] [Non-Patent Document 22] 22. 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.

[0075] [Non-Patent Document 23] 23. 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.

[0076] [Non-Patent Document 24] 24. Yoshida, A., Huang, IY, & 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. [Detailed description of the invention] [Technical problem]

[0077] Summary of the Invention [Problem to be solved by the invention]

[0078] In order to prepare the hangover relieving composition of the present invention, a strain capable of simultaneously and highly efficiently producing aldehyde dehydrogenase and glutathione was developed by first creating mutant strains using a chemical mutagenesis method, and then selecting adaptation mutant strains using a secondary selection factor.

[0079] The mutant strain that simultaneously overproduces glutathione and acetaldehyde dehydrogenase 2 has been reported to be GRAS (Generally Recognized As Safe), meaning that it can be used in foods, health foods, feed, cosmetics, and medicines without any problems. We selected and used wild-type Saccharomyces cerevisiae, which is already known to produce both glutathione and aldehyde dehydrogenase, although the production efficiency is low.

[0080] Thus, a new improved strain of Saccharomyces cerevisiae sp. was prepared through mutation, in which the glutathione production capacity and aldehyde dehydrogenase production capacity were increased. A dried powder, lysate, or ALDH-containing extract of this strain was prepared to complete the hangover reliever of the present invention. [Technical solution] [Means for solving the problem]

[0081] The active ingredient of the hangover reliever composition of the present invention is described in detail in Korean Patent Application No. 10-2020-0019858, and is a dry powder, lysate, or ALDH-containing extract of Saccharomyces cerevisiae Kwon P-1 (KCTC13925BP), Saccharomyces cerevisiae Kwon P-2 (KCTC14122BP), or Saccharomyces cerevisiae Kwon P-3 (KCTC14123BP), which are deposited at the Korean Council of Trade and Industry (KCTC).

[0082] The inventors have invented a new fermentation process that dramatically increases the ALDH production yield through a two-step process that includes a primary liquid-phase fermentation step using three strains, including Saccharomyces cerevisiae Kwon P-1 (Accession Number: KCTC13925BP), which has high ALDH production ability due to mutations and high glutathione production ability, either alone or in combination. The liquid-phase fermentation product is then added to fermented rice powder and subjected to a secondary solid-phase fermentation step.

[0083] In addition, in the new fermentation process of the present invention, one of three strains, including Saccharomyces cerevisiae Kwon P-1 (Accession No.: KCTC13925BP), or a mixture of these strains is used to perform a primary liquid-phase fermentation step and a secondary solid-phase fermentation step, thereby completing a fermentation process that can simultaneously produce glutathione, a powerful reducing agent, and ALDH enzyme in high yields.

[0084] The mass-cultured Saccharomyces cerevisiae KwonP-1 (Accession No. KCTC13925BP) was further inoculated onto rice and subjected to solid-state fermentation to culture the Saccharomyces cerevisiae strain that overproduces glutathione and acetaldehyde dehydrogenase on a larger scale in a two-step process, and the hangover relief composition of the present invention containing the dried powder, lysate, or extract powder of the strain was then prepared. [Brief explanation of the drawings]

[0085] [Figure 1] 1 is a graph showing changes in acetaldehyde content in the blood of experimental animals following administration of the composition of the present invention.

[0086] [Figure 2] 1 is a graph showing changes in acetaldehyde content in the blood of experimental animals following administration of the composition of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0087] [Best Mode for Carrying Out the Invention] Hereinafter, the configuration and effects of the present invention will be described in more detail through the following embodiments. These embodiments are for the purpose of illustrating the present invention only, and the scope of the present invention is not limited by these embodiments. Hereinafter, the configuration and effects of the present invention will be described in more detail through the following embodiments. These embodiments are for the purpose of illustrating the present invention only, and the scope of the present invention is not limited by these embodiments. [Mode for carrying out the invention]

[0088] Example 1: Preparation of yeast lysate containing glutathione and ALDH

[0089] Embodiment 1-1: Glutathione and ALDH-containing Saccharomyces cerevisiae yeast fermentation process

[0090] The ALDH-containing Saccharomyces cerevisiae yeast seed culture was cultured in a 200 mL flask using YPD medium (a medium containing yeast extract, peptone, and glucose) at 160 rpm and 30°C in an incubator for 24 hours. The main culture was then carried out in a 5 L fermentor (Marado-05D-PS, CNS, Korea) for 72 hours. After the culture was completed, the yeast was centrifuged using a high-speed centrifuge (Supra R22, Hanil, Korea).

[0091]

[0092] Embodiment 1-2: Preparation of glutathione- and ALDH-containing yeast lysate

[0093] The centrifuged ALDH-containing yeast was frozen in an ultra-low temperature freezer (CLN-52U, Nihon Freezer, Japan) for 2 days and then freeze-dried for 2 days in a freeze dryer (FDU-7006, Operon, Korea). 3 g of freeze-dried yeast powder was dissolved in 50 mL of phosphate-buffered saline (PBS) containing a protease inhibitor (A32955, Thermo Fisher, USA). The yeast was then homogenized with 10 g of 0.5 mm cell disruption glass beads (11079105, Biospec) in a bead homogenizer (Mixer Mill MM400, Retsch, Germany) for 2 minutes each, a total of three times. After centrifugation in a high-speed centrifuge (Supra R22, Hanil, Korea), the supernatant was isolated and freeze-dried for 2 days in a freeze dryer (FDU-7006, Operon, Korea).

[0094]

[0095] [Example 2] Mass production of Saccharomyces cerevisiae strains using a two-stage fermentation process

[0096] Saccharomyces cerevisiae KwonP-1 strain (KCTC13925BP) was inoculated into YPD medium containing 2% peptone, 1% yeast extract, and 2% glucose, and cultured at 30°C until OD 600nm The culture was fermented in a fermenter (Fermentor, Kobiotech) at 200 rpm and 1 vvm until the value reached 50. The culture medium was passed through a membrane filter to collect the bacterial cells.

[0097] The collected bacteria were mixed with sterilized fermented rice powder at a ratio of 10% to adjust the moisture content to 60%, and then cultured in a solid phase at 30°C for 2 days. After that, the mixture was dried at 50°C to adjust the final moisture content to 7%, producing yeast-fermented fermented rice powder.

[0098] The fermented composition of the present invention thus prepared contained a maximum of 600 units of ALDH / g. Considering that rice fermented powder using a previously known wild-type Saccharomyces cerevisiae yeast strain typically contains approximately 2 units of ALDH / g, it was confirmed that the ALDH content of the fermented composition of the present invention was increased by approximately 300 times.

[0099] Table 1 shows the results of evaluating the acetaldehyde decomposition ability of the compositions (1 to 4) of the present invention produced by the two-stage fermentation process of the present invention for 5 minutes.

[0100] [Table 1]

[0101]

[0102] The dried and ground powder of the fermented composition of the present invention thus prepared is added with crude ALDH enzyme NAD as an enzyme activator, and then mixed with lactic acid, magnesium stearate, DL methionine, vitamin C, and lactic acid bacteria (Lactobacillus plantium 10 7 / g), zinc oxide, and silicon dioxide were added to prepare the hangover reliever of the present invention.

[0103] Through animal experiments on the hangover reliever of the present invention, the blood acetaldehyde concentration after ingesting alcohol was measured, and it was confirmed that the hangover reliever of the present invention reduces the blood acetaldehyde concentration significantly faster than conventional hangover relievers.

[0104] In addition, for the human clinical trials of the hangover reliever of the present invention, the volunteers were divided into two experimental groups through genome testing: one with ALDH2, which breaks down aldehydes, and the other with ALDH2*2 mutant genes, which are genetically deficient in the ability to break down aldehydes.

[0105] A 15-hour hangover relief test in humans confirmed a significant difference in aldehyde decomposition ability between the ALDH2-positive and ALDH2*2 gene mutation experimental groups. The hangover reliever of the present invention was able to efficiently remove acetaldehyde in both experimental groups. In particular, it efficiently removed aldehyde even in the ALDH2*2 gene mutation experimental group, which has difficulty decomposing aldehydes, confirming the aldehyde decomposition and hangover relief effects of the hangover reliever of the present invention, which are due to the increased content of ALDH and glutathione.

[0106]

[0107] [Example 3] Measurement of the hangover relief effect of the composition of the present invention

[0108] Example 3-1: Changes in blood acetaldehyde over time Animal test

[0109] Table 2 shows the results of animal tests on the time course of changes in blood acetaldehyde after ethanol administration.

[0110] [Table 2]

[0111] The results of animal experiments on the cumulative amount of aldehyde in the blood (mg / L·hr) are shown in Table 3.

[0112] [Table 3]

[0113]

[0114] Example 3-2: Analysis of blood ethanol and acetaldehyde in human clinical trial supporters

[0115] The subjects selected for the human clinical trial were 43 healthy adult men in their 20s to 40s who could drink soju with an average alcohol content of 20% in one sitting. They entered the clinical trial hospital once a week on Friday evenings at 5pm for a total of four weeks, and conducted clinical trials for a total of 15 hours until 8am the following day. During the clinical trial, only 23 participants were able to complete the clinical trial due to personal reasons.

[0116] On the first day of the training camp, subjects drank 10 cups of soju, and then measured blood alcohol metabolism by time period, i.e., changes in alcohol concentration and changes in acetaldehyde concentration. On the second day of the training camp, subjects drank 73 mg / kg of the composition of the present invention, 30 minutes later drank 10 cups of soju, and then measured changes in blood alcohol metabolism. On the second day of the training camp, subjects drank 220 mg / kg of the composition of the present invention, 30 minutes later drank 10 cups of soju, and then measured changes in blood alcohol metabolism.

[0117] In the group that took the composition containing 500 mg / day of the two-stage fermented dry powder of the present invention and 1500 mg of fermented rice powder, the blood concentration of acetaldehyde, a substance that causes hangovers and is a potent carcinogen in the body, was significantly reduced in a dose-dependent manner compared to the group that took alcohol alone.In addition, the amount of residual alcohol in the blood was also significantly reduced in a dose-dependent manner.

[0118] The reduction in blood alcohol concentration in human clinical trial supporters is shown in Table 4.

[0119] [Table 4]

[0120] The reduction in blood acetaldehyde levels in human clinical trial supporters is shown in Table 5.

[0121] [Table 5]

[0122]

[0123] Example 3-3: Test to confirm changes in ethanol and acetaldehyde depending on whether or not the ALDH gene is mutated

[0124] To recruit human clinical trial supporters, 43 healthy adult men in their 20s to 40s who could drink soju with an average alcohol content of 20% in one sitting were selected as subjects. They entered the clinical trial hospital once a week on Friday evenings at 5pm for a total of four weeks, and conducted clinical trials for a total of 15 hours until 8am the following day. During the clinical trial, only 23 participants were able to complete the clinical trial due to personal reasons.

[0125] Of these, approximately 22 people participated in genetic testing related to alcohol metabolism and consent was obtained for the experiment and use of information. Through genome testing of three types of genes involved in alcohol metabolism in the body, ADH1B (Alcohol dehydrogenase 1B), ALDH2 (Aldehyde dehydrogenase 2), and CPY2E1 P450, it was confirmed that the blood levels of acetaldehyde, a substance that causes hangovers and is a potent carcinogen in the body, were dose-dependently reduced in both the ALDH2 non-mutant group and the ALDH2*2 mutant group compared to the group that only consumed alcohol.

[0126] It is known that individuals with ALDH2*2 gene mutations exhibit extremely high blood acetaldehyde levels even with small amounts of alcohol, and the reduction of blood acetaldehyde levels has never been observed or reported through conventional hangover relief drinks or conventional hangover relief foods and medicines. However, the reduction of blood acetaldehyde levels in individuals with ALDH2*2 gene mutations when administered with the hangover relief composition of the present invention is a remarkable result.

[0127] Table 6 shows the measured alcohol intake (g hr / L) for the normal ALDH gene carrier group and the ALDH gene mutation group.

[0128] [Table 6]

[0129] The mean blood acetaldehyde content (g hr / L) of the normal ALDH gene carrier group and the ALDH gene mutation group is shown in Table 7.

[0130] [Table 7]

[0131]

[0132] [Example 4] Toxicity test of the hangover relief composition of the present invention

[0133] Example 4-1. Preparation of experimental animals

[0134] Female and male ICR mice (7 weeks old) were provided as experimental animals and allowed to acclimate for 7 days. During the acclimatization period, general symptoms were observed and only healthy animals were used in the study. Food and water were provided ad libitum, and the animals were divided into groups of 10, with 5 males and 5 females per group, based on an average body weight of approximately 20g on the day before oral administration.

[0135]

[0136] Example 4-2. Administration of the hangover relief composition of the present invention

[0137] The test substances were dissolved in saline to give experimental animal doses of 0, 750, 3000, and 5000 mg / kg, based on the content of the yeast lysate containing GSH and ALDH of the present invention. The dosage standards followed the toxicity test manual of the Korea National Toxicology Program (KNTP) of the Ministry of Food and Drug Safety, and the maximum applicable dose of 5000 mg / kg as specified in the KNTP manual was used as the maximum concentration in this experiment. The samples prepared for each group were orally administered once to the test animals, and saline was administered to the normal group (G1).

[0138]

[0139] Example 4-3. Observation and autopsy

[0140] All animals in the test groups were observed for symptoms at least once daily from the date of acquisition until the date of necropsy, and symptoms were observed for 7 days after oral administration. After symptom observation was completed, necropsies were performed, and changes in each organ were observed with the naked eye at the time of necropsy.

[0141] As a result of a single-dose toxicity test using mice with the yeast lysate containing glutathione and ALDH of the present invention, no deaths were observed for 7 days at concentrations up to 5000 mg / kg, and no abnormalities were found in weight gain, feed intake, etc. Furthermore, no specific findings were found in the autopsy results performed after the end of the observation period.

[0142]

[0143] [Accession number]

[0144] Depository institution name: Korea Institute of Bioscience and Biotechnology [Accession number]

[0145] Accession number: KCTC13925BP

[0146] Date of acceptance: 20190822

[0147] TIFF2025163195000009.tif255168

[0148] Depository institution name: Korea Institute of Bioscience and Biotechnology

[0149] Accession number: KCTC14122BP

[0150] Date of acceptance: 20200130

[0151] TIFF2025163195000010.tif255170

[0152] Depository institution name: Korea Institute of Bioscience and Biotechnology

[0153] Accession number: KCTC14123BP

[0154] Date of acceptance: 20200130

[0155] TIFF2025163195000011.tif255170

Claims

1. A hangover relief composition containing glutathione and aldehyde dehydrogenase.

2. 2. The hangover relief composition according to claim 1, wherein the glutathione and aldehyde dehydrogenase are derived from any one selected from the group consisting of Saccharomyces cerevisiae yeast, Saccharomyces cerevisiae Kwon P-1 KCTC13925BP, Saccharomyces cerevisiae Kwon P-2 KCTC14122BP, Saccharomyces cerevisiae Kwon P-3 KCTC14123BP, or a mixture thereof.

3. A method for mass-cultivating a Saccharomyces cerevisiae strain, comprising: a first step of culturing the Saccharomyces cerevisiae strain in a liquid phase medium; and a second step of further culturing the Saccharomyces cerevisiae strain cultured in the first step in a solid phase medium.

4. 4. The method for mass-cultivating a Saccharomyces cerevisiae strain according to claim 3, wherein the solid phase medium is any one selected from the group consisting of rice, barley, wheat, corn, and beans, or a mixture thereof.

5. 5. The method for mass culturing Saccharomyces cerevisiae according to claim 3 or 4, wherein the Saccharomyces cerevisiae strain is selected from the group consisting of Saccharomyces cerevisiae Kwon P-1 (KCTC13925BP), Saccharomyces cerevisiae Kwon P-2 (KCTC14122BP), and Saccharomyces cerevisiae Kwon P-3 KCTC14123BP.

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