Hangover elimination agent containing glutathione and aldehyde dehydrogenase

Abstract

This invention relates to a hangover relief composition containing a dried powder, lysate, or extract of enzymes that produce glutathione and aldehyde dehydrogenase. Specifically, this invention relates to a hangover relief composition containing dried 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, which simultaneously produce glutathione and aldehyde dehydrogenase.

Description

Technical Field

[0001] This invention relates to a hangover remedy containing glutathione (GSH) and aldehyde dehydrogenase (hereinafter referred to as "ALDH"). Specifically, this invention relates to a hangover remedy containing both glutathione and aldehyde dehydrogenase derived from Saccharomyces cerevisiae Kwon P-1KCTC 13925BP, Saccharomyces cerevisiae Kwon P-2KCTC14122BP, or Saccharomyces cerevisiae Kwon P-3KCTC14123BP. Background Technology

[0002] While alcohol has been a source of enjoyment throughout human history, excessive drinking can lead to a hangover, causing physical and mental discomfort, nausea, vomiting, dizziness, thirst, weakness, drowsiness, and headaches. This can result in abnormalities in the nervous system (Alcohol Use Disorder, AUD) (Shao-Cheng Wang et al., 2020), becoming a social problem causing severe alcohol addiction and panic disorder (Choe Songsig 2013).

[0003] After drinking alcohol, 5% is absorbed in the mouth, 10-15% in the stomach, 80% in the small intestine before flowing into the bloodstream, 2-4% is broken down in the lungs, 2-4% in the kidneys, 2-6% through sweat, and 90% in the liver.

[0004] In the liver, alcohol is broken down by being oxidized into acetaldehyde by alcohol dehydrogenase (ADH), and then oxidized into non-toxic alcohol by aldehyde dehydrogenase (ALDH).

[0005] However, reports indicate that 15% to 50% of Asians with insufficient aldehyde dehydrogenase or who are born with the opposite gene for aldehyde dehydrogenase (ALDH2*2) experience alcohol flushing syndrome (Brooks, PJ et al. 2009) due to their inability to break down acetaldehyde produced during alcohol consumption. Furthermore, the accumulation of acetaldehyde increases the risk of alcohol poisoning or liver disease (Larson, HN et al. 2007). The inability to break down acetaldehyde and its subsequent retention in the body can lead to death or disability due to alcoholic hepatitis or liver cirrhosis (Gilpin, NWetal. 2008).

[0006] In addition, there have been reports that excessive residual aldehydes in the body generated by alcohol intake can lead to cardiovascular diseases, diabetes, neurodegenerative diseases, cancers of the upper digestive and respiratory organs, radiation dermatitis, Fanconi anemia, peripheral nerve damage, inflammation, osteoporosis, and aging caused by oxidation (Chen et al. 2014).

[0007] In addition, the report states that in most countries, the socioeconomic losses caused by alcohol consumption account for approximately 0.5-2.7% of GDP. In South Korea, the socioeconomic losses caused by alcohol consumption in 2000 were estimated at 14.9352 trillion won, of which the reduction in productivity and losses due to illness, accidents, and hangovers were estimated at 6.2845 trillion won (Jeog Wujinet al. 2006).

[0008] To address this social problem, research and experiments are conducted on various substances that can reduce the toxicity of ethanol or inhibit its expression, resulting in the development of various health supplement products. Alcohol entering the body is absorbed in the stomach or small intestine and enters the bloodstream, where it is transported to the liver for breakdown and detoxification.

[0009] Alcohol dehydrogenase (ADH) in liver cells first oxidizes alcohol to acetaldehyde. Acetaldehyde is then broken down into acetic acid by aldehyde dehydrogenase (ALDH) in liver cells and transported to muscle or adipose tissue throughout the body, ultimately decomposing into carbon dioxide and water. As the initial metabolite of ethanol, acetaldehyde is much more reactive and toxic than ethanol, and is a major cause of hangovers and alcoholic liver disease.

[0010] The report states that there are 19 types of aldehyde dehydrogenases in the human body (Marchitti et al. 2007, 2008), among which aldehyde dehydrogenase 2, which is mainly found in mitochondria, showed the lowest Km value (~0.2 μM) when acetaldehyde was used as the enzyme substrate compared to other types of aldehydes, and thus best oxidized and removed acetaldehyde derived from alcohol.

[0011] The efficient conversion of acetaldehyde, a hangover causative agent produced during ethanol metabolism in the body, using acetic acid to remove aldehydes is crucial for human health (Eriksson et al. 1977). Furthermore, aldehyde dehydrogenase 2 is used not only for acetaldehyde but also for the metabolism of other aldehydes such as aliphatic aldehydes, aromatic aldehydes, and polycyclic aldehydes to remove toxic substances from the body (Klyosove et al. 1996).

[0012] Representative examples include the removal of 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA), oxidized aldehydes produced during oxidative stress, and the removal of acrolein from cigarette smoke and vehicle exhaust (Chen et al. 2010, Yoval-Sancheze et al. 2012). Individuals with low expression of aldehyde dehydrogenase 2 or whose 487th amino acid residue has been mutated from glutamate to lysine exhibit facial flushing, are sensitive to even small amounts of alcohol, and, due to their inability to convert aldehydes, have high blood acetaldehyde concentrations after drinking (Yoshida et al. 1984).

[0013] In particular, those with homozygous ALDH2-2, which acts as aldehyde dehydrogenase 2, have lower alcohol tolerance. This genetic variation is almost absent in Westerners, but is found in 50% of the overall population of Koreans, Chinese, and Japanese. (Brookset al. 2009)

[0014] Although much research has been conducted on aldehyde dehydrogenase 2 promoters and inhibitors for medical purposes, thus increasing the importance of aldehyde dehydrogenase 2 (Budase et al. 2009, Chenet et al. 2014, M. Zelet et al. 2018), research on microbial breeding or large-scale production technologies for the overproduction of aldehyde dehydrogenase 2 is still unsatisfactory.

[0015] There have been reports of strains that overproduce aldehyde dehydrogenase 2, using protein expression systems with E. coli as the host to express human aldehyde dehydrogenase 1 and 2 proteins, with about 30% expressed as active, usable forms of the enzyme to produce 2 to 4 mg / L of protein (Zheng et al. 1993). In contrast, in the case of rat aldehyde dehydrogenase 2, although 95% was expressed as active, usable protein, only a very small amount of protein, 1 to 2 mg / L, was produced (Jeng et al. 1991).

[0016] However, there have been no reported cases of increased production of aldehyde dehydrogenase 2 using mutation methods, which are readily applicable due to fewer legal restrictions. Therefore, to expand the use of aldehyde dehydrogenase 2, it is urgent to develop microorganisms with high activity of aldehyde dehydrogenase 2 using mutation methods.

[0017] Ethanol absorbed into the body is oxidized to acetaldehyde by the enzyme ADH (alcohol dehydrogenase). In the process of decomposition / oxidation of acetaldehyde produced by ethanol oxidation, it is decomposed into carbon dioxide and water by the enzyme ALDH (aldehyde dehydrogenase) and excreted from the body.

[0018] ALDH not only breaks down Acetaldehyde, but also decomposes Nonnal (4-hydroxy-2-nonenal), HNE (4-hydroxy-trans-2-nonenal), Malondialdehyde, DOPAL (3,4-dihydroxy-phenylacetaldehyde), DOPEGAL (3,4-dihydroxy-phenylglycolaldehyde), 5-HIAL (5-hydroxy indole-acetaldehyde), Retinaldehyde (Arnold SL et al, 2015), etc., which exert oxidative stress on the human body.

[0019] The various types of aldehydes mentioned above damage DNA in the human body (Garaycoechea, JI et al, 2018), leading to mitochondrial dysfunction, an important energy-producing organ in cells (Gomes, KMetal, 2014), and thus causing serious diseases.

[0020] To break down these harmful aldehydes, ALDH derived from Saccharomyces yeast is primarily used. According to the yeast Genome Database, there are approximately six species of ALDH in Saccharomyces (Datta S. et al., 2017).

[0021] The binding site of the coenzyme NAD in ALDH2 is structurally similar to that of human ALDH (Mukhopadhyay, A. et al., 2013). NAD is used as a coenzyme not only in mitochondria but also in the cytoplasm of yeast. The specific activity of the enzyme is more than 20 times higher in yeast ALDH (yALDH) than in human ALDH (hALDH) (M.-F. Wang et al., 2009). Therefore, good results can be expected when used in humans.

[0022] While existing rice-derived yeast ALDH produced from solid-state culture in rice has the advantage of easy purification, its low yield limits its commercial-scale production as a hangover remedy. To improve these issues, a method for mass-producing rice-derived yeast ALDH has been developed. A method for ensuring the ALDH gene is used to manufacture recombinant yeast is described in Korean Patent Publication No. 10-2005-0052664 (PCT / EP2003 / 01049).

[0023] The technique for recombination of the aldehyde dehydrogenase gene (ALDH gene) derived from yeast is described in Patent No. 10-1664814. A method for producing an ethanol-oxidizing ADH enzyme based on gene recombination is described in Patent Application No. 10-2020-0045978.

[0024] In addition, efforts are being made to develop activators of the ALDH enzyme in the human body using various health food materials to prevent hangovers and liver damage caused by alcohol consumption. (US10,406,126B2 (2019), US Pub.NO US2020 / 0237716A1 (2020)).

[0025] In South Korea, a traditional Chinese medicine extract made by individually or in combination of Chinese herbs such as Ligusticum chuanxiong, licorice, kudzu root, tangerine peel, and Japanese raisin tree is disclosed as an activator (Korean Patent Application 10-2020-0142768).

[0026] In addition, Korean Patent Registration No. 10-0696589 describes a hangover relief composition containing extracts of yellow moss, Japanese raisin tree, mistletoe, and kudzu. Korean Patent Publication No. 10-2012-0123860 discloses a hangover relief composition containing turmeric, alder, Japanese raisin tree fruit stalk, concentrated Acanthopanax senticosus extract, fermented soybean extract without germ, milk thistle, and glutathione, disclosing the use of the reducing agent glutathione.

[0027] However, most current patented technologies focus on hangover relief rather than prevention, and in most cases, the hangover relief effect is minimal. Therefore, there is an urgent need in the field to develop hangover relief compositions containing ALDH, which can directly and rapidly degrade acetaldehyde, the root cause of hangovers.

[0028] The inventors have developed a hangover relief composition containing glutathione and ALDH that can rapidly decompose alcohol and aldehydes in the body, thereby rapidly decomposing various ROS that induce reactive oxygen species (ROS) generated during human metabolism. Its effect is sustained in the body, thus not only eliminating hangovers but also protecting human physiological functions.

[0029] The objective of this invention is to provide a novel hangover remedy composition with sufficient ALDH enzyme and glutathione content, thereby rapidly removing aldehyde toxins from the body and ensuring sustained efficacy. The hangover remedy composition according to this invention not only maintains aldehyde toxicity removal activity in the digestive organs but also maintains the removal activity against various endogenous aldehyde toxicities in the human body.

[0030] In addition, glutathione (γ-L-glutamyl-L-cysteinylglycine, GSH) is a physiologically active substance present in cells. It is a tripeptide composed of three amino acids: glutamate, cysteine, and glycine. It exists in the cells of animals, plants, and microorganisms at a concentration of 0.1 to 10 mM, accounting for more than 90% of the total non-protein active components of cells.

[0031] In vivo, glutathione increases immune activity by producing white blood cells, thus playing an important role as an antiviral agent. As the matrix of GST (glutathione S-transferase), it conjugates toxic substances such as xenobiotics that are harmful to organisms, playing an important role in detoxification.

[0032] In addition, glutathione helps prevent the necrosis of cell membranes, nucleic acids, and cell structures through oxidation, and mitigates the toxicity of reactive oxygen species (ROS), a cause of aging. ROS are formed during various metabolic processes in organisms and include superoxide, peroxide, and hydroxyl radicals. They can be categorized into endogenous ROS generated as metabolic byproducts of organisms and exogenous ROS from sources such as smoke and radiation.

[0033] Oxidative stress caused by reactive oxygen species can impair cognitive function (Liu et al. 2002), damage sperm DNA leading to male infertility (Wright et al. 2014), damage cellular proteins, lipids, and nucleic acids leading to cancer, and reduce physiological function, thus becoming a contributing factor to various diseases and aging. Therefore, antioxidants, which play a role in preventing disease, enhancing immunity, and preventing aging, are crucial for our bodies. The function of glutathione, which acts as an antioxidant within cells, has attracted attention in many medical fields, including enzymology, pharmacology, therapeutics, toxicology, endocrinology, and microbiology.

[0034] The aforementioned glutathione is primarily synthesized within the body. However, its absolute content decreases due to disease, weakened immunity, aging, and other abnormal states, leading to a decline in health. Therefore, externally supplied glutathione can maintain health and slow aging by removing intracellular reactive oxygen species. Due to its physiological activity in the human body, glutathione is now used in food, cosmetics, animal feed, and pharmaceuticals, and its usage is gradually increasing.

[0035] In addition, glutathione production currently utilizes edible microorganisms, but the inherent glutathione content produced by microorganisms is very low. Therefore, research is actively underway to increase the glutathione content of microorganisms through mutation and recombination technologies, so as to utilize the fermentation method of this technology to achieve large-scale production using high-glutathione-producing strains.

[0036] Therefore, the development of strains with high glutathione content is a fundamental material for improving economic value, enabling glutathione to have market competitiveness that can be widely used in health foods, pharmaceuticals, feed, etc.

[0037] However, strains developed through gene recombination technology are limited in their application due to the persistent problems related to GOM (glutathione) that have become a topic of discussion. In contrast, strains developed through mutation technology have fewer limitations and are easier to develop for various uses. Therefore, breeding techniques for high-glutathione-producing strains using mutation technology are suitable for the production of glutathione as an active ingredient in food or pharmaceuticals.

[0038] However, as mentioned above, the simultaneous use of glutathione and aldehyde dehydrogenase is highly efficient in removing various harmful substances accumulated in the body, especially reactive oxygen species or various aldehydes and other chemicals. But so far, strains that can simultaneously produce large quantities of glutathione and aldehyde dehydrogenase have not been commercialized.

[0039] [Patent Literature]

[0040] 1. Korean Patent Application No. 10-2020-0019858: Saccharomyces cerevisiae KwonP-1, 2, 3 producing glutathione and aldehyde dehydrogenase.

[0041] 2. Korean Patent Application Publication No. 10-2005-0052664

[0042] 3. Korean Patent No. 10-1664814

[0043] 4. Korean Patent Publication No. 10-2020-0045978

[0044] 5. United States Patent 10,406,126B2 ALDH2 ACTIVATOR

[0045] 6. United States Patent Application US2020 / 0237716A1

[0046] 7. Korean Patent Publication No. 10-2020-0142768

[0047] 8. Korean Patent No. 10-0696589

[0048] 9. Korean Patent Application Publication No. 10-2012-0123860

[0049] [Non-patent literature]

[0050] 1. Gilpin, NW; Koob, GFNeurobiology of alcohol dependence: Focus on motivational mechanisms. Alcohol Res. Health, 2008, 31, 185.

[0051] 2. Shao-Cheng Wang et al, Alcohol Addiction, Gut Microbiota, and Alcoholism Treatment: A Review.Int.J.Mol.Sci.2020,21,6413

[0052] 3. Choe Songsig, A Study on Relapse Prevention Strategies for Alcoholics in Korean Society, Korean National Culture No. 48, 2013, 307-348.

[0053] 4、Arnold SL,Kent T,Hogarth CA,Schlatt S,Prasad B,Haenisch M,Walsh T,Muller CH,Griswold MD,Amory JK,et al.Importance ofALDH1A enzymes indetermining human testicular retinoic acid concentrations.J Lipid Res.2015,56:342-357.

[0054] 5、Garaycoechea J.I.,G.P.Crossan,F.Langevin.,Alcohol and endogenousaldehydes damage chromosomes and mutate stem cells,Nature,553,2018,171e177

[0055] 6、Gomes K.M.,J.C.Campos,L.R.Bechara,et al.,Aldehydedehydrogenase2activation in heart failure restores mitochondrial function andimproves ventricular function and remodeling,Cardiovasc.Res.103,2014,498e508.

[0056] 7、Datta,S.,Annapure,U.S.and Timson,D.J.,Different specificities oftwo aldehyde dehydrogenases from Saccharomyces cerevisiae var.boulardii,BioscienceReports,2017,37BSR20160529

[0057] 8、Brooks,P.J.,Enoch,M.A.,Goldman,D.,Li,T.K.,&Yokoyama,A.,The alcoholflushing response:an unrecognized risk factor for esophageal cancer fromalcohol consumption.PLoS medicine,2009,6(3),e1000050.

[0058] 9、Larson,H.N.,Zhou,J.,Chen,Z.et al.,Structural and functionalconsequences ofcoenzyme binding to the inactive Asian variant ofmitochondrialaldehyde dehydrogenase:Roles of residues 475and487,J.Biol.Chem.Vol.282,2007pp.12940-12950、

[0059] 10、M.-F Wang et al.,Chemico-BiologicalInterrations 178,2009,36-39

[0060] 11、Budas,G.R.,Disatnik,M.H.,&Mochly-Rosen,D.(2009).Aldehydedehydrogenase 2in cardiac protection:a new therapeutic target.Trends incardiovascular medicine,19(5),158-164.

[0061] 12、Chen,C.H.,Ferreira,J.C.B.,Gross,E.R.,&Mochly-Rosen,D.(2014).Targeting aldehyde dehydrogenase 2:new therapeuticopportunities.Physiological reviews,94(1),1-34.

[0062] 13、Eriksson,C.J.(1977).Acetaldehyde metabolism in vivo duringethanoloxidation.Advances in experimental medicine andbiology,85,319-341.

[0063] 14、Hamad,G.M.,Taha,T.H.,Alshehri,A.M.&Hafez,E.E.,(2018).Enhancementof the Glutathione Production by Mutated Yeast Strains and itsPotential asFood Supplement and Preservative.Res.J.Microbiol.,13:28-36.

[0064] 15、Jeng,J.,&Weiner,H.(1991).Purification and characterizationofcatalytically active precursor of rat liver mitochondrial aldehydedehydrogenaseexpressed in Escherichia coli.Archives of biochemistry andbiophysics,289(1),214-222.

[0065] 16、Klyosov,A.A.,Rashkovetsky,L.G.,Tahir,M.K.,&Keung,W.M.(1996-1).Possible role of liver cytosolic and mitochondrial aldehydedehydrogenases inacetaldehyde metabolism.Biochemistry,35(14),4445-4456.

[0066] 17、Klyosov,A.A、(1996-2).Kinetics and specificity of humanliveraldehyde dehydrogenases toward aliphatic,aromatic,and fused polycyclicaldehydes.Biochemistry,35(14),4457-4467.

[0067] 18、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 brainmitochondrialdecay and RNA / DNA oxidation:Partial reversal by feedingacetyl-L-carnitineand / or R-α-lipoic acid.Proc.Natl.Acad.Sci.,99(4),2356-2361.

[0068] 19、Marchitti,S.A.,Deitrich,R.A.,&Vasiliou,V.(2007).Neurotoxicityandmetabolism ofthe catecholamine-derived 3,4-dihydroxyphenyla cetaldehydeand3,4-dihydroxyphenylglycolaldehyde:the role of aldehydedehydrogenase.Pharmacological reviews,59(2),125-150.

[0069] 20、Marchitti,S.A.,Brocker,C.,Stagos,D.,&Vasiliou,V.(2008).Non-P450aldehyde oxidizing enzymes:the aldehyde dehydrogenase superfamily.Expertopinion on drug metabolism&toxicology,4(6),697-720.

[0070] 21. Najafi, MBH, & Pezechki, P. (2013) Bacterial mutation; types, mechanisms and mutant detection methods: a review. European Scientific Journal, 4

[0071] 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.

[0072] 23. Wright, C., Milne, S., & Leeson, H. (2014). Sperm DNA damage caused by oxidative stress: modifiable clinical, lifestyle and nutritional factors inmale infertility. Reprod. BioMed. Online, 28 (6), 684-703.

[0073] 24. Yoshida, A., Huang, IY, & Ikawa, M. (1984). Molecular abnormality of aninactive aldehyde dehydrogenase variant commonly found in Orientals. Proceedings of the National Academy of Sciences, 81 (1), 258-261. Summary of the Invention

[0074] The problem the invention aims to solve

[0075] To manufacture the hangover remedy composition of the present invention, a mutant strain capable of simultaneously and efficiently producing aldehyde dehydrogenase and glutathione was first created using a chemical mutagenesis method, and a selection factor adaptation mutant strain was developed.

[0076] Mutant strains that can simultaneously overproduce glutathione and aldehyde dehydrogenase 2 were selected. Wild-type Saccharomyces cerevisiae strains that are reported as GRAS (Generally Recognized As Safe) and have low production efficiency were selected for use in food, health food, feed, cosmetics, and pharmaceutical applications.

[0077] This invention provides a hangover remedy by producing a new, improved strain of *Saccharomyces cerevisiae* sp. through mutation, which increases both glutathione and aldehyde dehydrogenase production. The resulting strain is then used to produce a dried powder, lysate, or ALDH-containing extract.

[0078] Problem Solving Methods

[0079] The active ingredients of the hangover remedy composition of the present invention are detailed in Korean Patent Application No. 10-2020-0019858, which are dried powder, lysate, or ALDH-containing extracts of Saccharomyces cerevisiae Kwon P-1 (KCTC13925BP), Saccharomyces cerevisiae Kwon P-2 (KCTC14122BP), or Saccharomyces cerevisiae Kwon P-3 (KCTC14123BP) deposited with the International Depository & Trust Company (KCTC).

[0080] The inventors have invented a new fermentation process that can significantly improve the production yield of ALDH by using three strains, including Saccharomyces cerevisiae Kwon P-1 (deposit number: KCTC13925BP), which has high ALDH and glutathione production capacity, either alone or in combination, for a first liquid fermentation step, followed by adding the liquid fermentation product to rice fermentation powder for a second solid fermentation step.

[0081] Furthermore, in the novel fermentation process of this invention, one or a mixture of three strains, such as Saccharomyces cerevisiae Kwon P-1 (deposit number: KCTC13925BP), is used to carry out a first liquid fermentation step and a second solid fermentation step, thereby completing a fermentation process that can simultaneously produce glutathione and ALDH enzyme, which are powerful reducing agents, with high efficiency.

[0082] After inoculating a large quantity of cultured Saccharomyces cerevisiae KwonP-1 (deposit number: KCTC13925BP) into rice for solid-state fermentation, thereby culturing a Saccharomyces cerevisiae strain that overproduces glutathione and aldehyde dehydrogenase on a larger scale in a two-step process, the hangover relief composition of the present invention, containing the strain as a dried powder, lysate, or extract powder, is produced. Attached Figure Description

[0083] Figure 1 A graph showing the changes in acetaldehyde levels in the blood of experimental animals using the compositions according to the present invention;

[0084] Figure 2 A graph showing the changes in acetaldehyde levels in the blood of experimental animals using the compositions according to the present invention.

[0085] Best practice

[0086] The structure and effects of the present invention will now be described in detail through the following embodiments. However, these embodiments are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to the following embodiments. Detailed Implementation

[0087] [Example 1] Preparation of yeast lysate containing glutathione and ALDH

[0088] Example 1-1: Fermentation process of Saccharomyces cerevisiae containing glutathione and ALDH

[0089] Saccharomyces cerevisiae containing ALDH was fermented in 200 mL Flask on YPD medium (containing yeast extract, peptone, and glucose) at 160 rpm and 30°C for 24 hours. This culture was further cultured in a 5L fermenter (Marado-05D-PS, CNS, Korea) for 72 hours. After the culture, the yeast was separated by centrifugation using a high-speed centrifuge (Supra R22, Hanil, Korea).

[0090] Examples 1-2: Preparation of yeast lysates containing glutathione and ALDH

[0091] ALDH-containing yeast, separated by centrifugation, was frozen for 2 days in an ultra-low temperature freezer (CLN-52U, Nihon freezer, Japan), and then freeze-dried for 2 days using a freeze dryer (FDU-7006, Opera, Korea). 3g of the freeze-dried yeast powder was dissolved in 50mL of phosphate-buffered saline (PBS) containing a yeast proteolytic inhibitor (A32955, Thermo Fisher, USA), followed by the addition of 10g of 0.5mm cell disruption glass beads (11079105, Biospec). The yeast was disrupted three times using a glass bead mill (MixerMill MM400, Retsch, Germany), each time for 2 minutes. After centrifugation using a high-speed centrifuge (Supra R22, Hanil, Korea), only the supernatant was separated and freeze-dried for 2 days using a freeze dryer (FDU-7006, Opera, Korea).

[0092] [Example 2] Mass production of Saccharomyces cerevisiae strains via a two-step fermentation process

[0093] The *Saccharomyces cerevisiae* strain KwonP-1 (KCTC13925BP) was inoculated into YPD medium containing 2% peptone, 1% yeast extract, and 2% glucose, and cultured at 30°C until OD (dose retardation) was achieved. 600nm Fermentation was carried out in a fermenter (Fermentor, KoBioTech) at 200 rpm and 1 vvm until the value reached 50. The cells were then recovered from the culture using a membrane filter.

[0094] The recovered microbial cells were mixed with sterilized rice fermentation powder at a ratio of 10%, and the moisture content was adjusted to 60%. After solid-state culture at 30°C for 2 days, the mixture was dried at 50°C to adjust the final moisture content to 7%, thus producing yeast-fermented rice fermentation powder.

[0095] The fermentation composition of the present invention prepared in this way contains a maximum of 600 units / g of ALDH. Considering that rice fermentation powder using wild-type Saccharomyces cerevisiae strains generally contains about 2 units / g of ALDH, the ALDH content of the fermentation composition of the present invention is increased by about 300 times.

[0096] Table 1 shows the results of evaluating the aldehyde decomposition capabilities of the compositions (1 to 4) produced by the two-step fermentation process of the present invention within 5 minutes.

[0097] Table 1

[0098]

[0099] The fermentation composition of the present invention, thus prepared, is dried and pulverized into a powder. After adding ALDH and coenzyme NAD as enzyme activators, citric acid, magnesium stearate, DL-methionine, vitamin C, and lactic acid bacteria (Lactobacillus plantium 10) are added. 7 The hangover remedy of the present invention is made of zinc oxide and silicon dioxide ( / g).

[0100] Animal experiments using the hangover remedy of this invention, which measured the concentration of acetaldehyde in the blood after alcohol consumption, showed that, compared with existing hangover remedies, the hangover remedy of this invention significantly and rapidly reduced the concentration of acetaldehyde in the blood.

[0101] In addition, for the human clinical trial of the hangover eliminator of the present invention, active volunteers in the clinical trial were divided into an ALDH2 preserving group, which can decompose aldehydes, and an ALDH2*2 variant gene preserving group, which has a genetic deficiency in aldehyde decomposition ability.

[0102] Results of a 15-hour hangover elimination experiment in humans showed significant differences in aldehyde decomposition ability between the ALDH2 preserving experimental group and the ALDH2*2 gene mutation experimental group. The hangover eliminator of this invention effectively removed acetaldehyde in both experimental groups. In particular, it effectively removed aldehydes even in the ALDH 2*2 gene mutation experimental group, where aldehydes are difficult to decompose, thus confirming the aldehyde decomposition and hangover elimination effects of the hangover eliminator of this invention achieved by increasing the content of ALDH and glutathione.

[0103] [Example 3] Measurement of the hangover relief effect of the composition of the present invention

[0104] Example 3-1: Animal experiment on the change of acetaldehyde in blood over time

[0105] The animal experimental results showing the changes in acetaldehyde in the blood over time after the use of ethanol are presented in Table 2.

[0106] Table 2

[0107]

[0108] The results of animal experiments on the accumulation of aldehydes in the blood (mg / L·hr) are shown in Table 3.

[0109] Table 3

[0110]

[0111]

[0112] Example 3-2: Analysis of ethanol and aldehyde in the blood of volunteers in human clinical trials

[0113] Forty-three healthy adult men aged 20 to 40 who could drink an average of 20% alcohol by one time were selected as volunteers for the human clinical trial. The trial was conducted weekly for 15 hours, from 5 p.m. to 8 a.m. the following week, in a group accommodation at the clinical trial hospital. Due to individual circumstances and other reasons, 23 of the participants eventually completed the trial.

[0114] On the first day of group accommodation, after drinking 10 cups of soju, the blood alcohol metabolism, i.e., changes in alcohol concentration and acetaldehyde concentration, was measured at different time intervals. On the second day of group accommodation, after taking 73 mg / kg of the composition of the present invention, 10 cups of soju were taken 30 minutes later, and the changes in blood alcohol metabolism were measured. On the third day of group accommodation, after taking 220 mg / kg of the composition of the present invention, 10 cups of soju were taken 30 minutes later, and the changes in blood alcohol metabolism were measured.

[0115] In the group taking the composition containing 500 mg / day of the two-step fermented dried powder and 1500 mg of fermented rice powder of the present invention, the blood concentration of acetaldehyde, a hangover causative agent and a potent carcinogen, was significantly reduced volume-related compared to the group taking alcohol alone. Furthermore, the amount of residual alcohol in the blood was also significantly reduced volume-related compared to the composition of the present invention.

[0116] The reduction in blood alcohol concentration in volunteers in human clinical trials is shown in Table 4.

[0117] Table 4

[0118]

[0119] The reduction in residual acetaldehyde in the blood of volunteers in human clinical trials is shown in Table 5.

[0120] Table 5

[0121]

[0122] Example 3-3: Confirmation test of changes in ethanol and acetaldehyde based on whether or not there is an ALDH gene mutation

[0123] To recruit volunteers for the human clinical trial, 43 healthy adult men aged 20 to 40 who could drink an average of 20% alcohol by volume of standard soju were selected. The clinical trial was conducted weekly for 15 hours, from 5 PM to 8 AM the following day, at the designated hospital. Due to individual circumstances and other reasons, 23 participants ultimately completed the trial.

[0124] Among them, about 22 participants underwent gene testing related to alcohol metabolism. With the consent of the participants, the three genomes related to alcohol metabolism, ADH1B (Alcohol dehydrogenase 1B), ALDH2 (Aldehydedehydrogenase 2), and CPY2E1 P450, were tested in vivo in these 22 participants. It was confirmed that compared with the alcohol-only intake group, the blood concentration of acetaldehyde, a hangover causative agent and a potent carcinogen, was significantly reduced in both the non-variant ALDH2 subunit and the ALDH2*2 variant subunit.

[0125] In cases of the ALDH2*2 gene mutation, even small amounts of alcohol consumption result in very high blood acetaldehyde concentrations, and there have been no reports of acetaldehyde reduction effects in the blood achieved through conventional hangover relief drinks or existing hangover relief foods and medicines. However, the acetaldehyde reduction effect in the blood caused by the ALDH2*2 gene mutation is noteworthy when using the hangover relief composition of the present invention.

[0126] The alcohol content (g hr / L) measurements of the normal ALDH gene preserving group and the ALDH gene variant group are shown in Table 6.

[0127] Table 6

[0128]

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

[0130] Table 7

[0131]

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

[0133] Example 4-1 Preparation of experimental animals

[0134] Experimental animals were male and female ICR mice (7 weeks old) that were acclimatized for 7 days. During the acclimatization period, general symptoms were observed, and only healthy animals were used for the experiment. Feed and water were provided freely. One day before oral administration, the mice were divided into groups of 10, with 5 males and 5 females in each group, based on a weight of approximately 20g.

[0135] Example 4-2: Use of the hangover relief composition of the present invention

[0136] The test substances were based on the content of the yeast lysate containing GSH and ALDH of this invention, dissolved in physiological saline to prepare dosages of 0, 750, 3000, and 5000 mg / kg. The dosage guidelines followed the Korea National Toxicology Program (KNTP) toxicity testing manual of the Ministry of Food and Drug Safety, using the maximum recommended dosage of 5000 mg / kg as the maximum concentration for this experiment. Each experimental animal was orally administered the prepared test materials once, while the control group (G1) received physiological saline.

[0137] Example 4-3 Observation and Autopsy

[0138] For all animals in the experimental group, symptom observation was conducted more than once daily from the date of administration until the necropsy, with symptom observation also performed for 7 days after oral administration. After the symptom observation period, necropsy was performed, during which changes in various organs were observed visually.

[0139] The present invention, using yeast lysate containing glutathione and ALDH, conducted a single-use toxicity test on mice. Results showed that at concentrations up to 5000 mg / kg, no deaths were observed within 7 days, and no unusual changes were observed such as weight gain or feed intake. Furthermore, no unusual conditions were observed in the post-observation autopsy.

[0140] [Collection Number]

[0141] Name of the depository: Korea Institute of Life Sciences

[0142] Collection Number: KCTC13925BP

[0143] Date of preservation: August 22, 2019

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[0145]

[0146]

[0147]

Claims

1. The use of a composition containing glutathione and aldehyde dehydrogenase in the preparation of a hangover remedy, wherein, The composition comprises a lysate of a mutant Saccharomyces cerevisiae, wherein the glutathione and aldehyde dehydrogenase are derived from the lysate of the mutant Saccharomyces cerevisiae; or, the use comprises extracting glutathione and aldehyde dehydrogenase from the mutant Saccharomyces cerevisiae; and The mutant brewer's yeast is selected from the group consisting of: brewer's yeast Kwon P-1 with accession number KCTC13925BP, brewer's yeast Kwon P-2 with accession number KCTC14122BP, and brewer's yeast Kwon P-3 with accession number KCTC14123BP, or a mixture thereof.

2. The use according to claim 1, wherein, The composition is characterized by further containing added NAD.

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