Food and pharmaceutical compositions for detoxifying endogenous aldehydes

Abstract

The present invention relates to a food and pharmaceutical composition for promoting the decomposition of an endogenous aldehyde produced by the oxidation of an endogenous amine compound and an alcohol, said food and pharmaceutical composition comprising an aldehyde dehydrogenase selected from the group consisting of Saccharomyces cerevisiae KCTC 13925BP, KCTC 14122BP, KCTC 14123BP, KCTC 14983BP, KCTC 14984BP, and KCTC 14985BP, or a mixture thereof. The food and pharmaceutical compositions of the present invention inhibit oxidative stress and autowine syndrome.

Description

Technical Field

[0001] The present invention relates to mutant Saccharomyces cerevisiae KCTC14983BP, KCTC14984BP, and KCTC14985BP. Furthermore, the present invention relates to an aldehyde dehydrogenase encoded by a gene having greater than 98% homology to the gene of SEQ ID NO: 1. Specifically, the present invention relates to an aldehyde dehydrogenase encoded by the gene of SEQ ID NO: 1, characterized in that it includes SEQ ID NO: 2.

[0002] In addition, the present invention relates to a food composition that suppresses physiological discomfort in the human body caused by various aldehydes derived from endogenous alcohol compounds. Specifically, it relates to a food or pharmaceutical composition that suppresses auto-brewery symptoms caused by endogenous acetaldehyde.

[0003] In addition, the present invention relates to a food composition and a pharmaceutical composition for inhibiting oxidative stress, comprising a lysate of any one selected from the group consisting of KCTC13925BP, KCTC 14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof. Background Art

[0004] The human body is subject to oxidative stress due to various factors, such as drinking, smoking, taking drugs, strenuous exercise, and physical and mental stress. Accordingly, various types of endogenous aldehydes are produced in human cells.

[0005] In addition, reactive oxygen species (ROS) are generated during energy production in mitochondria in cells. They generate various types of endogenous aldehydes through lipid peroxidation (LPO) in cell membranes.

[0006] The endogenous aldehydes produced in this way are very reactive. They readily react with surrounding proteins and modify DNA. This can degrade proteins, and their unique functions may be reduced or completely lost.

[0007] Therefore, the production and accumulation of endogenous aldehydes leads to accelerated cellular aging and may be the root cause of cancer, diabetes, cardiovascular disease, and neurodegenerative diseases.

[0008] The largest contributor to the increase in endogenous aldehydes in the human body is alcohol, which is consumed through drinking alcohol, taking medication, etc. For example, when drinking alcohol, 80%-90% of the alcohol undergoes a two-stage enzymatic metabolism.

[0009] In the first stage, alcohol is converted into the toxic metabolic intermediate acetaldehyde (Ach) by alcohol dehydrogenase (ADH). In the second stage, acetaldehyde (Ach) is detoxified into acetate by the action of aldehyde dehydrogenase (ALDH). Figure 1 ].

[0010] During alcohol metabolism in the body, excess acetaldehyde (ACh) generated due to genetic defects or excessive alcohol consumption directly modifies surrounding proteins and DNA. Consequently, it acts as a toxic substance that can cause cancer. The present invention relates to an aldehyde dehydrogenase that detoxifies endogenous acetaldehyde, preventing it from acting as a toxic substance.

[0011] Even people who don't drink alcohol can produce ethanol through their gut microbes. This endogenous ethanol is converted to acetaldehyde by ethanol decomposition enzymes. The production of acetaldehyde can lead to physiological discomfort, such as hangovers caused by alcohol consumption, and can cause auto-brewery syndrome (ABS) (Malik F et al., case report and literature review, "Auto-brewery syndrome: probably an underdiagnosed medical condition," BMJ Open Gastro 2019).

[0012] Auto-brewery syndrome results from endogenous ethanol fermentation. This so-called "gut fermentation syndrome" can cause hangover symptoms even if the person has not consumed any alcohol.

[0013] According to the U.S. National Library of Medicine and Makati Medical Hospital, enteric fermentation syndrome (IFS) occurs when fungi or bacteria in the gastrointestinal tract, oral cavity, and urinary system spontaneously produce ethanol. Even if a person doesn't drink alcohol, the yeast in the body produces endogenous alcohol, resulting in hangover symptoms. It is caused by the fermentation of alcohol by various yeast strains and bacteria in the human intestine.

[0014] Yeast in the intestines produces carbon dioxide and ethanol during food digestion, so even if people with auto-brewery syndrome don't consume alcohol, they experience hangover symptoms due to acetaldehyde produced during the breakdown of alcohol in the body.

[0015] While endogenous ethanol production is part of the normal digestive process, enteric fermentation syndrome is caused by yeast or bacteria in the body that produce alcohol. This primarily occurs in people with conditions such as diabetes, obesity, and Crohn's disease, but can also occur in healthy people. People with liver disorders such as chronic intestinal obstruction, gastrointestinal paralysis, nonalcoholic fatty liver disease, or nonalcoholic hepatitis may also experience auto-brewery syndrome.

[0016] Patients with auto-brewery syndrome may experience various symptoms such as vomiting, belching, dizziness, disorientation, fainting, irritable bowel symptoms, runny nose, cough, and sinusitis. Chronic fatigue syndrome can lead to health problems such as anxiety, depression, and reduced work efficiency.

[0017] At the same time, the increase of acetaldehyde in the human body directly or indirectly leads to lipid peroxidation (LPO), which in turn promotes the production of various endogenous harmful aldehydes, such as malondialdehyde (MDA) and nonenal (4-hydroxynonenal, 4-HNE).

[0018] Like acetaldehyde, which is classified as a major carcinogen by the International Agency for Research on Cancer (IARC) and the World Health Organization (WHO), nonenal and malondialdehyde also modify proteins and cells and cause cancer. 4-HNE has been found to be a biomarker associated with the development of Alzheimer's disease (AD), cataracts, atherosclerosis, diabetes, and cancer.

[0019] Malondialdehyde binds to deoxyadenosine or deoxyguanosine in DNA, permanently modifying it. In other words, it is a substance that causes cancer. The U.S. National Cancer Institute (NCI Glossary, NCIt) classifies malondialdehyde as a strong endogenous mutagen and uses it as a biomarker for cardiovascular disease and fatigue.

[0020] The human body generates energy through the mitochondria within its cells to sustain various life activities. During this process, reactive oxygen species (ROS) are inevitably generated during mitochondrial energy production or conversion. This leads to lipid peroxidation (LPO), which damages lipid membranes. This lipid peroxidation reaction produces and accumulates aldehydes, such as nonenal (4-HNE), malondialdehyde (MDA), and acetaldehyde (ACh), in cells.

[0021] Modified proteins, such as malondialdehyde-acetaldehyde adducts (MAA) and malondialdehyde-lysine adducts (M-lys), are formed through chain reactions between endogenous aldehydes and proteins. They accumulate in cells and increase oxidative stress in the human body. Figure 2 ].

[0022] Increased oxidative stress disrupts the smooth energy metabolism process in mitochondria, further increasing the amount of aldehydes in cells, such as methylglyoxal (MG) and advanced glycation end products (AGEs). As a result, the intracellular accumulation of aldehydes is accelerated.

[0023] Thus, cytotoxicity occurs when reactive aldehydes (such as HNE and MDA) or aldehydes (such as the glycolytic intermediate glyceraldehyde-3-phosphate (GA3P)) produced by lipid peroxidation (LPO) due to increased free radicals and oxidative stress are overproduced and accumulated in cells.

[0024] The intracellular accumulation of free radicals or reactive aldehydes weakens the cellular antioxidant defense system (such as glutathione). Disruption of energy metabolism and the accumulation of unfolded proteins (UP) ultimately lead to increased endoplasmic reticulum stress (ER stress). As a result, cytotoxicity occurs, accelerating aging and triggering various diseases.

[0025] At the same time, the human body contains monoamine substances that perform various physiological functions, such as dopamine (DA), serotonin (5-HT), norepinephrine (NE), epinephrine (Adr), γ-aminobutyric acid (GABA) and histamine.

[0026] Through the systemic action of monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), catechol-O-methyltransferase (COMT) and alcohol dehydrogenase (ADH), the amine group (-NH2) of the monoamine substance is converted into an aldehyde group (-CHO). The aldehyde group thus converted is finally converted into an acid radical (-CO2H) to complete the metabolism of the monoamine substance in the body [ Figure 3 ].

[0027] Dopamine (DA) is the raw material for adrenaline (Adrenaline), which regulates the autonomic nervous system. Dopamine is a representative neurotransmitter and is associated with the development of Parkinson's disease (PD).

[0028] L-Dopa, produced from L-phenylalanine, is converted into dopamine (DA) by enzymes in dopaminergic neurons in the substantia nigra pars compacta (SNpc), a specific part of the brain.

[0029] Dopamine (DA) thus secreted is metabolized by a non-enzymatic reaction in which it is converted into neuromelanin or quinone by auto-oxidation via free radicals. In addition, dopamine can be metabolized by the action of monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH) and catechol methyltransferase (COMT) [ Figure 3 ].

[0030] As explained above, the metabolic process of dopamine (DA) is divided into two metabolic pathways based on the order of the enzymes involved. In the metabolic pathway where monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), and catechol methyltransferase (COMT) act in sequence, dopamine is converted into 3,4-dihydroxyphenylacetaldehyde (DOPAL), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA).

[0031] In a metabolic pathway where catechol methyltransferase (COMT), monoamine oxidase (MAO), and aldehyde dehydrogenase (ALDH) act sequentially, dopamine is converted into 3-methoxytyramine (3-MT), 3-methoxy-4-hydroxyphenylacetaldehyde (MOPAL), and homovanillic acid (HVA). Through these two metabolic pathways, dopamine is ultimately converted into homovanillic acid (HVA).

[0032] Dopamine is also metabolized into norepinephrine (NE) and epinephrine (Adr), hormones that regulate the autonomic nervous system. In this metabolic process, dopamine is metabolized in the order of 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), 3,4-dihydroxymandelic acid (DOMA), and 4-hydroxy-3-methoxymandelic acid.

[0033] In addition, dopamine is converted into 3-methoxynorepinephrine (methoxy NE), 4-hydroxy-3-methoxyphenylethanolaldehyde (MOPEGAL) and 3-methoxy-4-hydroxymandelic acid through the sequential action of catechol methyltransferase (COMT), MAO and aldehyde dehydrogenase (ALDH).

[0034] As described above, the amine (-NH2) functional group of dopamine is converted to aldehyde (-CHO) and ultimately to acid (-CO2H) through the sequential enzymatic actions of monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), and catechol methyltransferase (COMT).

[0035] At the same time, serotonin (5-HT), produced from tryptophan (L-tryptophan), is closely related to mental health, such as learning and sleep. Serotonin is also known as the representative neurotransmitter. Through the sequential enzymatic action of monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), and catechol methyltransferase (COMT), serotonin is metabolized to 5-hydroxyindoleacetaldehyde (5-HIAL) and 5-hydroxyindoleacetic acid (5-HIAA).

[0036] Serotonin is converted into melatonin by the action of acetyltransferase (N-acetyltransferase) and catechol methyltransferase (COMT). Melatonin is converted into 5-methoxyindole-3-acetaldehyde (5-MIAL) by the action of monoamine oxidase (MAO). The 5-MIAL thus produced is metabolized into acid by the action of aldehyde dehydrogenase (ALDH). Figure 4 ].

[0037] In addition, 5-hydroxyindoleacetaldehyde (5-HIAL), produced during serotonin (5-HT) metabolism, has been reported to lead to modifications of α-synuclein (α-Syn) and the production of α-synuclein oligomers (Jinsmaa et al., 2015).

[0038] When drinking alcohol, the normal metabolism of serotonin is disrupted because serotonin binds to alcohol-metabolizing enzymes. As a result, serotonin aldehyde (5-HIAL) is converted to 5-hydroxytryptol (5-HTOL) and accumulates in cranial nerves (Shibata et al., 2014).

[0039] During the metabolism of alcohol into acetic acid, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are rapidly consumed. ALDH deficiency, which is required for the metabolism of serotonin, may occur.

[0040] Due to the lack of this aldehyde dehydrogenase (ALDH), the metabolite of serotonin (5-HT), serotonin aldehyde (5-HIAL), cannot be broken down normally and accumulates, leading to toxic effects and deforming proteins in brain nerve cells.

[0041] To mitigate the toxic effects of 5-HIAL, abnormal metabolic pathways are activated to rapidly convert 5-HIAL into 5-hydroxytryptol (5-HTOL).

[0042] After the breakdown of alcohol in the body is complete, the deficiency of aldehyde dehydrogenase is resolved, the normal metabolic pathway of serotonin is restored, and 5-hydroxytryptol (5-HTOL) is converted back into serotonin aldehyde (5-HIAL) and metabolized through the normal metabolism of serotonin.

[0043] Due to the toxic effects of alcohol decomposition, oxidative stress is increased and the function of alcohol dehydrogenase (ADH) is significantly reduced. Even after alcohol is metabolized in the body, 5-hydroxytryptol (5-HTOL) accumulates in the body and cannot be converted into serotonin aldehyde (5-HIAL).

[0044] With alcohol intake, the serotonin metabolism pathway is disrupted due to the deficiency of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), and 5-hydroxytryptol (5-HTOL) abnormally increases in brain nerve cells (Shibata et al., 2014).

[0045] It has been found that due to this abnormality in serotonin metabolism, 5-hydroxytryptol (5-HTOL) increases in the liver, ileum, and spleen, weakening the functions of related organs and leading to disease. It is happening.

[0046] In summary, 5-HIAL itself is a toxin that causes Parkinson's disease. In addition, 5-HIAL distorts the serotonin metabolic pathway and increases the amount of modified proteins, leading to various diseases such as abnormal liver function.

[0047] At the same time, GABA is converted to succinic semialdehyde (SSA) by monoamine oxidase (MAO), which may act as a toxic aldehyde to nerve cells[ Figure 5 If SSA is not converted into succinate and accumulates in

[0048] Succinic semialdehyde (SSA), a metabolic intermediate of GABA, is converted into non-toxic succinate by the action of succinic semialdehyde dehydrogenase (SSADH). If aldehyde dehydrogenase does not function properly, succinic semialdehyde (SSA) is converted into gamma-hydroxybutyrate (GHB) by the action of aldo-keto reductase (AKR).

[0049] Succinic semialdehyde (SSA), an endogenous aldehyde, is a representative cytotoxic substance that accumulates when aldehyde dehydrogenase (ALDH) function is impaired. This toxic substance induces the production and accumulation of gamma-hydroxybutyrate (GHB), thereby impairing physical functions such as liver function, speech, and walking (Buzzi, Andrea et al., 2006).

[0050] Histamine is also involved in allergic reactions and inflammation. Histamine is one of the substances secreted by the human body in response to external stimuli (stress). Histamine is secreted by immune cells (such as basophils and mast cells) in response to antigen-antibody reactions, leading to symptoms such as bronchoconstriction (allergic asthma), capillary dilation, runny nose, and edema.

[0051] Histamine is also metabolized by an organized enzyme system involving monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), and histamine-N-methyltransferase (HNMT). Histamine is converted to imidazoleacetaldehyde, imidazoleacetic acid, and ultimately to imidazoleacetic acid nucleoside. Figure 6 ].

[0052] Histamine is also converted into N-methylhistamine, N-methylimidazole acetaldehyde and N-methylimidazole acetic acid by the action of the enzyme system of histamine-N-methyltransferase (HNMT), MAO and aldehyde dehydrogenase in sequence.

[0053] During the metabolism of histamine, the amine group (-NH2) is metabolized into aldehyde (-CHO) and acid (-CO2H) through the detoxification process of monoamine oxidase (MAO) and aldehyde dehydrogenase (ALDH). Figure 6 ].

[0054] Imidazole acetaldehyde, which can be produced when histamine metabolism is abnormal, can cause various diseases through toxic effects that are more potent than histamine. The human body is designed to quickly detoxify secreted histamine using an enzyme system to minimize damage to the body.

[0055] When excessive amounts of histamine are secreted at once, or when histamine metabolism is abnormal due to weakened aldehyde dehydrogenase (ALDH) function, the body overreacts to antigens, causing discomfort and abnormal symptoms. These phenomena are called histamine intolerance (HIT) or allergic symptoms.

[0056] Imidazole acetaldehyde or N-methylimidazole acetaldehyde produced from histamine by the action of monoamine oxidase (MAO) should be converted into acid by the action of aldehyde dehydrogenase (ALDH).

[0057] When the function of the aldehyde dehydrogenase (ALDH) enzyme is impaired, aldehyde metabolites derived from histamine have toxic effects in cells and lead to allergic symptoms or histamine hypersensitivity (HIT). In severe cases, the monoamine aldehyde imidazole acetaldehyde produced from histamine disrupts the immune system and increases the toxic effects of histamine. This can lead to chronic itching, psoriasis, atopy, and asthma.

[0058] As discussed above, various monoamines (e.g., dopamine (DA), serotonin (5-HT), norepinephrine (NE), epinephrine (Adr), gamma-aminobutyric acid (GABA), and histamine) that act as hormones or neurotransmitters (monoaminergic neurotransmitters) in the human body can be converted into 3,4-dihydroxyphenylacetaldehyde (DOPAL), 3-methoxy-4-hydroxyphenylacetaldehyde (MOPAL), 3,4-dihydroxyphenylethanolaldehyde (DOPEGAL), 3-methoxy-4-hydroxyphenylethanolaldehyde (MOPEGAL), 5-hydroxyindoleacetaldehyde (5-HIAL), 5-methoxyindole-3-acetaldehyde (5-MIAL), imidazoleacetaldehyde, and N-methylimidazoleacetaldehyde through the action of monoamine oxidase.

[0059] When aldehyde dehydrogenase in the human body fails to function properly, various aldehydes produced in the body act as toxic substances to the human body. As a result, various pathological phenomena occur in the human body and aging is accelerated.

[0060] There is an urgent need to develop food or pharmaceutical compositions that can rapidly oxidize and detoxify various endogenous aldehydes, thereby inhibiting the accumulation of endogenous aldehydes in the body and causing various problems.

[0061] [Prior Art Document]

[0062] [Patent Document]

[0063] U.S. Patent Application Publication No. US2021-0254023-A1, dated August 21, 2021

[0064] [Non-patent document]

[0065] JRRoede, BJStewart, DRPetersen, Hepatotoxicity of ReactiveAldehydes, Comprehensive Toxicology(2 nd Edition)Vol 92010:581-594.

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[0067] Huang et al,″Association of alcohol dehydrogenase and aldehydedehydrogenase polymorphism with spontaneous deep intracerebral haemorrhage inthe Taiwan population″.Scientific Reports.2020;10:article 3641.

[0068] Bhatt et al,″Age-dependent protein abundance of cytocolic alcohol andaldehyde de-hydrogenascs in human liver″.Drug Metab Dispos.2007Sep;45(9):1044-1048.

[0069] Chen et al,″Targeting aldehyde dehydrogenase 2:new therapeuticopportunities″.Physiol Rev.2014;94:1-34.

[0070] Edenberg et al,″The Genetics of Alcohol Metabolism:Role of AlcoholDehy-drogenase and Aldehyde Dehydrogenase Variants◎Alcohol Research&Health2007;30(1):5-13.

[0071] Eisenhofer etal,″Catecholamine Metabolism:A Contemporary View withIm-plications for Physiology and Medicine.Pharmacol Rev.2004 Sep;56(3):331-49.

[0072] Cagle et al,″Biogenic aldehyde-mediated mcchanisms of toxicity inneurode-generative disease.Curr Opin Toxicol,2019 Feb;13:16-21.

[0073] Lv et al,″The Role of Serotonin beyond the Central Nervous Systemduring Em-bryogenesis◎Front.Cell.Neurosci.2017 March;13.

[0074] Erdag et al,″Biochemical and Pharmacological Properties of BiogenicAmines″.Biogenic Amines.Edited Vol 2018 Apr 21.

[0075] Erwin et al,″Brain Aldehyde Dehydrogenase:Localization,PurificationAnd Properties″.J.Biological Chem.1966 Aug 10;241(15):3533-3539.

[0076] Mackerell Jr et al.″Human Aldehyde Dehydrogenase:KineticIdentification of the Isozyme for Which Biogenic Aldehydes and AcetaldehydeCompete″.Alcohol Clin Exp Res.1986 Jun;10(3):266-270.

[0077] Masato et al,″Impaired dopamine metabolism in Parkinson's diseasepathogenesis″.Molecular Neurodegeneration.2019;14(35):14-35.

[0078] Wenzel et al,″Manganese Superoxide dismutase and aldehydedehydrogenase de-ficiency increase mitochondrial oxidative stress andaggravate age-dependent vascular dysfunction″.Cardiovascular Res.2008 Nov;80(2):280-289.

[0079] Jin et al,″Associations of Alcohol Dehydrogenase and AldehydeDehydrogenase Polymorphism With Cognitive Impairment Among the Oldest-Old inChina″.Front Aging Neurosci.2021;13:710966.

[0080] Ohsawa et al,″Genetic deficiency of a mitochondrial aldehydedehydrogenase increases serum lipid peroxides in community-dwelling females″.Journal of Human Genetics2003;48:404-409.

[0081] Shin et al,″Alcohol Consumption,Aldehyde Dehydrogenase 2 GenePolymorphisms and Cardiovascular Health in Korea″.Yonsei Med J.2017 Jul;58(4):689-696.

[0082] Malik F, et al. "Case report and literature review of auto-brewerysyndrome: probably an underdiagnosed medical condition" BMJ Open Gastro 2019;6:e000325.doi:10.1136 / bmjgast-2019-000325. Summary of the Invention Technical issues

[0083] Despite the various studies listed above, food compositions or pharmaceutical compositions that suppress increased oxidative stress, auto-brewery syndrome, chronic pruritus, psoriasis, atopy, and asthma caused by accumulation of endogenous aldehydes in the body by detoxifying various endogenous aldehydes have not yet been developed.

[0084] The basic object of the present invention is to provide mutant Saccharomyces cerevisiae KCTC14983BP, mutant Saccharomyces cerevisiae KCTC14984BP and mutant Saccharomyces cerevisiae KCTC14985BP, which can produce aldehyde dehydrogenase.

[0085] In addition, the main object of the present invention is to provide a food composition comprising an aldehyde dehydrogenase encoded by a gene having more than 98% homology to the gene of SEQ ID NO: 1, including SEQ ID NO: 2, which promotes the decomposition of endogenous aldehydes produced by oxidation of endogenous amine compounds or alcohols.

[0086] Another object of the present invention is to provide a food composition and a pharmaceutical composition for reducing oxidative stress in the human body, which contain aldehyde dehydrogenase, wherein the aldehyde dehydrogenase is contained in a lysate of any one selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

[0087] Another object of the present invention is to provide a food composition and a pharmaceutical composition for preventing auto-brewery syndrome, which contain aldehyde dehydrogenase contained in a lysate of any one selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

[0088] Another object of the present invention is to provide a food composition and a pharmaceutical composition for preventing various symptoms of diseases caused by endogenous aldehydes, which contain aldehyde dehydrogenase, and the aldehyde dehydrogenase is contained in a lysate of any one selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

[0089] Solution to the problem

[0090] The object of the present invention as described above can be achieved by providing a food composition and a pharmaceutical composition, wherein the composition comprises an aldehyde dehydrogenase, wherein the aldehyde dehydrogenase is contained in a lysate selected from any one of the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof (hereinafter referred to as KARC), and the aldehyde dehydrogenase can quickly decompose endogenous aldehydes.

[0091] Another object of the present invention can also be achieved by providing a food composition or a pharmaceutical composition for preventing auto-brewery syndrome, wherein the food composition or the pharmaceutical composition comprises a lysate of any one selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

[0092] Advantageous Effects of the Invention

[0093] KARC, a dried powder of lysate of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, comprising the aldehyde dehydrogenase encoded by SEQ ID NO: 1.

[0094] The food composition and the pharmaceutical composition of the present invention exhibit the effects of inhibiting oxidative stress in the human body and inhibiting auto-brewery syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] [ Figure 1 and Figure 2 ] shows the production and decomposition of endogenous aldehydes in the body.

[0096] Ethanol or ethanol derivatives (2-substituted alcohols, R-CH2CH2-OH) are reversibly converted into acetaldehyde derivatives (R-CH2-CHO) in vivo by alcohol dehydrogenase (ADH). Acetaldehyde derivatives (highly toxic substances) are irreversibly converted into relatively non-toxic acetic acid derivatives (R-CH2-CO2H).

[0097] Endogenous monoamines (R-C2H4-NH2) are irreversibly converted to highly toxic acetaldehyde (R-CH2-CHO) by monoamine oxidase (MAO) and to acetaldehyde by aldehyde dehydrogenase, and ultimately detoxified to acetic acid (R-CH2-CO2H) as in alcohol metabolism.

[0098] exist[ Figure 2 In the human body, dopamine (DA) is a representative monoamine neurotransmitter, which is converted into toxic aldehyde structures (DOPANAL, dopamine-induced aldehydes) such as DOPAL and MOPAL by monoamine oxidase (MAO). These are degraded by aldehyde dehydrogenase (ALDH) and ultimately metabolized into relatively low-toxic homovanillic acid (HVA).

[0099] Dopamine is also converted to DOPANALs via norepinephrine (NE) by monoamine oxidase, such as dopegal and mopegal (known toxic substances), which are ultimately broken down into acidic compounds by aldehyde dehydrogenase.

[0100] In addition, due to various reasons, such as decreased ALDH, dopamine metabolism is not well performed, resulting in an increase in DOPANOL (dopamine-induced alcohol), such as DOPOL via DOPANAL, because DOPANAL is not converted into less toxic acidic compounds.

[0101] This is known. Due to the toxicity of DOPANAL (dopamine-induced aldehyde), it is temporarily converted into a relatively less toxic alcohol, DOPANOL (dopamine-induced alcohol), and stored. When dopamine metabolism returns to its original state, the representative DOPANOL stored in the body, DOPET, is metabolized and broken down into acid through the activation of alcohol-metabolizing enzymes (i.e., alcohol dehydrogenase and aldehyde dehydrogenase).

[0102] Despite the existence of various enzymatic dopamine metabolic pathways, when enzymatic dopamine metabolism is not well performed, dopamine is metabolized through non-enzymatic reactions, in which it is spontaneously converted into quinone derivatives by reactive oxygen species (ROS) and then into neuromelanin. In this case, it is also known that the disruption of homeostasis due to rapid changes in melanin distribution can cause various diseases.

[0103] [ Figure 3 ] is a chemical formula showing the production and decomposition process of dopamine in the body.

[0104] [ Figure 4 ] is a chemical formula showing the production and decomposition process of serotonin in the body.

[0105] [ Figure 5 ] is a chemical formula showing the decomposition process of GABA in the body.

[0106] [ Figure 6 ] is a chemical formula showing the production and decomposition process of histamine in the body.

[0107] Monoamine neurotransmitters like dopamine (DA), serotonin (5-HT), GABA, and histamine have a common structure of two carbon chains and one amine group (R-CH2-CH2-NH2). They are oxidized by monoamine oxidase (MAO) and converted into endogenous aldehydes (-CHO), such as DOPAL, 5-HIAL, SSA, 4-imidazoleacetaldehyde, and 1-methylimidazoleacetaldehyde, which bind to and denature surrounding proteins. As a result, the accumulation of denatured proteins in the endoplasmic reticulum acts as a cytotoxic agent to induce cell death. Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 ].

[0108] [ Figure 7 ] is a graph showing the ability of KARC of the present invention to decompose endogenous acetaldehyde. These results show that the composition of the present invention decomposes endogenous acetaldehyde and suppresses auto-brew symptoms.

[0109] [ Figure 8 ] is a graph showing the malondialdehyde decomposition ability of KARC.

[0110] In the case of endogenous toxic aldehyde malondialdehyde in the blood caused by alcohol intake [ Figure 8 ]In animal experiments, the aldehyde-reducing and oxidative stress-reducing effects of KARC were confirmed.

[0111] [ Figure 9 ] show that the contents of DOPAL, DOPAC, and HVA in the brain of a Parkinson's disease (PD) animal model are changed by administration of the KARC of the present invention.

[0112] [ Figure 10 ] show the results of dopamine turnover index in Parkinson's disease (PD) animal model.

[0113] When Parkinson's disease is induced in animals using rotenone, dopamine secretion decreases. Dopamine catabolism is abnormally inhibited, leading to a sharp decrease in the production of DOPAC and HVA, while increasing the abnormal metabolite DOPET. In the group administered with the KARC of the present invention, DA, DOPAC, and HVA increased, while the abnormal dopamine metabolite DOPET decreased. It is speculated that the KARC of the present invention restores normal dopamine secretion and dopamine catabolism in the body.

[0114] [ Figure 11 ] is a graph showing the ability of KARC of the present invention to decompose acetaldehyde in the human body. It shows that the composition containing KARC of the present invention suppresses auto-brewery syndrome.

[0115] [ Figure 12 ] is a graph showing the ability of KARC to decompose malondialdehyde in the human body.

[0116] [ Figure 13 ] is a graph showing the stabilization of malondialdehyde in the human body by KARC.

[0117] In the demonstration of endogenous blood acetaldehyde reduction in humans[ Figure 11 ] and blood malondialdehyde reduction [ Figure 12 ], the acetaldehyde and malondialdehyde reducing effects of KARC administration were shown. Figure 13 ], under conditions of increased oxidative stress due to medication, etc., after taking KARC, malondialdehyde, a biomarker of oxidative stress and reactive oxygen species, decreased. This confirmed the effect of KARC in reducing oxidative stress.

[0118] [ Figure 14 ] shows changes in enzyme activity when the KwonP-1 strain contained in the KARC of the present invention is orally administered.

[0119] [ Figure 15 ] shows changes in enzyme activity when the KwonP-2 strain contained in the KARC of the present invention is orally administered.

[0120] [ Figure 16 ] shows changes in enzyme activity when the KwonP-3 strain contained in the KARC of the present invention is orally administered.

[0121] [ Figure 17 ] shows changes in enzyme activity when the PicoYP strain contained in the KARC of the present invention is orally administered.

[0122] [ Figure 18 ] shows changes in enzyme activity when the PicoYP-01 strain contained in the KARC of the present invention is orally administered.

[0123] [ Figure 19Shows the change in enzyme activity when the PicoYP-02 strain contained in the KARC of the present invention is orally administered.

[0124] Figure 14 、 Figure 15 、 Figure 16 、 Figure 17 、 Figure 18 、 Figure 19 Shows that the KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01 and PicoYP-02 strains are orally administered for 90 minutes under conditions similar to the digestion process in the human stomach (1 < pH < 5). The change in ALDH enzyme activity is measured. At pH = 5 (similar to the conditions observed during food intake), the ALDH enzyme activity remains at a minimum of 37.29 units / g and a maximum of 52.24%. It is confirmed that the enzyme activity is maintained when the KARC is orally administered.

[0125] Figure 20 Shows the growth curve and enzyme activity when the KwonP-1 strain contained in the KARC of the present invention is cultured in a 5L fermenter.

[0126] Figure 21 Shows the growth curve and enzyme activity when the KwonP-2 strain contained in the KARC of the present invention is cultured in a 5L fermenter.

[0127] Figure 22 Shows the growth curve and enzyme activity when the KwonP-3 strain is cultured in a 5L fermenter.

[0128] Figure 23 Shows the growth curve and enzyme activity when the PicoYP strain contained in the KARC of the present invention is cultured in a 5L fermenter.

[0129] Figure 24 Shows the growth curve and enzyme activity when the PicoYP-01 strain is cultured in a 5L fermenter.

[0130] Figure 25 Shows the growth curve and enzyme activity when the PicoYP-02 strain contained in the KARC of the present invention is cultured in a 5L fermenter.

[0131] At Figure 20 、 Figure 21 、 Figure 22 、 Figure 23 、 Figure 24 、 Figure 25 ​​​​​​​], the new mutant strains KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-01 were cultured in 5 L fermentors using YPD medium under the same conditions. This was carried out at 30°C and 200 rpm for 48 hours. When the growth curve (OD660nm) and ALDH enzyme activity of each strain were compared with the model strain, the ALDH enzyme activity was at least 10.5 times and at most 18.75 times higher. PicoYP-01 had the highest ALDH activity at 52.68 units / g, and KwonP-3 had the lowest value at 29.5 units / g.

[0132] [ Figure 26 ] is the HPLC spectrum of a mixture of distilled water and acetaldehyde.

[0133] [ Figure 27 ] is an HPLC spectrum of a mixture of KARC of the present invention and acetaldehyde kept at 30°C for 1 hour.

[0134] [ Figure 28 ] is an HPLC spectrum of a mixture of KARC and acetaldehyde of the present invention maintained at 30°C for 3 hours.

[0135] [ Figure 29 ] is an HPLC spectrum of a mixture of KARC of the present invention and acetaldehyde kept at 37°C for 1 hour.

[0136] [ Figure 30 ] is an HPLC spectrum of a mixture of KARC and acetaldehyde kept at 37°C for 3 hours.

[0137] When acetaldehyde was treated with KARC for 1 hour, acetaldehyde, a known representative endogenous aldehyde and carcinogen, was oxidized 100% not only at 30°C but also at 37°C. Figure 26 、 Figure 27 、 Figure 28 、 Figure 29 、 Figure 30 ]

[0138] [ Figure 31 ] is the HPLC spectrum of a mixture of distilled water and glyoxal.

[0139] [ Figure 32 ] is an HPLC spectrum of a mixture of KARC and glyoxal of the present invention maintained at 30°C for 1 hour.

[0140] [ Figure 33 ] is an HPLC spectrum of the mixture of KARC and glyoxal of the present invention after being kept at 30°C for 3 hours.

[0141] [ Figure 34] is an HPLC spectrum of the mixture of KARC of the present invention and glyoxal after being kept at 37°C for 1 hour.

[0142] [ Figure 35 ] is an HPLC spectrum of a mixture of KARC and glyoxal kept at 37°C for 3 hours.

[0143] Glyoxal (a representative aldehyde produced during energy metabolism in the body) was reduced by 20.4% within 1 hour and 25.3% within 3 hours at 30°C by KARC treatment. Meanwhile, it was reduced by 23.8% within 1 hour and 23.8% within 3 hours at 37°C. Figure 31 、 Figure 32 、 Figure 33 、 Figure 34 、 Figure 35 ]

[0144] [ Figure 36 ] is the HPLC spectrum of a mixture of distilled water and succinic semialdehyde (SSA).

[0145] [ Figure 37 ] is an HPLC spectrum of the mixture of KARC and SSA of the present invention after being kept at 37°C for 1 hour.

[0146] [ Figure 38 ] is an HPLC profile of a mixture of KARC and SSA maintained at 37°C for 3 hours.

[0147] exist[ Figure 36 、 Figure 37 、 Figure 38 In the case of KARC, the level of GABA decreased by 55% in 1 hour and by 74.9% in 3 hours when treated at 37° C. KARC oxidizes SSA, a metabolite of GABA.

[0148] [ Figure 39 ] is the HPLC spectrum of a mixture of distilled water and trans-cinnamaldehyde.

[0149] [ Figure 40 ] is an HPLC spectrum of the mixture of KARC and trans-cinnamaldehyde of the present invention after being kept at 30°C for 1 hour.

[0150] [ Figure 41 ] is an HPLC spectrum of the mixture of KARC and trans-cinnamaldehyde of the present invention after being kept at 30°C for 3 hours.

[0151] [ Figure 42 ] is an HPLC spectrum of the mixture of KARC and trans-cinnamaldehyde of the present invention after being kept at 37°C for 1 hour.

[0152] [ Figure 43] is an HPLC profile of a mixture of KARC and trans-cinnamaldehyde maintained at 37°C for 3 hours.

[0153] When treated with KARC, trans-cinnamaldehyde was reduced by 35.9% in 1 hour and 97.4% in 3 hours at 30°C, and converted by 82.4% in 1 hour and 99.6% in 3 hours at 37°C. Figure 39 、 Figure 40 、 Figure 41 、 Figure 42 、 Figure 43 ]

[0154] [ Figure 44 ] is the HPLC spectrum of a mixture of distilled water and benzaldehyde.

[0155] [ Figure 45 ] is an HPLC spectrum of a mixture of KARC and benzaldehyde of the present invention maintained at 30°C for 1 hour.

[0156] [ Figure 46 ] is an HPLC spectrum of a mixture of KARC and benzaldehyde of the present invention maintained at 30°C for 3 hours.

[0157] [ Figure 47 ] is an HPLC spectrum of a mixture of KARC of the present invention and benzaldehyde kept at 37°C for 1 hour.

[0158] [ Figure 48 ] is an HPLC spectrum of a mixture of KARC and benzaldehyde kept at 37°C for 3 hours.

[0159] When treated with KARC, benzaldehyde was reduced by 12.2% in 1 hour and 32.0% in 3 hours at 30°C, and converted by 57.4% in 1 hour and 97.1% in 3 hours at 37°C. Figure 44 、 Figure 45 、 Figure 46 、 Figure 47 、 Figure 48 ]

[0160] [ Figure 49 ] is the HPLC spectrum of a mixture of distilled water and DOPAL.

[0161] [ Figure 50 ] is an HPLC spectrum of the mixture of KARC and DOPAL of the present invention after being kept at 30°C for 1 hour.

[0162] [ Figure 51 ] is an HPLC spectrum of the mixture of KARC and DOPAL of the present invention after being kept at 30°C for 3 hours.

[0163] [ Figure 52] is an HPLC profile of the mixture of KARC and DOPAL of the present invention after being kept at 37°C for 1 hour.

[0164] [ Figure 53 ] is an HPLC profile of a mixture of KARC and DOPAL maintained at 37°C for 3 hours.

[0165] exist[ Figure 49 、 Figure 50 、 Figure 51 、 Figure 52 、 Figure 53 ], when KARC was treated at 30°C, DOPAL decreased by 4.7% at 1 hour and by 15.7% at 3 hours; at 37°C, DOPAL decreased by 13.4% at 1 hour and by 24.4% at 3 hours.

[0166] DOPAC increased at 6 minutes, thus confirming that KARC oxidized DOPAL and converted it into DOPAC. DETAILED DESCRIPTION

[0167] Hereinafter, the method for producing the KARC dry powder of the present invention, i.e., the lysate of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, KCTC14985BP, will be described in more detail.

[0168] These examples are only for the purpose of illustrating compositions that can achieve the purpose of the present invention, and therefore, the scope of the present invention is not limited to the compositions described in the following examples.

[0169] Example

[0170] [Example 1] Screening of wild yeast parent strains for mutation.

[0171] In the present invention, each Makgeolli (Korean traditional liquor) suspension was prepared by mixing various types of Makgeolli with a 0.9% NaCl solution. The Makgeolli suspension was stirred at 200 rpm for 1 hour. The supernatant containing the wild yeast strain was diluted with YPD (yeast extract peptone dextrose broth) medium. The diluted solution was prepared to be 10% of the original solution. -6 times.

[0172] The diluted solution was spread onto YPD agar medium. The agar medium was incubated statically at 30°C under aerobic conditions for one week. Preliminary screening of Saccharomyces cerevisiae was performed based on colony morphology, growth characteristics in YPD agar, and microscopic observation.

[0173] The ALDH activity and glutathione content of the screened Saccharomyces cerevisiae were measured. Parent strains were selected based on ALDH activity and glutathione production.

[0174] 1-1: Measurement of aldehyde dehydrogenase

[0175] Acetaldehyde reacts with dinitrophenylhydrazine (DNPH) to form an acetaldehyde-hydrazone (Ach-DNPH) compound. The Ach-DNPH compound is detected by HPLC equipped with a C18 column at 360 nm. The amount of Ach-DNPH compound detected quantifies the amount of aldehyde reduced by the decomposition reaction by aldehyde dehydrogenase (ALDH).

[0176] The enzyme reaction was performed at 30°C by adding 10 μL of yeast lysate to 990 μL of a reaction mixture [50 mM potassium phosphate buffer (pH 8.0), 1.5 mM acetaldehyde, and 3 mM NADP+]. After the enzyme reaction was complete, 50 μL of 10 mM DNPH was added to induce the formation of Ach-DNPH. Ach-DNPH formation was performed at 22°C for 1 hour.

[0177] The formation of Ach-DNPH was terminated by adding 3M sodium acetate (pH 9). The formed Ach-DNPH compound was isolated by adding two volumes of acetonitrile. The isolated Ach-DNPH compound (in ACN) was analyzed by injection HPLC.

[0178] The concentration of Ach-DNPH compounds was analyzed by HPLC at a wavelength of 360 nm using a mobile phase (acetonitrile, water) developed on a C18 column at a rate of 1 mL / min. The area values of the chromatograms obtained from the HPLC results were converted using a standard curve for aldehyde-DNPH (Sigma-Aldrich) to quantify the concentration of Ach-DNPH compounds. A decrease of 1 mM in Ach-DNPH concentration per minute was calculated as 1 unit of ALDH. ALDH activity was normalized to units per mg of protein.

[0179] 1-2: Glutathione measurement

[0180] Yeast cells were harvested by centrifuging 1 mL of Saccharomyces cerevisiae culture medium. A suspension was prepared by adding 1 mL of water to the harvested yeast cells. Glutathione was extracted by stirring the suspension at 85°C and 1,000 rpm for 2 hours. The suspension was centrifuged to remove the yeast cells, and the supernatant was filtered through a 0.22 μm filter to obtain a glutathione-containing sample.

[0181] The glutathione concentration in the sample was analyzed by HPLC (Shimazu LC-20AD) equipped with a C18 column. Glutathione concentration was analyzed at a wavelength of 210 nm under conditions of developing a mobile phase (2.02 g / L sodium 1-heptanesulfonate monohydrate, 6.8 g / L potassium dihydrogen phosphate, pH 3.0, methanol mixture) at a rate of 1 mL / min. The area value of the chromatogram obtained as the HPLC result was analyzed using a standard curve analysis of glutathione.

[0182] The ALDH activity and glutathione content of 200 different yeasts obtained from Korean Makgeolli were analyzed. The 10 yeasts listed in [Table 1] showed higher ALDH activity or glutathione production capacity than other yeasts.

[0183] Table 1

[0184] Yeast #97 had an ALDH activity of 0.10 units / mg protein, the second highest overall. Yeast #97 also had a glutathione content of 0.42%, the highest of all strains. Yeast #97 was selected as the parent strain and subjected to the mutation induction procedure.

[0185] [Example 2] Identification of parent strains used in the mutagenesis process

[0186] Identification was performed to confirm the exact species of the wild-type parent strain (yeast #97, wild-type yeast). To ensure sufficient yeast cells for DNA extraction, only a single yeast colony was plated on YPD agar medium. DNA was extracted using a genomic DNA preparation kit (HiGene™, BIOFACT Co., Ltd., Daejeon, South Korea) according to the manufacturer's instructions.

[0187] To amplify the rRNA gene in the yeast ITS region, polymerase chain reaction (PCR) was performed on yeast chromosomal DNA using ITS5 (forward) and ITS4 (reverse) primers. PCR results were analyzed by DNA sequencing.

[0188] The DNA sequence of the parent strain was isolated using the Bioedit program. The reverse strand of the PCR result was converted to a paired base sequence using the reverse completion process.

[0189] The forward strand sequence matched its reverse strand counterpart using the Cluster X program. Using the BLAST database provided by the National Center for Biotechnology Information (NCBI), the parent strain was identified as matching the sequence identified in the experimental procedure. The rRNA in the parent strain's ITS was found to be 100% identical to that of Saccharomyces cerevisiae.

[0190] [Example 3] Selection of mutants with improved aldehyde dehydrogenase production

[0191] The mutagenesis procedure for the wild-type S. cerevisiae parent strain was performed according to the methods described in US Patent Application No. 17 / 176,365.

[0192] To induce mutations in the yeast parent strain, wild yeast strains that produce both ALDH and glutathione were treated with ethyl methanesulfonate (EMS) or nitrosoguanidine (NGD). The induced mutant yeast strains were exposed to varying concentrations of methylglyoxal. Mutants with excellent adaptability to methylglyoxal were screened. The selected yeast strains were exposed to varying concentrations of lysine. Mutants with excellent adaptability to lysine were screened. 30 mutants with excellent adaptability to both methylglyoxal and lysine were obtained. Each of the 30 yeast strains was evaluated for five properties: growth curve, ALDH activity, ADH activity, coenzyme content, and glutathione content.

[0193] 3-1: Growth characteristics

[0194] Saccharomyces cerevisiae is a Crabtree-positive microorganism that produces ethanol while growing under aerobic conditions. Cultivating high-yield yeast requires Saccharomyces cerevisiae with high ethanol tolerance.

[0195] YPD medium with different ethanol concentrations (no ethanol, 5%, 7% and 10%) was prepared. Saccharomyces cerevisiae (yeast) medium adjusted to OD=1 at 660nm was prepared. Each mixture of the prepared YPD medium and yeast medium was diluted in a ratio of 99:1. Finally, YPD medium containing yeast with four different alcohol concentrations was prepared. Each YPD medium mixed with yeast was shaken at 30°C and 200rpm. The growth curve of the mutant strain was measured every 3 hours for 48 hours. The growth curve of each mutant strain was evaluated by three characteristics: the time (or period) of the lag phase, the specific growth rate (OD660nm / hr) in the exponential phase and the maximum density (OD660nm).

[0196] [Table 2]

[0197] The higher the ethanol concentration in YPD medium, the longer the lag phase. The maximum density and specific growth rate decreased. Comparing the maximum density of mutant strains at low (5%) and high (10%) ethanol concentrations revealed that, for nine mutant strains, the high concentration maintained 50% growth compared to the low concentration. The nine mutant strains are distinguished from other strains by their short lag phase and high specific growth rate.

[0198] 3-2: Activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH)

[0199] The activity of alcohol dehydrogenase (ADH) was measured by adding 10 μL of yeast lysate to 990 μL of a reaction mixture consisting of 50 mM potassium phosphate buffer (pH 8.0), 2 mM NAD+, and 1% ethanol. The activity of aldehyde dehydrogenase (ALDH) was measured by adding 10 μL of yeast lysate to 990 μL of a reaction mixture consisting of 50 mM potassium phosphate buffer (pH 8.0), 3 mM NAD+, and 1.5 mM acetaldehyde. The enzyme reaction of ADH and ALDH was carried out at 30°C for 5 minutes, and the concentration of NAD(P)H generated by the enzyme reaction was measured by absorbance at 340 nm.

[0200] The enzyme activities of the nine mutant strains (K-1 to K-9) selected in the present invention were measured. The ADH activity of the mutant strains was as low as 382.69 units / g and as high as 975.29 units / g. Compared with the model strain (reference yeast, Saccharomyces cerevisiae KCTC7296), the ADH activity of the mutant strains increased by at least 5.1 times and as much as 13.1 times. The ALDH activity of the mutant strains was as low as 15.23 units / g and as high as 72.16 units / g. Compared with the enzyme activity of the model strain, the ALDH activity of the mutant strains increased by at least 5.3 times and as much as 24.9 times.

[0201] Six mutant strains (K-1, K-4, K-6, K-7, K-8, and K-9) showed similar rates of increase in ADH and ALDH enzyme activities compared to the model strain. The ALDH enzyme activities in three mutant strains (K-2, K-3, and K-5) were 18.3-, 23.2-, and 24.9-fold higher than those in the model strain, respectively. The ADH enzyme activities in three mutant strains (K-2, K-3, and K-5) were 9.7-, 11.6-, and 13.1-fold higher than those in the model strain, respectively. The rate of increase in ALDH enzyme activity in the three mutant strains (K-2, K-3, and K-5) was twice that of ADH.

[0202] The present inventors named three new mutants (K-2, K-3, and K-5) adapted to increase aldehyde dehydrogenase (ALDH) activity PicoYP, PicoYP-01, and PicoYP-02, respectively. These three new mutants were deposited with the Bioresource Center of the Korea Institute of Bioscience and Biotechnology under the accession numbers KCTC14983BP, KCTC14984BP, and KCTC14985BP, respectively.

[0203] 3-3: Content of coenzymes (NAD and NADP)

[0204] NAD total and NADP total were measured in lysates extracted from the mutant strain using the NADH / NAD+ assay kit and the NADPH / NADP+ assay kit, respectively. NAD(P) in the sample was converted to NAD(P)H using an NAD(P) cycling buffer and an NAD(P) cycling enzyme mix. A chromogenic reaction was induced with an NAD(P) chromogen, and the absorbance at 450 nm was measured. The chromogenic reaction was measured by absorbance at 450 nm. The sample absorbance was substituted into the equation corresponding to the standard curve to calculate the NAD(P) total in the yeast lysate.

[0205] The coenzyme content of the nine mutant strains (K-1 to K-9) selected in the present invention was measured. The NADtotal of the mutant strains was as low as 126 nmol / g and as high as 195 nmol / g. Compared with the model strain, the NADtotal of the mutant strain increased by at least 7.3 times and as much as 10.8 times. The NADPtotal content of the mutant strain was as low as 2.4 nmol / g and as high as 5.8 nmol / g. Compared with the model strain, the NADPtotal content of the mutant strain increased by at least 11.4 times and as much as 27.6 times.

[0206] In the six mutants (K-1, K-4, K-6, K-7, K-8, and K-9), the rate of increase in NADPtotal was less than twice the rate of increase in NADtotal. The rates of increase in total NADP content in the three new mutants (PicoYP, PicoYP-01, and PicoYP-02) were 25.7-fold, 22.9-fold, and 27.6-fold, respectively. The rates of increase in total NAD content in the three new mutants were 10.8-fold, 9.9-fold, and 11.3-fold, respectively. The rate of increase in NADPtotal in the three new mutants was greater than twice the rate of increase in NADtotal.

[0207] 3-4: Glutathione (GSH) content

[0208] Glutathione content was measured in nine mutant strains using the same method as in Examples 1-2. Glutathione content ranged from a low of 0.85% to a high of 1.05%. Compared to the model strain, glutathione content increased by at least 2.7 times and as much as 3.3 times in the mutants. Three of the new mutants (PicoYP, PicoYP-01, and PicoYP-02) showed higher increases in ALDH activity and coenzyme content than in the other strains.

[0209] The three new mutant yeasts (PicoYP, PicoYP-01, and PicoYP-02) have similar glutathione production capabilities to existing deposited strains (Kwon P-1, Kwon P-2, and Kwon P-3). Compared to existing deposited strains, the three new mutant yeasts have significantly increased ADH and ALDH enzyme activities and coenzyme content.

[0210] [Table 3]

[0211] [Table 4]

[0212] [Example 4] Comparison of carbon source preferences

[0213] The carbon source preferences of three mutants (KwonP-1, KwonP-2, and KwonP-3) with elevated ALDH and glutathione levels were investigated, for which a domestic patent application was filed on February 18, 2020. The carbon source preferences of a reference yeast strain (KCTC7296) were measured. To determine the maximum capacity for ALDH production, the carbon source preferences of three new mutants (PicoYP, PicoYP-01, and PicoYP-02) were investigated.

[0214] The carbon source preference of the strains was analyzed for specificity and novelty using the API 50 CHL kit (API Systems, BIOMERIEUX, SA, France).

[0215] Prepare 15 mL conical tubes containing 8 mL of YPD medium. Inoculate each of the seven mutant strains into the prepared conical tubes.

[0216] After incubating the inoculated conical tubes at 30°C and 200 rpm for 24 hours, each of the seven mutant strains was isolated from the exponential growth phase. To eliminate the effects of residual carbon source in the YPD medium, the yeast was washed three times using a centrifuge. A yeast suspension at a 2 McFarland concentration was prepared using API50 CHL medium. The prepared yeast suspension was placed in the tubes of the test strips. The test strips, with the suspensions dispensed, were incubated at 30°C for 24 hours.

[0217] API 50 CHL medium, used for API testing, is purple. As acids are produced through energy metabolism, API 50 CHL medium turns blue, then green, and finally yellow. The carbon source used by the mutant is recorded based on the color change, such as purple x, blue +, green ++, and yellow +++.

[0218] All seven mutant strains tested used 19 carbon sources for energy production and growth: L-arabinose, ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, N-acetylglucosamine, arbutin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, raffinose, and gentiobiose.

[0219] Only three mutants used rhamnose: KwonP-1, PicoYP-01, and PicoYP-02. Four mutants used sorbitol: KwonP-1, KwonP-3, PicoYP-01, and PicoYP-02. Four mutants used α-methyl-D-mannoside: the model strain, KwonP-1, KwonP-2, and PicoYP-02. Six mutants used amygdalin: KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02. Four mutants used D-turanose: the model strain, KwonP-1, KwonP-3, and PicoYP-02. Three mutants used D-tagatose: the model strain, KwonP-3, and PicoYP-3. Only the model strain used gluconate.

[0220] Mannitol and sorbitol, corresponding to alcoholic carbon sources, had a significant effect on yeast growth. The three new mutants differed from the other four yeast strains in the types of sugars they utilized for growth. The three new mutants (PicoYP, PicoYP-01, and PicoYP-02) showed slight differences in their preferred alcoholic carbon sources [Table 5].

[0221] [Table 5]

[0222] [Example 5] Changes in ALDH activity of mutant strains in gastric juice

[0223] When KARC is administered orally, in order to maintain enzyme activity in the intestine, the enzyme activity must pass safely without being destroyed by gastric acid, which secretes strong proteolytic enzymes such as pepsin.

[0224] NaOH solution was added to artificial gastric fluid at pH 1.17 to create two simulated solutions at pH 3 and pH 5, similar to the human gastric environment during food digestion. 1g of KARC was added to 7mL of artificial gastric fluid and 7mL of each simulated solution and mixed at 36.5°C for 5, 30, 60, and 90 minutes, respectively. NaOH solution was added to each reaction mixture to adjust the acidity to pH 7. 10mL samples were collected from each of the adjusted pH 7 solutions for analysis. ALDH activity was assayed for each sample.

[0225] At pH 1.17, ALDH activity in samples decreased by over 92.88% compared to the control within 5 minutes of reaction. At pH 1.17, ALDH activity in samples decreased by an average of 98.89% over 90 minutes. At pH 3, ALDH activity decreased by an average of 96.66% after 90 minutes, and at pH 5, it decreased by 56.83%. Finally, ALDH activity at pH 3 and 5 remained relatively higher than at pH 1.17 during the 90-minute reaction.

[0226] Specifically, at pH 1.17, the ALDH activity of KwonP-1 (KCTC13925BP) decreased by 90.94% to 5.57 units / g after 5 minutes of reaction compared to the control group. The ALDH activity of KwonP-1 decreased by 98.57% to 0.88 units / g after 90 minutes of reaction. Figure 14 The enzyme activity at pH = 3 and pH = 5 remained relatively higher than that at pH = 1.17. After 90 minutes of reaction, the ALDH activity of KwonP-1 decreased by 96.66% to 5.57 units / g at pH = 3, and decreased by 98.57% to 0.88 units / g at pH = 5.

[0227] At pH = 1.17, the ALDH activity of KwonP-2 (KCTC14122BP) decreased by 91.18% to 5.43 units / g after 5 minutes of reaction. The ALDH activity of KwonP-2 decreased by 98.81% to 0.73 units / g after 90 minutes of reaction. Figure 15 At pH 3 and pH 5, higher enzyme activity was maintained than at pH 1.17. After 90 minutes of reaction, ALDH activity decreased by 97.62% to 1.47 units / g at pH 3, and decreased by 56.11% to 26.99 units / g at pH 5.

[0228] At pH = 1.17, the ALDH activity of KwonP-3 (KCTC14123BP) decreased by 89.99% to 6.16 units / g after 5 minutes of reaction. The ALDH activity of KwonP-3 decreased by 97.85% to 1.32 units / g after 90 minutes of reaction. Figure 16 At pH 3 and pH 5, higher enzyme activity was maintained than at pH 1.17. After 90 minutes of reaction, ALDH activity decreased by 92.61% to 4.55 units / g at pH 3, and decreased by 62.31% to 22.18 units / g at pH 5.

[0229] At pH = 1.17, the ALDH activity of PicoYP (KCTC14983BP) decreased by 92.84% to 4.40 units / g after 5 minutes of reaction. The ALDH activity of PicoYP decreased by 98.33% to 1.03 units / g after 90 minutes of reaction. Figure 17 High enzyme activity was maintained at both pH 3 and pH 5. After 90 minutes of reaction, ALDH activity decreased by 96.66% at pH 3 to 2.05 units / g, and decreased by 53.97% at pH 5 to 28.31 units / g.

[0230] At pH = 1.17, the ALDH activity of PicoYP-01 (KCTC14984BP) decreased by 95.71% to 2.64 units / g after 5 minutes of reaction. The ALDH activity of PicoYP-01 decreased by 99.76% to 0.15 units / g after 90 minutes of reaction. Figure 18 At pH 3 and pH 5, higher enzyme activity was maintained than in gastric juice. After 90 minutes of reaction, ALDH activity decreased by 98.21% at pH 3 to 1.10 units / g, and decreased by 58.74% at pH 5 to 25.38 units / g.

[0231] At pH = 1.17, the ALDH activity of PicoYP-02 (KCTC14985BP) decreased by 96.66% to 2.05 units / g after 5 minutes of reaction. The ALDH activity of PicoYP-02 decreased by 99.76% to 0.15 units / g after 90 minutes of reaction. Figure 19 At pH 3 and pH 5, higher enzyme activity was maintained than in gastric juice. After 90 minutes of reaction, at pH = 3, ALDH activity decreased by 98.21% to 1.10 units / g, and at pH = 5, ALDH activity decreased by 62.08% to 23.32 units / g.

[0232] The pH of secreted gastric juice is 1.17. When food is consumed, the pH of the original gastric juice and food mix in the stomach, raising it from 3 to 5, making it unlikely to reach a pH of 1.17. Despite this, ALDH activity in the mutant strain is maintained even under this extreme pH condition.

[0233] Finally, the ALDH enzyme activity of the new mutants (PicoYP, PicoYP-01, PicoYP-02) remained at 2 to 5 units / g, despite a 92% to 97% decrease under the strongly acidic conditions of pH 1.17. Maintaining 2-5 units of enzyme activity is sufficient to function in the intestine. Compared to pH 1.17, it was even higher at pH 3 and pH 5. This leads to the conclusion that the new mutants (PicoYP, PicoYP-01, PicoYP-02) can be administered orally.

[0234] [Example 6] Growth characteristics of 5L fermenter culture

[0235] Be inoculated in YPD substratum (2% peptone, 1% yeast extract, 2% glucose) respectively, and carry out primary seed culture 18 hours at 30 ℃ and 200rpm.The seed of 20mL culture is inoculated in 1980mL YPD substratum, and in 5L, cultivate again.The cultivation in 5L culture tank was carried out 48 hours at 30 ℃ and 200rpm.Use the 10mL sample analysis OD660nm growth curve and the enzyme activity under collecting from secondary culture.

[0236] The maximum density (OD660nm) of KwonP-1 (KCTC13925BP) was 134.4. The maximum density of KwonP-1 was 4.35% higher than that of the model strain (KCTC7296). The growth curve characteristics and specific growth rate (OD660nm / hr) of KwonP-1 were similar to those of the model strain. The ALDH activity of KwonP-1 was 33.6 units / g. The ALDH activity of KwonP-1 was 11.96 times higher than that of the model strain. Figure 20 ].

[0237] The maximum density (OD660nm) of KwonP-2 (KCTC14122BP) was 133.8. The maximum density of KwonP-2 was 3.88% higher than that of the model strain. The growth of KwonP-2 ended earlier than that of the model strain. The specific growth rate (OD660nm / hr) of KwonP-2 was 14.8% higher than that of the model strain. The ALDH activity of KwonP-2 was 31.5 units / g. The ALDH activity of KwonP-2 was 11.21 times higher than that of the model strain. Figure 21 ].

[0238] The maximum density (OD660nm) of KwonP-3 (KCTC14123BP) was 134.1. The maximum density of KwonP-3 was 4.12% higher than that of the model strain. The growth of KwonP-3 ended earlier than that of the model strain. The specific growth rate (OD660nm / hr) of KwonP-3 was 6.08% higher than that of the model strain. The ALDH activity of KwonP-3 was 29.5 units / g. The ALDH activity of KwonP-3 was 10.5 times higher than that of the model strain. Figure 22 ].

[0239] The maximum density (OD660nm) of PicoYP (KCTC14983BP) was 123.8. The maximum density of PicoYP was 3.88% higher than that of the model strain. The growth curve characteristics of PicoYP were similar to those of the model strain. The specific growth rate (OD660nm / hr) of PicoYP was 6.22% higher than that of the model strain. The ALDH activity of PicoYP was 44.2 units / g. The ALDH activity of PicoYP was 15.73 times higher than that of the model strain. Figure 23 ].

[0240] The maximum density (OD660nm) of PicoYP-01 (KCTC14984BP) was 126.9. The maximum density of PicoYP-01 was 1.47% higher than that of the model strain. The growth curve characteristics of PicoYP-01 were similar to those of the model strain. The specific growth rate (OD660nm / hr) of PicoYP-01 was 2.14% higher than that of the model strain. The ALDH activity of PicoYP-01 was 47.1 units / g. The ALDH activity of PicoYP-01 was 16.76 times higher than that of the model strain. Figure 24 ].

[0241] The maximum density (OD660nm) of PicoYP-02 (KCTC14985BP) was 148.1. The maximum density of PicoYP-02 was 14.99% higher than that of the model strain. Compared with the model strain, the growth curve of PicoYP-02 was at the top. The specific growth rate (OD660nm / hr) of PicoYP-02 was 9.64% lower than that of the model strain. The ALDH activity of PicoYP-02 was 52.68 units / g. The ALDH activity of PicoYP-02 was 18.75 times higher than that of the model strain. Figure 25 ].

[0242] [Example 7] Preparation of mutant strain lysate (KARC)

[0243] To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysate, proteases are removed and inhibited. To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysate, cell debris is removed. The dried products or lysates of the mutant strains are mixed to prepare a KARC composition.

[0244] The mutant strain and the culture medium containing it contain a variety of substances, such as yeast metabolites and proteolytic enzymes secreted by the yeast. To extract and preserve the ALDH, coenzymes, and glutathione present in the yeast, it is necessary to fully remove substances outside the yeast. To this end, a washing procedure was performed. Washing of the mutant strain was performed by dispensing 40 mL of culture medium into a 50 mL conical tube, centrifuging at 13,000 rpm for 15 minutes, and removing the supernatant.

[0245] As a result of centrifugation, the yeast cells aggregated into a pellet containing residual culture medium. Add 30 mL of purified water and vortex to loosen the pellet. Repeat this procedure three times to fully remove the residual culture medium.

[0246] Yeast is known to have an ethanol tolerance of up to 13%, and yeast will die when exposed to high ethanol concentrations. Dissolve the washed pellet thoroughly in 10 mL of 20% ethanol solution to induce yeast death. Stir the pellet in ethanol at 100 rpm for 30 minutes to induce yeast death. At the end of the reaction time, add 30 mL of purified water to bring the ethanol concentration to 5%. Repeat this washing procedure three times to fully remove the ethanol.

[0247] To prevent the degradation of ALDH and ADH by proteases present in yeast cells, 10 mL of 1X PBS was prepared by dissolving two protease inhibitor tablets (Pierce Protease Inhibitor Tablets, EDTA-free, Thermo Scientific). The solution was added to the washed yeast pellet and distributed thoroughly.

[0248] To prepare the lysate of the mutant strain prepared in the present invention, 4 g of glass beads were added and stirred to disrupt the yeast cell walls. To prevent heat generated during yeast disruption from denaturing the enzyme, vortexing for 30 seconds and incubating on ice for 30 seconds were repeated 6 times.

[0249] After the yeast cell wall was broken, 10 mL of 100 mM potassium phosphate buffer was added and mixed by vortexing for 3-5 seconds. Centrifugation was performed at 13,000 rpm for 15 minutes to remove cell structures such as yeast cell walls and glass beads. The supernatant was filtered through a 0.2 μm filter (Minisart The KARC composition was prepared by filtration through a syringe filter (Sartorius, Goettingen, Germany).

[0250] To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysates, remove and inhibit intracellular proteases, and remove cellular debris such as cell walls, KARC compositions were prepared using lysates of six mutant strains (KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, PicoYP-02) or mixtures thereof in a free ratio [Table 6].

[0251] KARC 1 is made from Kwon P-1. KARC 1's ADH and ALDH enzyme activities are 461.4 units / g and 28.6 units / g, respectively. The NAD and NADP total coenzyme contents in KARC 1 are 176.2 nanomoles / g and 5.1 nanomoles / g, respectively. KARC 1 also contains 0.98 wt% GSH.

[0252] KARC 2 is made from Kwon P-2. KARC 2's ADH and ALDH enzyme activities are 482.1 units / g and 29.8 units / g, respectively. KARC 2's NAD total and NADP total coenzyme contents are 175.4 nanomoles / g and 5.2 nanomoles / g, respectively. KARC 2 also contains 0.96 wt% GSH.

[0253] KARC 3 is made from Kwon P-3. KARC 2 has ADH and ALDH enzyme activities of 477.5 units / g and 28.1 units / g, respectively. KARC 3 has NAD total and NADP total coenzyme contents of 177.2 nanomoles / g and 5.1 nanomoles / g, respectively. KARC 3 also has a GSH content of 1.00 wt%.

[0254] KARC 4 is made from PicoYP. KARC 2 has ADH and ALDH enzyme activities of 586.8 units / g and 33.8 units / g, respectively. KARC 4 has NAD total and NADP total coenzyme contents of 184.3 nanomoles / g and 5.7 nanomoles / g, respectively. KARC 4 also has a GSH content of 0.84 wt%.

[0255] KARC 5 is produced from PicoYP-01. KARC 5 exhibits ADH and ALDH enzyme activities of 621.6 units / g and 38.2 units / g, respectively. The NAD and NADP total coenzyme contents in KARC 5 are 186.9 nanomoles / g and 5.6 nanomoles / g, respectively. KARC 5 also contains 0.84 wt% GSH.

[0256] KARC 6 is made from PicoYP-02. KARC 5 has ADH and ALDH enzyme activities of 664.1 units / g and 41.6 units / g, respectively. KARC 6 has NAD total and NADP total coenzyme contents of 195.0 nanomoles / g and 5.8 nanomoles / g, respectively. KARC 6 also has a GSH content of 0.88 wt%.

[0257] KARC was prepared by freely mixing dry powders and lysates prepared from six deposited strains. The average enzyme activities of ADH and ALDH in the KARC composition were 547.6 units / g and 33.1 units / g, respectively. The average contents of the coenzyme NAD total and coenzyme NADP total in the KARC composition were 180.4 nanomoles / g and 5.4 nanomoles / g, respectively. The average glutathione content in the KARC composition was 0.84 wt%.

[0258] The aldehyde decomposition ability of KARC was maintained during the lysate production process. KARC exhibited the ability to remove endogenous aldehydes such as HNE, MDA, and 3,4-dihydroxyphenylacetaldehyde (DOPAL).

[0259] [Table 6]

[0260] [Example 8] Sequence analysis of ALDH contained in mutant strains.

[0261] The differences in ALD (yeast aldehyde dehydrogenase) between the mutants and the parent strain were investigated. Whole-genome sequencing was performed on the parent strain and mutants KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02. Mutant cells were isolated by culturing pure strains on solid culture medium. The resulting mutant genome sequences were then analyzed.

[0262] In the ALD (yeast aldehyde dehydrogenase) of the new mutant strain, ALD2 (SEQ ID NO: 3) and ALD3 (SEQ ID NO: 4) were found to be densely distributed on chromosome 13. A 689-nucleotide noncoding region was located between the ALD2 and ALD3 encoding genes.

[0263] ALD2 and ALD3 coexist in the same genome. ALD2 and ALD3 encode separate aldehyde dehydrogenases. The ALD2 gene is nearly identical to ALD3, consisting of 1,521 nucleotides and 506 amino acids, but differs by 8.2% in sequence. ALD2 and ALD3 were identified as separate aldehyde dehydrogenases, differing by 125 bases (8.2%).

[0264] In six mutant strains (KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02), the ALD2 sequence lacks a stop codon, allowing protein synthesis to proceed. Consequently, a new, larger ALDH enzyme was created by linking a portion of ALD2 to ALD3 [SEQ ID NO: 1].

[0265] ALD2 [SEQ ID NO. 3] of the model strain (KCTC7296) consists of a 30-nucleotide sequence (5'-GTTCACATAAATCTCTCTTTGGACAACTAA-3') encoding 9 amino acids (N-VHINLSLDN-C) at the terminal, excluding the stop codon.

[0266] ALD2 of the six mutants consists of a specific 42-nucleotide sequence (5'-AGATATAGATTATACACATTTAGAAAATTAGCCAAAAGAAAA-3') encoding 14 amino acids (N-RYRLYTFRKLAKRK-C) between the 5' end of ALD2 and ALD3 [SEQ ID NO. 2].

[0267] The ALD2 gene encoding ALD2 is deleted from nucleotide 1492 to nucleotide 647 in the noncoding region, eliminating the termination codon. Ultimately, the six deposited mutants contain a novel mutant gene consisting of 3,054 bases encoding a novel ALD2 gene [SEQ ID NO: 1].

[0268] [Example 9] Effect of Oral Administration of KARC on Reducing Acetaldehyde (Ach) and Malondialdehyde (MDA) in Vivo

[0269] For the acetaldehyde and MDA animal experiments, 5-week-old male Sprague Dawley (SD) rats were used. The KARC composition was orally administered to the rats at 10 units / kg or 20 units / kg, and alcohol (3 g / kg) was orally administered to the rats 30 minutes after KARC injection.

[0270] After administration, blood samples were collected from the tail vein at 0, 1, 3, 5, and 8 hours after KARC injection, and the plasma was stored at −80°C after centrifugation. Figure 7 、 Figure 8 ].

[0271] Seven-week-old male Wistar rats (7 weeks old, 250 g, n=8-10) were used. Rotenone solution (2.5 mg rotenone / mL, 20 μL DMSO / mL) was prepared using natural oil (medium-chain triglycerides). Rotenone solution (2.5 mg / kg) was administered intraperitoneally to mice daily for 60 days.

[0272] To confirm the preventive and therapeutic effects of KARC on Parkinson's disease, two administration methods were used. KARC (20 units / kg) was orally administered simultaneously with rotenone to observe the preventive effect on Parkinson's disease. KARC (20 units / kg) or levodopa was orally administered two weeks after rotenone administration to observe the therapeutic effect on Parkinson's disease. To quantify dopamine, brain tissue was isolated and stored in liquid nitrogen at -80°C. Figure 9 、 Figure 10 ].

[0273] 9-1: Acetaldehyde-reducing effect of KARC by oral administration

[0274] *The total acetaldehyde-reducing effect of oral administration of KARC was evaluated using an acetaldehyde assay kit (LSBio, Seattle, WA, USA). 20 μL of each sample was dispensed into two wells of a 96-well plate. 80 μL of working reagent (75 μL assay buffer, 8 μL NAD / MTT, 1 μL enzyme A, 1 μL enzyme B) was dispensed into one well. In the remaining wells, 80 μL of blank working reagent (75 μL assay buffer, 8 μL NAD / MTT, 1 μL enzyme B) was dispensed. The dispensed plate was gently mixed and allowed to react at room temperature for 30 minutes. After completion of the reaction, the absorbance was measured at 565 nm (520-600 nm).

[0275] In the KARC composition-administered group, acetaldehyde concentrations reached their maximum 1 hour after ethanol administration and then showed a downward trend. In the KARC-administered group, acetaldehyde concentrations were significantly lower 1, 3, and 5 hours after ethanol administration compared to the control group (vehicle). In the high-dose KARC-administered group (F), blood acetaldehyde concentrations were 0.356, 0.224, and 0.091 mM, respectively, representing decreases of 39.2%, 58.4%, and 72.1% compared to the control group. Figure 7 ].

[0276] 9-2: MDA-reducing effect of oral administration of KARC

[0277] According to the manufacturer's protocol (ZeptoMetric, Buffalo, NY, USA), the total malondialdehyde content in the blood was analyzed using the OxiTecTM TBARS assay kit. 100 μL of sample, 100 μL of 8.1% SDS solution and 4 mL of color indicator (TBA, 10% NaOH solution, 20% acetic acid) were added to a conical tube and then reacted in a 95°C thermostatic water bath for 60 minutes. After the reaction was complete, the sample was centrifuged at 4°C and 1,600 rpm for 10 minutes and stabilized at room temperature for 30 minutes. 150 μL of supernatant was transferred to a 96-well plate and the absorbance was measured at 530-540 nm.

[0278] In the control group (vehicle), the MDA concentration in the blood reached its maximum 3 hours after ethanol administration, while in the KARC administration group, it reached its maximum 1 hour after ethanol administration. The MDA concentration in the blood decreased 3 and 5 hours after ethanol administration, showing a significant difference from the control group. The blood MDA concentrations in the high-dose KARC administration group (F) were 0.232 and 0.137 μM, respectively, which were decreased by 80.4% and 86.3% compared to the control group. Figure 8 ].

[0279] These results indicate that oral administration of KARC is effective in reducing multiple endogenous aldehydes such as acetaldehyde and malondialdehyde in the blood.

[0280] 9-3: Effect of oral administration of KARC on DOPAL reduction

[0281] To measure the effect of oral administration of KARC on reducing dopamine-derived DOPAL, DOPAL content in rat brain nigrostriatal samples was measured by HPLC / MMS. The samples were dissolved in trichloroacetic acid (3.0 M / 100 μL), and then pre-treated by centrifugation using a Toyopak SP kit (Toso, Tokyo, Japan).

[0282] In order to dissolve the adsorbed amine compounds, 0.6 M KCl-acetonitrile (1:1, 2 mL) was treated, and DPE reagent was added to the solution to induce fluorescence. The final solution produced as a result of the reaction was injected into HPLC to measure dopamine.

[0283] To measure DOPAC and HVA content in samples, samples were dissolved in HClO4 (300 μL) and homogenized and centrifuged (50,000 g, 4°C, 15 minutes) to obtain a supernatant. The supernatant was filtered and DOPAC and HVA content was measured by HPLC / MMS. DOPAL content was calculated in ng / g tissue.

[0284] To investigate the changes in dopamine metabolism in the brains of PD model animals treated with rotenone, DA, DOPAL, DOPAC, and HVA were measured using HPLC. Figure 9 ].

[0285] The brain levels of DA, DOPAL, DOPAC, and HVA were measured at 1542 ng / g tissue weight, 22 ng / g tissue weight, 620 ng / g tissue weight, and 970 ng / g tissue weight, respectively, in the control group. In the group treated with rotenone to induce PD, DA, DOPAC, and HVA levels decreased compared to the control group, reaching 1021 ng / g tissue weight, 234 ng / g tissue weight, and 102 ng / g tissue weight, respectively. However, DOPAL levels increased to 70 ng / g weight.

[0286] In the group receiving the reference drug levodopa, brain DA and DOPAL levels increased compared to the control group, reaching 1816 ng / g tissue weight and 96 ng / g tissue weight, respectively. However, DOPAC and HVA levels decreased, reaching 281 ng / g tissue weight and 126 ng / g tissue weight, respectively. Similar results were observed in the group receiving rotenone.

[0287] On the other hand, in the group administered KARC in the induced Parkinson's disease model, brain tissue levels of DA, DOPAL, DOPAC, and HVA were 1290 ng / g tissue weight, 21 ng / g tissue weight, 510 ng / g tissue weight, and 790 ng / g tissue weight, respectively. DA, DOPAC, and HVA increased compared to the rotenone-administered group, while DOPAL decreased.

[0288] In particular, in the KARC pre-administration group for preventive purposes, DA and DOPAL increased to 1522 ng / g tissue weight and 18 ng / g tissue weight, respectively, compared with the control group; and DOPAC and HVA also increased to 590 and 860 ng / g tissue weight. As a result, all levels of DA, DOPAL, DOPAC and HVA were almost the same as those of the control group.

[0289] The dopamine turnover index ((DOPAC+HVA) / DA) ratio was used to indirectly examine the amount of DOPAL remaining unmetabolized in DA metabolism, because DA is metabolized through DOPAL and DOPAC and ultimately metabolized to HVA. Figure 10 ].

[0290] As for the results of calculating the dopamine conversion index [(DOPAC+HVA) / DA], it was 103.1% in the control group, 100.8% in the KARC pre-administration group, and 95.3% in the KARC post-administration group, which means that the in vivo dopamine metabolism progressed well in the three groups.

[0291] On the other hand, compared with the three groups, 32.9% was decreased in the rotenone group, and 22.4% was decreased in the levodopa group, which means that Parkinson's disease abnormally causes dopamine metabolic dysfunction.

[0292] This suggests that the metabolic intermediate DOPAL is accumulating in the body. KARC administration inhibits the accumulation of the neurotoxin DOPAL, restores dopamine metabolism to normal, and accelerates the production of DOPAC and HVA, which are relatively less toxic than DOPAL. Therefore, KARC restores DA metabolism and has the potential to prevent and treat Parkinson's disease.

[0293] [Example 10] Effect of reducing oxidative stress.

[0294] When drinking alcohol, excessive acetaldehyde (ACh) is produced by alcohol dehydrogenase (ADH), leading to increased reactive oxygen species and oxidative stress. Aldehyde dehydrogenase (ALDH) converts ACh into acetic acid and excretes it from the body. Excessive aldehyde production due to mutations in the ALDH gene or from excessive alcohol consumption can lead to lipid peroxidation.

[0295] The resulting acetaldehyde and malondialdehyde exacerbate oxidative stress and interfere with mitochondrial energy metabolism. Endoplasmic reticulum stress is caused by the accumulation of denatured proteins in cells, leading to cell death.

[0296] Blood acetaldehyde concentrations were measured over time after drinking. Figure 11 The area under the curve (AUC) for blood acetaldehyde (Ach) was 13.02 ± 1.18 mg·h / dL for alcohol consumption alone. When administered at a dose of 10 units / kg of KARC, the area under the curve (AUC) for blood acetaldehyde (Ach) decreased significantly by 26.13% compared to alcohol consumption alone, measuring 9.39 ± 1.07 mg·h / dL (P = 0.005).

[0297] When administered at a dose of 20 units / kg, the AUC of blood acetaldehyde (ACh) significantly decreased by 55.71% compared to alcohol consumption alone, reaching 5.22 ± 0.99 mg·h / dL (P < 0.001). Compared to the group administered 10 units / kg of KARC, the 20 units / kg group showed a significant decrease in blood acetaldehyde (ACh) (P = 0.034). KARC demonstrated a dose-dependent reduction in total blood acetaldehyde (ACh) over time.

[0298] The reduction in blood acetaldehyde (Ach) concentration due to KARC administration has a positive impact on reducing oxidative stress and promoting health.

[0299] Measurement of blood malondialdehyde (MDA) concentration during chemotherapy Figure 12 The blood MDA concentration in the control group was 0.607±0.161 μM. The group receiving KARC treatment showed a significant 63.3% decrease in blood MDA concentration compared to the control group, measuring 0.223±0.033 μM (P<0.001).

[0300] In the control group, blood MDA concentration ranged from 0.427 μM to 0.885 μM, showing a wide variation. In the KARC-administered group, the range was significantly narrowed, with values ranging from 0.158 μM to 0.269 μM. This confirms not only the effect of lowering blood MDA concentration but also stabilizing it, as shown in [ Figure 13 ] confirmed.

[0301] Various factors, including drug ingestion, stress, and intense physical exercise, lead to an increase in intracellular reactive oxygen species (ROS). This triggers lipid peroxidation and the oxidation of endogenous amines (such as dopamine, norepinephrine, serotonin, and histamine). Reactive aldehyde compounds, including 4-hydroxynonenal (HNE), malondialdehyde (MDA), acetaldehyde (ACh), and dopamine-induced aldehydes, accumulate within cells, exacerbating oxidative stress.

[0302] These aldehydes then react with surrounding proteins and undergo secondary metabolic processes to form stable end products such as malondialdehyde-acetaldehyde adducts (MAA) and malondialdehyde-lysine adducts (M-lys adducts), known as advanced lipid peroxidation end products. The accumulation of these products has toxic effects on various cells, further exacerbating oxidative stress.

[0303] This cumulative oxidative stress disrupts mitochondrial energy metabolism within the cell and leads to the accumulation of aldehyde intermediates in aldose sugar metabolism, including methylglyoxal (MG) and glyceraldehyde-3-phosphate (GA3P). Chain reactions involving aldehydes lead to the accumulation of stable final sugar oxidation products, known as advanced glycation end products (AGEs), which weaken intracellular antioxidant defense systems such as glutathione (GSH). These processes increase endoplasmic reticulum (ER) stress, leading to increased apoptosis in neuronal cells.

[0304] Increased reactive oxygen species and oxidative stress are associated with elevated levels of reactive aldehydes, such as HNE and MDA, and modified proteins, such as advanced glycation end products (AGEs) and advanced lipid peroxidation end products (ALEs). This series of events is known to reinforce and amplify each other, leading to increased endoplasmic reticulum stress (ER stress).

[0305] KARC administration effectively regulated malondialdehyde, a marker of reactive oxygen species and oxidative stress, showing the potential to reduce oxidative stress and improve endoplasmic reticulum (ER) stress stability. KARC significantly reduced malondialdehyde concentrations in the bloodstream, indicating its ability to reduce reactive oxygen species and oxidative stress.

[0306] By reducing acetaldehyde and malondialdehyde levels in human blood, KARC has demonstrated the potential to prevent and correct ER stress by reducing reactive oxygen species and oxidative stress. This suggests that by modulating intracellular reactive oxygen species and oxidative stress, KARC inhibits neuronal apoptosis, thereby suppressing and preventing Parkinson's disease. This results in improved behavior and motor function.

[0307] [Example 11] Acute oral administration test

[0308] 11-1. Preparation of experimental animals

[0309] The experimental animals were female and male ICR mice (7 weeks old). The received ICR mice were adapted for 7 days. The general symptoms of the adopted mice during the adaptation period were observed, and only healthy animals were used for short-term toxicity testing. Feed and water were available freely. Based on an average body weight of about 20g the day before oral administration, the group was divided into 10 groups of 5 mice each.

[0310] 11-2. Administration of test substance

[0311] The test substances were prepared by dissolving in physiological saline so that the doses for experimental animals were 0, 750, 3,000, and 5,000 mg / kg, respectively, based on the content of the mutant yeast lysate KARC of the present invention.

[0312] The dosage standards were in accordance with the Korea National Toxicology Program (KNTP) Toxicity Test Manual of the Ministry of Food and Drug Safety. The maximum dose of 5,000 mg / kg, as specified in the KNTP Manual, was used as the maximum concentration for this experiment. Samples prepared for each group were administered orally once to each test animal. The normal group (G1) received normal saline.

[0313] 11-3. Observation and autopsy

[0314] For all test groups, mice were observed for symptoms at least once daily from the date of acquisition until the date of necropsy. Symptoms were observed for 7 days after oral administration. Necropsy was performed after observing the symptoms of the rats. During the rat necropsy, changes in various organs were observed visually.

[0315] Single-dose toxicity testing of the ALDH-containing KARC composition of the present invention was conducted in mice. Results showed that no mouse mortality was observed within 7 days at concentrations of up to 5,000 mg / kg of mutant yeast KARC. No abnormalities, such as weight gain or changes in feed intake, were observed in the mice. Post-observation autopsies revealed no abnormal findings.

[0316] [Example 12] Observation of in vitro metabolism of various aldehydes by KARC

[0317] The present invention demonstrates the effectiveness of KARC in reducing exogenous and endogenous aldehydes. When KARC (300 mg / mL) was reacted with various aldehydes (1 mM) at 37°C for 3 hours, 3,4-dihydroxyphenylacetaldehyde (DOPAL) decreased by 24.4%, succinic semialdehyde (SSA) by 74.9%, glyoxal by 23.8%, cinnamaldehyde by 99.6%, and benzaldehyde by 97.1%. Acetaldehyde was even reduced by 100.0% after 1 hour at 30°C. Figure 26-Figure 53 ].

[0318] 12-1: Reaction of KARC with various aldehydes

[0319] Potassium chloride (KCl) was dissolved in 50 mM HEPES buffer at pH 7.5 to 200 mM. For experiments with acetaldehyde, glyoxal, DOPAL, cinnamaldehyde, and benzaldehyde, 935 μL of buffer, 15 μL of 100 mM EDTA solution, 30 μL of 100 mM NADP+ solution, 10 μL of 100 mM aldehyde in deionized water (DW) or acetonitrile, and 10 μL of 300 mg / mL KARC were dispensed into microtubes. As a negative control, 935 μL of buffer, 15 μL of 100 mM EDTA solution, 30 μL of 100 mM NADP+ solution, 10 μL of 100 mM aldehyde in DW or acetonitrile, and 10 μL of DW were dispensed into microtubes.

[0320] For the SSA experiment, 845 μL of buffer, 15 μL of 100 mM EDTA in water, 30 μL of 100 mM NADP+ in water, 10 μL of 10 mM SSA in acetonitrile, and 10 μL of 300 mg / mL KARC were dispensed into a microtube. As a negative control, 845 μL of buffer, 15 μL of 100 mM EDTA in water, 30 μL of 100 mM NADP+ in water, 100 μL of 10 mM SSA in acetonitrile, and 10 μL of DW were dispensed into a microtube. The reaction was shaken at 30°C or 37°C for 1 or 3 hours.

[0321] The reaction was shaken using a thermoshaker at 30°C or 37°C for 1 hour or 3 hours.

[0322] 12-2: Pre-processing before HPLC analysis

[0323] For experiments using representative fatty aldehydes (SSA and acetaldehyde), 500 μL of each reaction was aliquoted into a microtube at the end of the reaction. 470 μL of methanol, 20 μL of 50 mM DNPH in acetonitrile, and 10 μL of 6N HCl were additionally dispensed into the microtube containing the reaction solution and heated at 70°C for 40 minutes. Alternatively, 480 μL of methanol, 10 μL of 100 mM DHBA in acetonitrile, and 10 μL of 6N HCl were added and heated at 70°C for 40 minutes. After cooling the heated solution, 10 μL was aliquoted and injected into the HPLC for analysis.

[0324] For experiments with DOPAL, cinnamaldehyde, and benzaldehyde (representative aromatic aldehydes), 10 μL of the solution reacted with KARC was aliquoted without heating with DNPH or DHBA and injected into HPLC for analysis.

[0325] 12-3: HPLC analysis

[0326] Analyses were performed using an HPLC system (Waters Alliance 2690 / 2695 HPLC with a Waters 2996 PDA detector). The analytical column was 150 mm x 4.6 mm i.d., packed with C18, 5 μm particle size (Shimadzu Scientific Instruments, Kyoto, Japan).

[0327] The gradient started from 80% water (1 v / v% trifluoroacetic acid) and after 15 minutes was adjusted to 20% in reverse phase.The absorbance was analyzed with a UV detector at a wavelength of 254 nm, 310 nm or 360 nm.

[0328] This result was confirmed by the progress of the reaction of consuming aldehyde by reduction of DNPH-aldehyde conjugate or DHBA-aldehyde conjugate in the experimental groups compared with the negative control group.

[0329] [Example 13] Preparation of food and pharmaceutical compositions for preventing and recovering from auto-brewery syndrome caused by the decomposition of endogenous ethanol in the body.

[0330] Food and pharmaceutical compositions containing KARC as an active ingredient for alleviating auto-brewery syndrome have been prepared. Food or pharmaceutical compositions containing KARC powder can be prepared in various composition ratios. For example, the powder composition according to the present invention suppresses auto-brewery syndrome and oxidative stress by taking 13 g of the composition twice daily. The weight ratios of the components and phases of the food or pharmaceutical compositions containing the powder composition are shown in Table 7.

[0331] Table 7 Industrial Applicability

[0332] KARC dry powder, excipients, and natural sweeteners such as oligofructose, enzyme-treated stevia, anhydrous citric acid, isomaltodextrin, and xylitol, citrus juice powder, and citrus seasoning powder are added to food and pharmaceutical compositions. The processing and testing of the raw materials and final products of the food or pharmaceutical compositions are carried out according to general test methods and the Health Functional Food Act described in the Korean Food Code. Food or pharmaceutical compositions containing KARC decompose endogenous aldehydes and show the effect of inhibiting auto-brewery syndrome and oxidative stress. Food or pharmaceutical compositions containing KARC can prevent or improve irritable bowel syndrome.

[0333] The above examples describe in detail the mutant yeast composition KARC containing aldehyde dehydrogenase, including its production methods, pharmacological effects, administration methods, therapeutically effective doses for disease models, acute toxicity following short-term administration, and representative examples of food or pharmaceutical compositions. While the above examples describe the efficacy of KARC in detail, they are merely illustrative of the present invention.

[0334] Those skilled in the art can easily derive various modifications and other embodiments equivalent to the present invention from the above-described embodiments of the present invention.

[0335] Even foods or therapeutic agents containing modified forms of aldehyde dehydrogenase that embody the technical gist of the present invention as described in the patent claims fall within the scope of legal protection of the present invention. Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure International Form Certificate of receipt in case of original deposit issued in accordance with Article 7(1) to: Quan Xingze 167 Songpa-daero, Songpa-gu, Seoul, South Korea, Kwon Heung-taek Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure International Form Certificate of receipt in case of original deposit issued in accordance with Article 7(1) to: Quan Xingze 167 Songpa-daero, Songpa-gu, Seoul, South Korea, Kwon Heung-taek Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure International Form Certificate of receipt in case of original deposit issued in accordance with Article 7(1) to: Quan Xingze 167 Songpa-daero, Songpa-gu, Seoul, South Korea, Kwon Heung-taek Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure International Form In the case of an original deposit, a certificate of receipt is issued under Article 7(1) To: Pico Entech Co. Ltd Pico Entech Co. Ltd., 14, 288 Seongnam Violet-ro, Jungwon-gu, Seongnam-si, Gyeonggi-do, South Korea Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure International Form In the case of an original deposit, a certificate of receipt is issued under Article 7(1) To: Pico Entech Co. Ltd Pico Entech Co. Ltd., 14, 288 Seongnam Violet-ro, Jungwon-gu, Seongnam-si, Gyeonggi-do, South Korea Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure International Form In the case of an original deposit, a certificate of receipt is issued under Article 7(1) To: Pico Entech Co. Ltd Pico Entech Co. Ltd., 14, 288 Seongnam Violet-ro, Jungwon-gu, Seongnam-si, Gyeonggi-do, South Korea

Claims

A composition for promoting the decomposition of endogenous aldehydes, comprising an aldehyde dehydrogenase encoded by a gene having more than 98% homology to the gene of SEQ ID NO:

1.

2. The composition for promoting the decomposition of endogenous aldehydes according to claim 1, wherein It comprises an aldehyde dehydrogenase encoded by the gene of SEQ ID NO: 1 including SEQ ID NO:

2.

3. The composition for promoting the decomposition of endogenous aldehydes according to claim 1 or claim 2, wherein: The endogenous aldehyde is an endogenous aldehyde generated by oxidation of endogenous amine compounds or alcohols.

4. The composition for promoting the decomposition of endogenous aldehydes according to claim 3, wherein The endogenous amine compound is selected from the group consisting of dopamine, norepinephrine, serotonin and gamma-aminobutyric acid (GABA).

5. The composition for promoting the decomposition of endogenous aldehydes according to claim 3, wherein The endogenous aldehyde is selected from the group consisting of formaldehyde, acetaldehyde, 4-hydroxy-2-nonenal, non-2-enal, 4-hydroxy-hexanal (hexanal), 4-oxo-nonenal, malondialdehyde (MDA), propionaldehyde, hexanal, palmitaldehyde, succinaldehyde and acrolein.

6. The composition for promoting the decomposition of endogenous aldehydes according to claim 1 or 2, wherein: The aldehyde dehydrogenase is contained in any one selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

7. A composition for promoting the decomposition of endogenous aldehydes, comprising any one selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

8. A food composition for inhibiting oxidative stress, comprising any one selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

9. A pharmaceutical composition for inhibiting oxidative stress, comprising any one selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

10. A food composition for suppressing auto-brewery syndrome, comprising any one selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

11. A pharmaceutical composition for inhibiting auto-brewery syndrome, comprising any one selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

12. Mutant Saccharomyces cerevisiae KCTC14983BP.

13. Mutant Saccharomyces cerevisiae KCTC14984BP.

14. Mutant Saccharomyces cerevisiae KCTC14985BP.

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