Food and pharmaceutical compositions for detoxifying endogenous aldehydes

Abstract

The present invention relates to food and pharmaceutical compositions for promoting the degradation of endogenous aldehydes produced by the oxidation of alcohol or endogenous amine compounds, which contain aldehyde dehydrogenase contained in a lysate of any one or a mixture of Saccharomyces cerevisiae strains, including KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP. The food and pharmaceutical compositions of the present invention suppress oxidative stress and auto-brewery symptoms.

Description

[Technical Field]

[0001] The present invention relates to mutant Saccharomyces cerevisiae KCTC14983BP, KCTC14984BP, and KCTC14985BP. In addition, the present invention relates to an aldehyde dehydrogenase encoded by a gene having more 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 contains 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, the present invention relates to a food composition 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 suppressing oxidative stress, which contain a lysate of any one or a mixture thereof selected from the group consisting of KCTC13925BP, KCTC 14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP. [Background technology]

[0004] The human body experiences oxidative stress due to various causes, such as alcohol consumption, smoking, medication, strenuous exercise, and physical and mental stress, and therefore various types of endogenous aldehydes are produced in the cells of the human body.

[0005] In addition, reactive oxygen species (ROS) are generated during the energy production process in mitochondria in cells, and various types of endogenous aldehydes are produced by lipid peroxidation (LPO) in the cell membrane caused by reactive oxygen species (ROS).

[0006] The endogenous aldehydes produced in this way are highly reactive. They readily react with surrounding proteins and modify DNA, resulting in protein degradation and the reduction or complete loss of their unique functions.

[0007] Consequently, the production and accumulation of endogenous aldehydes causes accelerated cellular aging or is an underlying cause of cancer, diabetes, cardiovascular disease, and neurodegenerative diseases.

[0008] Alcohol, whether consumed through drinking or taking drugs, is the main cause of increased endogenous aldehydes in the human body. For example, when a person drinks alcohol, 80% to 90% of the alcohol undergoes two stages of enzymatic metabolic degradation.

[0009] In the first step, alcohol is converted to the toxic metabolic intermediate acetaldehyde (Ach) by alcohol dehydrogenase (ADH), and in the second step, acetaldehyde (Ach) is detoxified to acetic acid by the action of aldehyde dehydrogenase (ALDH) [Figure 1].

[0010] During this in vivo alcohol metabolism process, excess acetaldehyde (Ach), produced due to genetic deficiency or excessive alcohol consumption, directly modifies surrounding proteins or DNA, thereby acting as a toxic substance that can cause cancer. The present invention relates to an aldehyde dehydrogenase enzyme that detoxifies endogenous acetaldehyde to prevent it from acting as a toxic substance.

[0011] On the other hand, even people who do not drink alcohol can produce ethanol in their bodies through intestinal microorganisms. This endogenous ethanol is also converted to acetaldehyde by ethanolases. The production of acetaldehyde causes physiological discomfort similar to that seen in alcohol-induced hangovers, resulting in auto-brewery syndrome (ABS) (Malik F, et al., "Case report and literature review of 'auto-brewery syndrome: probably an underdiagnosed medical condition'" BMJ Open Gastro 2019).

[0012] Auto-brewery syndrome is caused by endogenous ethanol fermentation, resulting in the so-called "gastrointestinal fermentation syndrome," which manifests as alcohol hangover symptoms even when no alcohol has been consumed.

[0013] According to data from the US National Library of Medicine and Makati Med Hospital, gut fermentation syndrome (GUF) is a condition that occurs when ethanol is spontaneously produced in the body by fungi or bacteria in the gastrointestinal, oral, and urinary systems. Even if a person does not drink alcohol, endogenous alcohol production occurs due to the action of yeast within the human body, resulting in symptoms of an alcohol hangover. This is caused by alcohol fermentation by various yeast strains and bacteria in the human intestine.

[0014] Because yeast in the intestine produces carbon dioxide and ethanol during food digestion, even if a patient with auto-brewing syndrome does not consume alcohol, the patient experiences 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, gut fermentation symptoms are caused by alcohol-producing yeast or bacteria in the human body. This is primarily seen in people with diseases such as diabetes, obesity, and Crohn's disease, but can also occur in healthy people. People with liver dysfunction, such as chronic intestinal obstruction, gastrointestinal paralysis, nonalcoholic fatty liver disease, or nonalcoholic hepatitis, may also experience auto-brewery symptoms.

[0016] Patients with auto-brewery syndrome may exhibit a variety of symptoms, such as vomiting, lightheadedness, chronic fatigue syndrome, auto-brewery syndrome, dizziness, disorientation, fainting, irritable bowel symptoms, runny nose, cough, and sinusitis. Chronic fatigue syndrome can lead to health problems such as anxiety, depression, and decreased productivity.

[0017] On the other hand, increased acetaldehyde in the human body directly or indirectly induces 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, leading to cancer. 4-HNE has been found to be a biomarker involved in 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 cancer-causing substance. The National Cancer Institute (NCI) Thesaurus (NCI) classifies malondialdehyde as a strong endogenous mutagen and uses it as a biomarker for cardiovascular disease and fatigue.

[0020] The human body sustains various vital functions by generating energy from mitochondria in cells. Reactive oxygen species (ROS) are inevitably generated during mitochondrial energy production or conversion. This leads to lipid peroxidation (LPO), which destroys lipid membranes. Through this LPO, aldehydes such as nonenal (4-HNE), malondialdehyde (MDA), and acetaldehyde (Ach) are produced and accumulate 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, and accumulate in cells, increasing oxidative stress in the human body [Figure 2].

[0022] Increased oxidative stress disrupts the smooth energy metabolism process in mitochondria, further increasing aldehyde substances such as methylglyoxal (MG) and advanced glycation end products (AGEs) in cells, resulting in the intracellular accumulation of aldehyde substances.

[0023] Thus, when reactive aldehydes such as HNE and MDA, which are produced by lipid peroxidation (LPO) due to increased free radicals and oxidative stress, or aldehyde substances such as glyceraldehyde-3-phosphate (GA3P), an intermediate of glycolysis, are produced in excess and accumulate in cells, causing cytotoxicity.

[0024] The intracellular accumulation of free radicals or reactive aldehydes weakens the intracellular antioxidant defense system, such as glutathione. Disruption of energy metabolism and the accumulation of misfolded proteins (UPs) ultimately lead to increased endoplasmic reticulum stress (ER stress), resulting in cytotoxicity, which accelerates aging and induces various diseases.

[0025] Meanwhile, within the human body, there are substances that perform various physiological functions, such as dopamine (DA), serotonin (5-HT), norepinephrine (NE), epinephrine (Adr), gamma-aminobutyric acid (GABA), and histamine.

[0026] Through the systematic actions of monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), catechol-O-methyltransferase (COMT), and alcohol dehydrogenase (ADH), the amine (-NH2) group of monoamine substances is converted to an aldehyde (-CHO) group, which is then finally converted to an acid (-CO2H) group, completing the in vivo metabolism of monoamine substances [Figure 3].

[0027] Dopamine (DA) is a precursor of epinephrine (Adr), which regulates the autonomic nervous system. Dopamine is a major neurotransmitter involved in the pathogenesis of Parkinson's disease (PD).

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

[0029] Dopamine (DA) secreted in this way is metabolized non-enzymatically and converted to neuromelanin or quinones via radical autoxidation. In addition, dopamine can be metabolized by the action of monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), and catechol methyltransferase (COMT) enzymes [Figure 3].

[0030] As explained above, the dopamine (DA) metabolic process can be divided into two metabolic pathways depending on the order of the enzymes involved: in the metabolic pathway involving the sequential actions of monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), and catechol methyltransferase (COMT), dopamine is converted successively into 3,4-dihydroxyphenylacetaldehyde (DOPAL), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA).

[0031] In the metabolic pathways where catechol methyltransferase (COMT), monoamine oxidase (MAO), and aldehyde dehydrogenase (ALDH) act consecutively, dopamine is converted successively to 3-methoxytyramine (3-MT), 3-methoxy-4-hydroxyphenylacetaldehyde (MOPAL), and homovanillic acid (HVA). Through these two metabolic pathways, dopamine is finally converted to homovanillic acid (HVA).

[0032] Dopamine is also metabolized to norepinephrine (NE) and epinephrine (ADR), hormones used to regulate the autonomic nervous system. During this metabolic process, dopamine is metabolized in the following order: 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), 3,4-dihydroxymandelic acid (DOMA), and 4-hydroxy-3-methoxymandelic acid.

[0033] In addition, dopamine is converted sequentially to 3-methoxynorepinephrine (methoxy-NE), 4-hydroxy-3-methoxyphenylglycolaldehyde (MOPEGAL), and 3-methoxy-4-hydroxymandelic acid by the sequential actions of catechol methyltransferase (COMT), MAO, and aldehyde dehydrogenase (ALDH).

[0034] As mentioned above, through the sequential enzymatic actions of monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), and catechol methyltransferase (COMT), the amine (-NH) functional group of dopamine is converted to an aldehyde (-CHO) and finally to an acid (-COH).

[0035] On the other hand, serotonin (5-HT), which is produced from tryptophan (L-tryptophan), is closely related to mental health, such as learning and sleep. Serotonin is also known as a representative neurotransmitter. Serotonin is sequentially metabolized to 5-hydroxyindoleacetaldehyde (5-HIAL) and 5-hydroxyindoleacetic acid (5-HIAA) through the sequential enzymatic actions of monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), and catechol methyltransferase (COMT).

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

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

[0038] When alcohol is consumed, serotonin binds to alcohol-metabolizing enzymes, disrupting normal serotonin metabolism. As a result, serotonin aldehyde (5-HIAL) is converted to 5-hydroxytryptopol (5-HTOL), which accumulates in the brain and nerves (Shibata et al. 2014).

[0039] In the process of metabolizing alcohol to acetic acid, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are rapidly consumed. A deficiency of aldehyde dehydrogenase (ALDH) that should be used to metabolize serotonin can occur.

[0040] Due to this lack of aldehyde dehydrogenase (ALDH), serotonin aldehyde (5-HIAL), a metabolic product of serotonin (5-HT), accumulates without being broken down normally, resulting in toxic effects that deform proteins in brain neurons.

[0041] To alleviate the toxic effects of 5-HIAL, an aberrant metabolic pathway is activated to rapidly convert 5-HIAL to 5-hydroxytryptophol (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-hydroxytryptophol (5-HTOL) is converted back into serotonin aldehyde (5-HIAL), which is then metabolized via the normal metabolism of serotonin.

[0043] Due to the toxic effects during the alcohol breakdown process, oxidative stress is already increased and the function of alcohol dehydrogenase (ADH) is significantly reduced. Even after alcohol is metabolized in the body, 5-hydroxytryptopol (5-HTOL) accumulates in the human body without being converted into serotonin aldehyde (5-HIAL).

[0044] Alcohol consumption causes abnormal increases in 5-hydroxytryptopol (5-HTOL) in brain neurons due to impaired serotonin metabolic pathways caused by a lack of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) (Shibata et al. 2014).

[0045] It has been found that this abnormality in serotonin metabolism leads to an increase in 5-hydroxytryptopol (5-HTOL) in the liver, ileum, and spleen, impairing the function of the associated organs and causing disease.

[0046] In conclusion, 5-HIAL itself is a toxin that causes Parkinson's disease. In addition, 5-HIAL distorts the serotonin metabolic pathway, increases modified proteins, and causes various diseases such as liver dysfunction.

[0047] On the other hand, GABA is converted by monoamine oxidase (MAO) to succinic semialdehyde (SSA), which can act as a toxic aldehyde to neurons [Figure 5]. If SSA is not converted to succinic acid and accumulates in the brain,

[0048] Succinic semialdehyde (SSA), a metabolic intermediate of GABA, should be converted to non-toxic succinic acid by the action of succinic semialdehyde dehydrogenase (SSADH).When aldehyde dehydrogenase does not function normally, succinic semialdehyde (SSA) is converted to gamma-hydroxybutyrate (GHB) by the action of aldoketone reductase (AKR).

[0049] The endogenous aldehyde succinic semialdehyde (SSA) is a typical cytotoxic substance that accumulates when aldehyde dehydrogenase (ALDH) function is impaired. This toxic substance induces the production and accumulation of gamma-hydroxybutyrate (GHB), which impairs bodily functions such as liver function, speech, and walking (Buzzi, Andrea et al. 2006).

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

[0051] Histamine is also metabolized by an orchestrated enzyme system including monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), and histamine N-methyltransferase (HNMT). Histamine is converted to imidazole acetaldehyde, imidazole acetic acid, and finally imidazole acetic acid riboside [Figure 6].

[0052] Histamine is also converted sequentially to N-methylhistamine, N-methylimidazoleacetaldehyde, and N-methylimidazoleacetic acid by the action of the enzyme systems histamine-N-methyltransferase (HNMT), MAO, and aldehyde dehydrogenase.

[0053] In the metabolic process of histamine, the amine group (-NH2) is metabolized to an aldehyde (-CHO) and an acid (-CO2H) through detoxification processes mediated by monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), etc. [Figure 6].

[0054] Imidazole acetaldehyde, which can be produced when histamine metabolism is abnormal, causes various diseases through a more potent toxic effect than histamine. The human body is designed to quickly detoxify secreted histamine using enzyme systems to minimize damage to the body.

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

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

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

[0058] As discussed above, various monoamines, such as dopamine (DA), serotonin (5-HT), norepinephrine (NE), epinephrine (Adr), gamma-aminobutyric acid (GABA), and histamine, which 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-dihydroxyphenylglycolaldehyde (DOPEGAL), 3-methoxy-4-hydroxyphenylglycolaldehyde (MOPEGAL), 5-hydroxyindoleacetaldehyde (5-HIAL), 5-methoxyindole-3-acetaldehyde (5-MIAL), imidazoleacetaldehyde, and N-methylimidazoleacetaldehyde by the action of monoamine oxidase.

[0059] When aldehyde dehydrogenase does not function normally in the human body, various aldehydes produced in the body act as toxic substances to the human body, resulting in various pathological phenomena and accelerated aging.

[0060] There is an urgent need to develop a food or pharmaceutical composition that can rapidly oxidize and detoxify various endogenous aldehydes and suppress various problems caused by the accumulation of endogenous aldehydes in the body. [Prior art documents] [Patent documents]

[0061] [Patent Document 1] U.S. Patent Application Publication No. 2021-0254023A1, dated August 21, 2021 [Non-patent literature]

[0062] TIFF2025540042000001.tif93109TIFF2025540042000002.tif98109 Summary of the Invention [Problem to be solved by the invention]

[0063] Disclosure of the Invention Technical issues Despite the various studies listed above, no food or pharmaceutical composition has yet been developed that can suppress the increase in oxidative stress, auto-brewery symptoms, chronic pruritus, psoriasis, atopy, and asthma caused by the accumulation of endogenous aldehydes in the body through the detoxification of various endogenous aldehydes.

[0064] A basic object of the present invention is to provide mutant Saccharomyces cerevisiae KCTC14983BP, mutant Saccharomyces cerevisiae KCTC14984BP, and mutant Saccharomyces cerevisiae KCTC14985BP that are capable of producing aldehyde dehydrogenase.

[0065] Additionally, a primary object of the present invention is to provide a food composition containing 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 degradation of endogenous aldehydes produced by the oxidation of alcohol or endogenous amine compounds.

[0066] 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 comprises aldehyde dehydrogenase contained in a lysate of any one or a mixture thereof selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP and KCTC14985BP.

[0067] Yet another object of the present invention is to provide a food composition and a pharmaceutical composition for preventing automatic brewery symptoms, comprising aldehyde dehydrogenase contained in a lysate of any one or a mixture thereof selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP and KCTC14985BP.

[0068] Yet another object of the present invention is to provide a food composition and a pharmaceutical composition for preventing the symptoms of various pathologies caused by endogenous aldehydes, comprising aldehyde dehydrogenase contained in a lysate of any one or a mixture thereof selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP and KCTC14985BP. [Means for solving the problem]

[0069] Solutions to problems The above-mentioned object of the present invention can be achieved by providing a food composition and a pharmaceutical composition containing an aldehyde dehydrogenase capable of rapidly decomposing endogenous aldehydes, which 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 (hereinafter abbreviated as KARC).

[0070] Another object of the present invention can also be achieved by providing a food composition or pharmaceutical composition for preventing automatic brewer's symptoms, which contains a lysate of any one or a mixture of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP. [Effects of the Invention]

[0071] Advantageous Effects of the Invention KARC, a dry powder of lysate of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, comprising the aldehyde dehydrogenase encoded by SEQ ID NO:1.

[0072] The food composition and pharmaceutical composition of the present invention exhibit the effect of suppressing oxidative stress in the human body and suppressing auto-brewery symptoms. [Brief explanation of the drawings]

[0073] Detailed Description of the Drawings [Figure 1] [Figures 1 and 2] show the production and degradation of endogenous aldehydes in vivo.

[0074] Ethanol or ethanol derivatives (2-substituted ethanol, R-CHCH-OH) are reversibly converted in vivo by alcohol dehydrogenase (ADH) to acetaldehyde derivatives (R-CH-CHO). The highly toxic acetaldehyde derivatives are irreversibly converted to the relatively non-toxic acetic acid derivatives (R-CH-COH).

[0075] Endogenous monoamines (R-C2H4-NH2) are irreversibly converted by monoamine oxidase (MAO) to the highly toxic acetaldehyde (R-CH2-CHO), which is then converted back to acetaldehyde by aldehyde dehydrogenase, and finally detoxified to acetate (R-CH2-CO2H), similar to alcohol metabolism.

[0076] [Figure 2] In Figure 2, dopamine (DA), a typical monoamine neurotransmitter, is converted by monoamine oxidase (MAO) into toxic aldehyde structures (DOPANAL, a dopamine-derived aldehyde) such as DOPAL and MOPAL. These are then decomposed by aldehyde dehydrogenase (ALDH) and ultimately metabolized to the relatively less toxic homovanillic acid (HVA).

[0077] Dopamine is also converted by monoamine oxidase to dopanal via norepinephrine (NE), which is finally decomposed into acid compounds by aldehyde dehydrogenase, as are the known toxic substances dopegal and mopegal.

[0078] Additionally, dopamine metabolism does not proceed well in vivo via DOPANAL to increase DOPANOL (dopamine-derived alcohols) such as DOPOL for various reasons, including a decrease in ALDH, because DOPANAL is not converted to less toxic acid compounds.

[0079] It is known that due to the toxicity of DOPANAL (a dopamine-derived aldehyde), it is temporarily converted to and stored as a relatively less toxic alcohol, DOPANOL (a dopamine-derived alcohol). When dopamine metabolism returns to its original state, DOPET, a representative DOPANOL stored in the body, is metabolized and decomposed into an acid via the activation of the alcohol-metabolizing enzymes alcohol dehydrogenase and aldehyde dehydrogenase.

[0080] Although various enzymatic dopamine metabolic pathways exist, when enzymatic dopamine metabolism fails, dopamine is metabolized via a non-enzymatic reaction in which reactive oxygen species (ROS) spontaneously convert dopamine into a quinone derivative, which is then converted into neuromelanin. In this case, rapid changes in melanin distribution disrupt homeostasis, leading to various diseases.

[0081] [Figure 3] [Figure 3] is a chemical formula showing the production and degradation process of dopamine in vivo.

[0082] [Figure 4] [Figure 4] is a chemical formula showing the in vivo production and degradation process of serotonin.

[0083] [Figure 5] [Figure 5] is a chemical formula showing the degradation process of GABA in vivo.

[0084] [Figure 6] [Figure 6] is a chemical formula showing the in vivo histamine production and degradation process.

[0085] Monoamine neurotransmitters, such as dopamine (DA), serotonin (5-HT), GABA, and histamine, share a common structure: a two-carbon chain and one amine (R-CH2-CH2-NH2). These are oxidized by monoamine oxidase (MAO) enzymes to endogenous aldehydes (-CHO), such as DOPAL, 5-HIAL, SSA, 4-imidazoleacetaldehyde, and 1-methylimidazoleacetaldehyde, which bind to and denature surrounding proteins. The resulting accumulation of unfolded proteins in the endoplasmic reticulum acts as a cytotoxic agent, inducing cell death (Figures 3, 4, 5, and 6).

[0086] [Figure 7] [Figure 7] is a graph showing the ability of the KARC of the present invention to decompose endogenous acetaldehyde. These results demonstrate that the composition of the present invention decomposes endogenous acetaldehyde and suppresses self-brewing symptoms.

[0087] [Figure 8] [Fig. 8] is a graph showing the malondialdehyde decomposition ability of KARC.

[0088] In animal experiments in which blood levels of malondialdehyde, an endogenous toxic aldehyde [Figure 8], were increased by alcohol consumption, KARC's effects of reducing aldehydes and oxidative stress were confirmed.

[0089] [Figure 9] [Figure 9] shows that administration of the KARC of the present invention changes the contents of DOPAL, DOPAC, and HVA in the brain of an animal model of Parkinson's disease (PD).

[0090] [Figure 10] [Figure 10] shows the results of the dopamine turnover index in an animal model of Parkinson's disease (PD).

[0091] When Parkinson's disease was induced in animals using rotenone, dopamine secretion decreased. Dopamine degradation metabolism was abnormally suppressed, resulting in a sharp decrease in the production of DOPAC and HVA and an increase in the abnormal metabolite DOPET. In the group administered with the KARC of the present invention, DA, DOPAC, and HVA increased, and DOPET, an abnormal metabolite of dopamine, decreased. It is expected that the KARC of the present invention will restore normal dopamine secretion and dopamine degradation metabolism in vivo.

[0092] [Figure 11] 11 is a graph showing the ability of the KARC of the present invention to decompose acetaldehyde in the human body. The composition containing the KARC of the present invention is shown to suppress the symptoms of brewery.

[0093] [Figure 12] [Figure 12] is a graph showing the ability of KARC to decompose malondialdehyde in the human body.

[0094] [Figure 13] FIG. 13 is a graph showing the stabilization of malondialdehyde in the human body by KARC.

[0095] Tests to confirm the reduction of endogenous blood acetaldehyde in the human body [Figure 11] and blood malondialdehyde [Figure 12] demonstrated the effect of KARC administration in reducing acetaldehyde and malondialdehyde. [Figure 13] In a state where oxidative stress was increased due to medication, etc., malondialdehyde, a biomarker of oxidative stress and reactive oxygen species, decreased after taking KARC. This confirmed the effect of KARC in reducing oxidative stress.

[0096] [Figure 14]

[0043] FIG. 14 shows the change in enzyme activity when the KwonP-1 strain included in the KARC of the present invention was orally administered.

[0097] [Figure 15][Figure 15] shows the changes in enzyme activity when the KwonP-2 strain contained in the KARC of the present invention was orally administered.

[0098] [Figure 16] [Figure 16] shows the changes in enzyme activity when the KwonP-3 strain contained in the KARC of the present invention was orally administered.

[0099] [Figure 17] [Figure 17] shows the changes in enzyme activity when the PicoYP strain contained in the KARC of the present invention was orally administered.

[0100] [Figure 18] [Figure 18] shows the changes in enzyme activity when the PicoYP-01 strain contained in the KARC of the present invention was orally administered.

[0101] [Figure 19] [Figure 19] shows the changes in enzyme activity when the PicoYP-02 strain contained in the KARC of the present invention was orally administered.

[0102] [Figures 14, 15, 16, 17, 18, and 19] show that KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02 were orally administered for 90 minutes under conditions similar to those of the human gastric digestion process (1 < pH < 5). The changes in ALDH enzyme activity were measured. The ALDH enzyme activity was maintained at a minimum of 37.29 units / g and a maximum of 52.24% at pH = 5 (similar to the conditions observed during food intake). When KARC was orally administered, it was confirmed that the enzyme activity was maintained.

[0103] [Figure 20] [Figure 20] shows the growth curve and enzyme activity when the KwonP-1 strain contained in the KARC of the present invention was cultured in a 5 L fermenter.

[0104] [Figure 21]

[0043] FIG. 21 shows the growth curve and enzyme activity when the KwonP-2 strain included in the KARC of the present invention was cultured in a 5-L fermenter.

[0105] [Figure 22] [Fig. 22] shows the growth curve and enzyme activity when the KwonP-3 strain was cultured in a 5 L fermenter.

[0106] [Figure 23] [Figure 23] shows the growth curve and enzyme activity when the PicoYP strain included in the KARC of the present invention was cultured in a 5 L fermenter.

[0107] [Figure 24] [Fig. 24] shows the growth curve and enzyme activity when the PicoYP-01 strain was cultured in a 5 L fermenter.

[0108] [Figure 25] [Figure 25] shows the growth curve and enzyme activity when the PicoYP-02 strain included in the KARC of the present invention was cultured in a 5 L fermenter.

[0109] In Figures 20, 21, 22, 23, 24, and 25, the new mutant strains KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-01 were cultured in 5-L fermenters using YPD medium under the same conditions. The cultures were grown at 30°C and 200 rpm for 48 hours. The growth curves (OD660nm) and ALDH enzyme activity of each strain were compared with those of the type strain, revealing that the ALDH enzyme activity was at least 10.5-fold and up to 18.75-fold higher. PicoYP-01 had the highest ALDH activity at 52.68 units / g, while KwonP-3 had the lowest at 29.5 units / g.

[0110] [Figure 26] [Figure 26] is an HPLC spectrum of a mixture of distilled water and acetaldehyde.

[0111] [Figure 27] [Figure 27] is an HPLC spectrum of a mixture of KARC of the present invention and acetaldehyde after being kept at 30°C for 1 hour.

[0112] [Figure 28] [Figure 28] is an HPLC spectrum of a mixture of KARC of the present invention and acetaldehyde after being kept at 30°C for 3 hours.

[0113] [Figure 29] [Figure 29] is an HPLC spectrum of a mixture of KARC of the present invention and acetaldehyde after being kept at 37°C for 1 hour.

[0114] [Figure 30] [Figure 30] shows the HPLC spectrum of a mixture of KARC and acetaldehyde after being kept at 37°C for 3 hours.

[0115] When acetaldehyde was treated with KARC for 1 hour, acetaldehyde, a representative endogenous aldehyde and known carcinogen, was oxidized 100% not only at 30°C but also at 37°C. [Figures 26, 27, 28, 29, 30]

[0116] [Figure 31] [Figure 31] is an HPLC spectrum of a mixture of distilled water and glyoxal.

[0117] [Figure 32] [Figure 32] is an HPLC spectrum of a mixture of KARC of the present invention and glyoxal after being kept at 30°C for 1 hour.

[0118] [Figure 33] [Figure 33] is an HPLC spectrum of a mixture of KARC of the present invention and glyoxal after being kept at 30°C for 3 hours.

[0119] [Figure 34][Figure 34] is an HPLC spectrum of a mixture of KARC of the present invention and glyoxal after being kept at 37°C for 1 hour.

[0120] [Figure 35] [Figure 35] shows the HPLC spectrum of a mixture of KARC and glyoxal after being kept at 37°C for 3 hours.

[0121] Glyoxal, a typical aldehyde produced during energy metabolism in vivo, was reduced by 20.4% within 1 hour and 25.3% within 3 hours after KARC treatment at 30°C. It was also reduced by 23.8% within 1 hour and 23.8% within 3 hours at 37°C [Figures 31, 32, 33, 34, 35].

[0122] [Figure 36] [Figure 36] is an HPLC spectrum of a mixture of distilled water and succinic semialdehyde (SSA).

[0123] [Figure 37] FIG. 37 shows an HPLC spectrum of a mixture of KARC and SSA of the present invention after being maintained at 37° C. for 1 hour.

[0124] [Figure 38] [Figure 38] shows the HPLC spectrum of a mixture of KARC and SSA after being kept at 37°C for 3 hours.

[0125] In Figures 36, 37, and 38, when KARC was added at 37°C, there was a 55% reduction in 1 hour and a 74.9% reduction in 3 hours. KARC oxidized SSA, a metabolite of GABA.

[0126] [Figure 39] [Figure 39] is an HPLC spectrum of a mixture of distilled water and trans-cinnamaldehyde.

[0127] [Figure 40][Figure 40] is an HPLC spectrum of a mixture of KARC of the present invention and trans-cinnamaldehyde after maintaining it at 30°C for 1 hour.

[0128] [Figure 41] [Figure 41] is an HPLC spectrum of a mixture of KARC of the present invention and trans-cinnamaldehyde after maintaining it at 30°C for 3 hours.

[0129] [Figure 42] [Figure 42] is an HPLC spectrum of a mixture of KARC of the present invention and trans-cinnamaldehyde after being kept at 37°C for 1 hour.

[0130] [Figure 43] [Figure 43] shows an HPLC spectrum of a mixture of KARC and trans-cinnamaldehyde after being kept at 37°C for 3 hours.

[0131] When treated with KARC, trans-cinnamaldehyde was reduced by 35.9% in 1 hour and 97.4% in 3 hours at 30°C, and was converted by 82.4% in 1 hour and 99.6% in 3 hours at 37°C. [Figures 39, 40, 41, 42, 43]

[0132] [Figure 44] [Figure 44] is an HPLC spectrum of a mixture of distilled water and benzaldehyde.

[0133] [Figure 45] [Figure 45] is an HPLC spectrum of a mixture of KARC of the present invention and benzaldehyde after keeping it at 30°C for 1 hour.

[0134] [Figure 46] [Figure 46] is an HPLC spectrum of a mixture of KARC of the present invention and benzaldehyde after being kept at 30°C for 3 hours.

[0135] [Figure 47][Figure 47] is an HPLC spectrum of a mixture of KARC of the present invention and benzaldehyde after being kept at 37°C for 1 hour.

[0136] [Figure 48] [Figure 48] shows the HPLC spectrum of a mixture of KARC and benzaldehyde after keeping it at 37°C for 3 hours.

[0137] When treated with KARC, benzaldehyde was reduced by 12.2% in 1 hour and 32.0% in 3 hours at 30°C, and was converted by 57.4% in 1 hour and 97.1% in 3 hours at 37°C. [Figures 44, 45, 46, 47, 48]

[0138] [Figure 49] [Figure 49] is an HPLC spectrum of a mixture of distilled water and DOPAL.

[0139] [Figure 50] [Figure 50] is an HPLC spectrum of a mixture of KARC and DOPAL of the present invention after being kept at 30°C for 1 hour.

[0140] [Figure 51] [Figure 51] is an HPLC spectrum of a mixture of KARC and DOPAL of the present invention after being kept at 30°C for 3 hours.

[0141] [Figure 52] [Figure 52] is an HPLC spectrum of a mixture of KARC and DOPAL of the present invention after being kept at 37°C for 1 hour.

[0142] [Figure 53] [Figure 53] shows the HPLC spectrum of a mixture of KARC and DOPAL after being kept at 37°C for 3 hours.

[0143] In Figures 49, 50, 51, 52, and 53, when KARC was treated at 30°C, DOPAL decreased by 4.7% at 1 hour and 15.7% at 3 hours. At 37°C, DOPAL decreased by 13.4% at 1 hour and 24.4% at 3 hours.

[0144] DOPAC increased at 6 minutes, confirming that KARC oxidizes DOPAL and converts it to DOPAC. DETAILED DESCRIPTION OF THE INVENTION

[0145] BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a method for producing a dry powder of the KARC of the present invention, which is a lysate of Saccharomyces celloviciae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, or KCTC14985BP, will be described in more detail.

[0146] These examples are for illustrative purposes only of compositions that can achieve the objectives of the present invention, and therefore the scope of the present invention is not limited to only the compositions illustrated in the following examples. [Example]

[0147] Mode of Invention [Example 1] Screening of wild yeast parent strains to promote mutations. In this study, various types of makgeolli (traditional Korean wine) were mixed with a 0.9% NaCl solution to prepare makgeolli suspensions. The makgeolli suspensions were stirred at 200 rpm for 1 hour. The supernatant containing the wild-type yeast strain was diluted with YPD (yeast extract peptone dextrose broth) medium. The diluted solutions were prepared to a concentration 106 times the original solution. The diluted solutions were then spread onto YPD agar medium. The agar medium was statically cultured at 30°C under aerobic conditions for 1 week. Saccharomyces celloviciae was initially screened based on colony morphology, growth characteristics on YPD medium, and microscopic observation.

[0148] The ALDH activity and glutathione content of the screened Saccharomyces celloviciae strains were measured. The parent strains were selected based on ALDH activity and glutathione production.

[0149] 1-1: Measurement of aldehyde dehydrogenase Acetaldehyde reacted with dinitrophenylhydrazine (DNPH) to form acetaldehyde hydrazone (Ach-DNPH) compounds. The Ach-DNPH compounds were detected at 360 nm by HPLC equipped with a C18 column. The amount of aldehyde reduced by the degradation reaction catalyzed by aldehyde dehydrogenase (ALDH) was quantified via the amount of detected Ach-DNPH compounds.

[0150] The enzymatic reaction was carried out at 30°C by adding 10 μl of yeast lysate to 990 μl of reaction mixture (50 mM potassium phosphate buffer (pH 8.0), 1.5 mM acetaldehyde, and 3 mM NADP+). After the enzymatic reaction was completed, 50 μl of 10 mM DNPH was added to induce the formation of Ach-DNPH. The formation of Ach-DNPH was allowed to proceed for 1 hour at 22°C.

[0151] The formation of Ach-DNPH was stopped by adding 3 M sodium acetate (pH 9). The formed Ach-DNPH compound was separated by adding two volumes of acetonitrile. The separated Ach-DNPH compound (in ACN) was analyzed by injection into HPLC.

[0152] The concentration of Ach-DNPH compounds was analyzed by HPLC under conditions of 360 nm wavelength, 1 ml / min flow rate, and acetonitrile / water mobile phase development on a C18 column. The area values ​​of the resulting chromatograms were converted using a standard curve of aldehyde-DNPH (Sigma-Aldrich) to quantify the concentration of Ach-DNPH compounds. A reduction of 1 mM Ach-DNPH per minute was calculated as 1 unit of ALDH. ALDH activity was standardized as units / mg of protein.

[0153] 1-2: Measurement of glutathione Yeast cells were harvested by centrifuging 1 ml of Saccharomyces celloviciae 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 1,000 rpm for 2 hours at 85°C. The suspension was centrifuged to remove the yeast cells, and the supernatant was filtered through a 0.22 μm filter to obtain a sample containing glutathione.

[0154] The glutathione concentration of the samples was analyzed by HPLC (Shimazu LC-20AD) equipped with a C18 column. The glutathione concentration was analyzed at a wavelength of 210 nm using a mobile phase (2.02 g / L sodium 1-heptanesulfonate monohydrate, 6.8 g / L potassium dihydrogen phosphate, pH 3.0, methanol mixture) developed at a rate of 1 ml / min. The area values ​​of the resulting chromatograms were analyzed using a glutathione standard curve.

[0155] Two hundred different types of yeast obtained from Korean makgeolli were analyzed for ALDH activity and glutathione content. The 10 types of yeast listed in Table 1 had higher ALDH activity or glutathione production capacity than the other yeasts.

[0156] The ALDH activity of yeast #97 was 0.10 units / mg protein, the second highest overall. The glutathione content of yeast #97 was 0.42%, the highest of all. Yeast #97 was selected as the parent strain and the mutagenesis procedure was performed. [Table 1]

[0157] [Example 2] Identification of parent strains used in the mutagenesis process 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 single yeast colonies were plated on YPD agar medium. DNA was extracted using a Genomic DNA prep kit (HiGene™, BIOFACT Co., Ltd., Daejeon, Korea) according to the manufacturer's instructions.

[0158] To amplify the yeast ITS region of the rRNA gene, polymerase chain reaction (PCR) was performed on yeast chromosomal DNA using ITS5 (forward) and ITS4 (reverse) primers. PCR results were analyzed by DNA sequencing.

[0159] The DNA sequence of the parent strain was isolated using the Bioedit program. The reverse strands of the PCR results were converted into paired sequences through the reverse completion process.

[0160] The Cluster X program confirmed that the sequence of the forward strand matched the sequence of the reverse strand. Parent strains that matched the sequence information confirmed through the above experimental process were identified using the BLAST database provided by the US National Center for Biotechnology Information (NCBI). As a result of the identification, it was found that the rRNA of the parent strain ITS was 100% identical to that of Saccharomyces celloviciae.

[0161] [Example 3] Selection of mutant strains with improved aldehyde dehydrogenase production The mutagenesis process was carried out on the wild-type Saccharomyces celloviciae parent strain according to the method described in US Patent Application No. 17 / 176,365.

[0162] To induce mutations in the parent yeast strain, a wild-type yeast strain that produces both ALDH and glutathione was treated with ethyl methanesulfonate (EMS) or nitrosoguanidine (NGD). The mutated yeast strains were exposed to various concentrations of methylglyoxal. Mutants with superior adaptation to methylglyoxal were selected. The selected yeast strains were then exposed to various concentrations of lysine. Mutants with superior adaptation to lysine were selected. Thirty mutant strains with superior adaptation to methylglyoxal and lysine were obtained. Each of the 30 yeast strains was evaluated via five characteristics: growth curve, ALDH activity, ADH activity, coenzyme content, and glutathione content.

[0163] 3-1: Growth characteristics Saccharomyces celloviciae is a crab tree-positive microorganism that grows under aerobic conditions and simultaneously produces ethanol. To cultivate yeast with high yields, Saccharomyces celloviciae with high ethanol tolerance is required.

[0164] YPD media with different ethanol concentrations were prepared (no ethanol, 5%, 7%, and 10%). Culture media for Saccharomyces celloviciae (yeast) were adjusted to an OD of 1 at 660 nm. Each mixture of prepared YPD medium and yeast culture medium was diluted 99:1. Finally, four YPD media containing yeast with different alcohol concentrations were prepared. Each YPD medium mixed with yeast was cultured at 30°C with shaking at 200 rpm. Growth curves of the mutant strains were measured every 3 hours for 48 hours. The growth curves of each mutant strain were evaluated based on three characteristics: the time (or duration) of the lag phase, the specific growth rate (OD / h) of the exponential phase, and the maximum density (OD).

[0165] The higher the ethanol concentration in YPD medium, the longer the lag phase. The maximum density and specific growth rate decreased. Comparing the maximum densities of the mutants at low (5%) and high (10%) ethanol concentrations, it was found that in the case of nine mutants, growth was maintained at 50% of the growth rate at the high concentration compared to the growth at the low concentration. The growth characteristics of the nine mutants that distinguished them from the other strains were their short lag phase and high specific growth rate. [Table 2] TIFF2025540042000005.tif351083-2: Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities Alcohol dehydrogenase (ADH) activity was measured by adding 10 μl of yeast lysate to a 990 μl reaction mixture containing 50 mM potassium phosphate buffer (pH 8.0), 2 mM NAD+, and 1% ethanol. Aldehyde dehydrogenase (ALDH) activity was measured by adding 10 μl of yeast lysate to a 990 μl reaction mixture containing 50 mM potassium phosphate buffer (pH 8.0), 3 mM NAD+, and 1.5% acetaldehyde. The enzymatic reactions of ADH and ALDH were carried out at 30°C for 5 minutes, and the concentration of NAD(P)H produced as a result of the enzymatic reactions was measured via absorbance at 340 nm.

[0166] The enzyme activities of nine mutant strains (K-1 to K-9) selected in this study were measured. The ADH activity of the mutant strains was a minimum of 382.69 units / g and a maximum of 975.29 units / g. The ADH activity of the mutant strains was increased by at least 5.1 times and a maximum of 13.1 times compared to the type strain (reference yeast, Saccharomyces celloviciae KCTC7296). The ALDH activity of the mutant strains was a minimum of 15.23 units / g and a maximum of 72.16 units / g. The ALDH activity of the mutant strains was increased by at least 5.3 times and a maximum of 24.9 times compared to the enzyme activity of the type strain.

[0167] The six mutant strains (K-1, 4, 6, 7, 8, and 9) showed similar rates of increase in ADH and ALDH enzyme activities compared with the type strain. The ALDH enzyme activity in three mutant strains (K-2, 3, and 5) was 18.3, 23.2, and 24.9 times higher, respectively, than that of the type strain. The ADH enzyme activity in three mutant strains (K-2, 3, and 5) was 9.7, 11.6, and 13.1 times higher, respectively, than that of the type strain. The rate of increase in ALDH enzyme activity in three mutant strains (K-2, 3, and 5) was twofold higher than that of ADH.

[0168] We have identified three novel mutant strains (K-2, 3, and 5) that have been adapted to increase aldehyde dehydrogenase (ALDH) activity and named them PicoYP, PicoYP-01, and PicoYP-02, respectively. The three novel mutant strains were deposited at the Biological Resources Center of the Korea Research Institute of Bioscience and Biotechnology and assigned the accession numbers KCTC14983BP, KCTC14984BP, and KCTC14985BP, respectively.

[0169] 3-3: Coenzyme (NAD and NADP) content NAD in lysates extracted from mutant strains 合計 and NADP 合計 was measured with the NADH / NAD+ assay kit and the NADPH / NADP+ assay kit, respectively. NAD(P) in the samples was converted to NAD(P)H using the NAD(P) cycling buffer and the NAD(P) cycling enzyme mix. The colorimetric test reaction was induced with the NAD(P) developer and measured as the absorbance at 450 nm. The colorimetric test reaction was measured as the absorbance at 450 nm. The absorbance of the samples was substituted into the equation corresponding to the standard curve to determine the NAD(P) of the yeast lysate. 合計 was calculated.

[0170] The coenzyme contents of the nine mutants (K-1 to K-9) selected in this study were measured. 合計 The NAD of the mutant strains was 126 nmole / g at the minimum and 195 nmole / g at the maximum. 合計 The NADP of the mutant strain increased by at least 7.3 times and up to 10.8 times compared to the type strain. 合計 The minimum and maximum NADP contents were 2.4 nmole / g and 5.8 nmole / g, respectively. 合計 The content increased by at least 11.4-fold and up to 27.6-fold compared to the type strain.

[0171] In six mutants (K-1, 4, 6, 7, 8, and 9), NADP 合計 The rate of increase in NAD 合計 The NADP of the three novel mutants (PicoYP, PicoYP-01, and PicoYP-02) increased less than twofold. 合計 The increase rates of NAD content were 25.7, 22.9, and 27.6 times, respectively. 合計 The increase rates of NADP content were 10.8, 9.9, and 11.3 times, respectively. 合計 The increase rate is NAD 合計 The rate of increase was more than double.

[0172] 3-4: Glutathione (GSH) content The glutathione contents of the nine mutant strains were measured in the same manner as in Examples 1 and 2. The glutathione contents of the mutant strains ranged from a minimum of 0.85% to a maximum of 1.05%. The glutathione contents of the mutant strains increased by at least 2.7-fold and up to 3.3-fold compared to the type strain. The three novel mutant strains (PicoYP, PicoYP-01, and PicoYP-02) showed higher increases in ALDH activity and coenzyme content than the others.

[0173] The three novel mutant yeast strains (PicoYP, PicoYP-01, and PicoYP-02) had glutathione production capabilities similar to those of the existing deposited strains (Kwon P-1, Kwon P-2, and Kwon P-3). The three novel mutant yeast strains had significantly increased ADH and ALDH enzyme activities and coenzyme contents compared to the existing deposited strains. [Table 3] [Table 4]

[0174] [Example 4] Comparison of carbon source selection A domestic patent application for this study was filed on February 18, 2020. We investigated the carbon source selection for the growth of three mutant strains (KwonP-1, KwonP-2, and KwonP-3) with high ALDH and glutathione levels. Various carbon sources used for growth by the reference yeast strain (KCTC7296) were measured. To determine the maximum capacity for ALDH production, we investigated the carbon source selection for the growth of three new mutant strains (PicoYP, PicoYP-01, and PicoYP-02).

[0175] The characteristics and novelty of the carbon source selection of the strains were analyzed by API 50 CHL kit (API systems, BIOMERIEUX, SA, France).

[0176] A 15 ml conical tube was prepared containing 8 ml of YPD medium. Each of the seven mutant strains was inoculated into the prepared conical tube.

[0177] After culturing 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 and extracted. To eliminate the influence of the carbon source contained in the residual YPD medium, the yeast was washed three times using a centrifuge. A yeast suspension with a 2 McFarland concentration was prepared using API 50 CHL medium. The prepared yeast suspension was then filled into strip tubes. The strips containing the suspension were cultured at 30°C for 24 hours.

[0178] The API 50 CHL medium used for API testing was purple in color. As acid was produced via energy metabolism, the medium turned blue, green, and finally yellow. Finally, the type of carbon source used by the mutant strain was recorded based on the color change: purple x, blue +, green ++, and yellow +++.

[0179] All seven mutants tested used 19 different carbon sources for energy production and growth: L-arabinose, ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, N-acetyl-glucosamine, arbutin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, raffinose, and gentiobiose.

[0180] Rhamnose was used by only three mutants: KwonP-1, PicoYP-01, and PicoYP-02. Sorbitol was used by four mutants: KwonP-1, KwonP-3, PicoYP-01, and PicoYP-02. α-Methyl-D-mannoside was used by four mutants: the type strain, KwonP-1, KwonP-2, and PicoYP-02. Amygdalin was used by six mutants: KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02. D-Turanose was used by four mutants: the type strain, KwonP-1, KwonP-3, and PicoYP-02. D-tagatose was used by three mutant strains: the type strain, KwonP-3, and PicoYP-3. Gluconate was used only by the type strain.

[0181] Mannitol and sorbitol, which correspond to alcohol carbon sources, had significant effects on yeast growth. The three new mutant strains differed from the other four yeast strains in the type of sugars they used for growth. The preferred alcohol carbon source usage differed slightly among the three new mutant strains (PicoYP, PicoYP-01, and PicoYP-02) [Table 5]. [Table 5]

[0182] [Example 5] Changes in ALDH activity of mutant strains in gastric juice When KARC is administered orally, to maintain enzymatic activity in the intestine, it must pass safely without being destroyed by stomach acid, which secretes potent proteolytic enzymes such as pepsin.

[0183] NaOH solution was added to artificial gastric juice at pH 1.17 to artificially generate two simulated solutions at pH 3 and pH 5, which mimic the human gastric environment during food digestion. 1 g of KARC was added to 7 ml of artificial gastric juice and 7 ml of the two simulated solutions and mixed at 36.5°C for 5, 30, 60, and 90 minutes, respectively. NaOH solution was added to the reaction mixture to adjust the acidity to pH 7. 10 ml samples were taken from each of the solutions adjusted to pH 7 for analysis. ALDH activity was analyzed from each sample.

[0184] Under pH = 1.17 conditions, the ALDH activity of the samples decreased by more than 92.88% compared to the control group during a 5-minute reaction. Under pH = 1.17 conditions, the ALDH activity of the samples decreased by an average of 98.89% over 90 minutes. Over 90 minutes, the ALDH activity of the samples decreased by an average of 96.66% at pH = 3 and 56.83% at pH = 5. Ultimately, over the 90-minute reaction, the ALDH activity at pH = 3 and 5 remained relatively higher than that at pH = 1.17.

[0185] Specifically, the ALDH activity of KwonP-1 (KCTC13925BP) at pH = 1.17 decreased by 90.94% to 5.57 units / g compared to the control after 5 minutes of incubation. The ALDH activity of KwonP-1 decreased by 98.57% to 0.88 units / g after 90 minutes (Figure 19). The enzyme activity at pH = 3 and 5 remained relatively higher than that at pH = 1.17. After 90 minutes of incubation, the ALDH activity of KwonP-1 decreased by 96.66% to 5.57 units / g at pH = 3 and by 98.57% to 0.88 units / g at pH = 5.

[0186] The ALDH activity of KwonP-2 (KCTC14122BP) at pH 1.17 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 (Figure 20). At pH 3 and pH 5, higher enzyme activity was maintained than at pH 1.17. After 90 minutes of reaction, the ALDH activity decreased by 97.62% to 1.47 units / g at pH 3 and by 56.11% to 26.99 units / g at pH 5.

[0187] The ALDH activity of KwonP-3 (KCTC14123BP) at pH = 1.17 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 (Figure 21). At pH 3 and pH 5, higher enzyme activity was maintained than at pH 1.17. After 90 minutes of reaction, the ALDH activity decreased by 92.61% to 4.55 units / g at pH = 3 and by 62.31% to 22.18 units / g at pH = 5.

[0188] The ALDH activity of PicoYP (KCTC14983BP) at pH = 1.17 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 22]. Higher enzyme activity was maintained at pH 3 and pH 5. After 90 minutes of reaction, the ALDH activity decreased by 96.66% to 2.05 units / g at pH = 3 and by 53.97% to 28.31 units / g at pH = 5.

[0189] The ALDH activity of PicoYP-01 (KCTC14984BP) at pH = 1.17 decreased by 95.71% to 2.64 units / g after 5 minutes of incubation. The ALDH activity of PicoYP-01 decreased by 99.76% to 0.15 units / g after 90 minutes (Figure 23). At pH 3 and pH 5, the enzyme activity remained higher than that of gastric juice. After 90 minutes of incubation, the ALDH activity decreased by 98.21% to 1.10 units / g at pH = 3 and by 58.74% to 25.38 units / g at pH = 5.

[0190] The ALDH activity of PicoYP-02 (KCTC14985BP) at pH = 1.17 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 (Figure 24). At pH 3 and pH 5, higher enzyme activity was maintained than in gastric juice. After 90 minutes of reaction, the ALDH activity decreased by 98.21% to 1.10 units / g at pH = 3 and by 62.08% to 23.32 units / g at pH = 5.

[0191] pH 1.17 is the pH secreted by unprocessed gastric juice. When humans eat food, the unprocessed gastric juice and food mix in the stomach, raising the pH from 3 to 5, making it unlikely that a pH of 1.17 will be reached. Nevertheless, ALDH activity in the mutant strain was retained even at the extreme condition of pH 1.17.

[0192] Finally, the ALDH enzyme activity of the new mutants (PicoYP, PicoYP-01, and PicoYP-02) decreased from 92% to 97% under strongly acidic conditions at pH 1.17, but remained at 2 to 5 units / g. This means that 2 to 5 units of enzyme activity remained, sufficient for function in the intestine. It remained higher even at pH 3 and 5 compared to pH 1.17. This led to the conclusion that the new mutants (PicoYP, PicoYP-01, and PicoYP-02) could be administered orally.

[0193] [Example 6] Growth characteristics of 5L fermenter culture Each was inoculated into YPD medium (2% peptone, 1% yeast extract, 2% glucose), and the primary seed culture was carried out at 30°C and 200 rpm for 18 hours. 20 ml of the cultured seed was inoculated into 1980 ml of YPD medium and cultured again in 5 L. Cultivation in a 5 L culture tank was carried out at 30°C and 200 rpm for 48 hours. Growth curves at OD660nm and enzyme activities were analyzed using 10 ml samples collected from the secondary culture.

[0194] 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 type strain (KCTC7296). The growth curve characteristics and specific growth rate (OD660nm / h) of KwonP-1 were similar to those of the type strain. The ALDH activity of KwonP-1 was 33.6 units / g. The ALDH activity of KwonP-1 was 11.96-fold higher than that of the type strain (Figure 25).

[0195] 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 type strain. Growth of KwonP-2 terminated earlier than that of the type strain. The specific growth rate (OD660nm / h) of KwonP-2 was 14.8% higher than that of the type strain. The ALDH activity of KwonP-2 was 31.5 units / g. The ALDH activity of KwonP-2 was 11.21-fold higher than that of the type strain (Figure 26).

[0196] 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 type strain. Growth of KwonP-3 terminated earlier than that of the type strain. The specific growth rate (OD660nm / h) of KwonP-3 was 6.08% higher than that of the type strain. The ALDH activity of KwonP-3 was 29.5 units / g. The ALDH activity of KwonP-3 was 10.5-fold higher than that of the type strain (Figure 27).

[0197] The maximum density (OD660nm) of PicoYP (KCTC14983BP) was 123.8. The maximum density of PicoYP was 3.88% higher than that of the type strain. The growth curve characteristics of PicoYP were similar to those of the type strain. The specific growth rate (OD660nm / h) of PicoYP was 6.22% higher than that of the type strain. The ALDH activity of PicoYP was 44.2 units / g. The ALDH activity of PicoYP was 15.73-fold higher than that of the type strain [Figure 28].

[0198] 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 type strain. The growth curve characteristics of PicoYP-01 were similar to those of the type strain. The specific growth rate (OD660nm / h) of PicoYP-01 was 2.14% higher than that of the type strain. The ALDH activity of PicoYP-01 was 47.1 units / g. The ALDH activity of PicoYP-01 was 16.76-fold higher than that of the type strain [Figure 29].

[0199] 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 type strain. The growth curve of PicoYP-02 was located at the top compared to the type strain. The specific growth rate (OD660nm / h) of PicoYP-02 was 9.64% lower than that of the type strain. The ALDH activity of PicoYP-02 was 52.68 units / g. The ALDH activity of PicoYP-02 was 18.75-fold higher than that of the type strain (Figure 30).

[0200] [Example 7] Preparation of mutant strain lysate (KARC) To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysate, proteases were removed and inhibited. To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysate, cell debris was removed. The dried products or lysates of the mutant strains were mixed to prepare the KARC composition.

[0201] The mutant strain and the culture medium contained various 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 was necessary to thoroughly remove substances external to the yeast. A washing process was performed to wash the mutant strain by dispensing 40 ml of culture medium into a 50 ml conical tube, centrifuging it at 13,000 rpm for 15 minutes, and removing the supernatant.

[0202] As a result of centrifugation, residual medium remained inside the pellet produced by the aggregated yeast bacteria. After adding 30 ml of purified water, the pellet was thoroughly loosened by vortexing, and the previous process was repeated three times to thoroughly remove the residual medium.

[0203] The ethanol resistance of yeast is known to be up to 13%, and yeast bacteria die when exposed to high concentrations of ethanol. The washed pellet was thoroughly dissolved using 10 ml of 20% ethanol solution to induce yeast bacterial death. The ethanol-dissolved pellet was stirred at 100 rpm for 30 minutes to allow the yeast to die. When the reaction time was complete, 30 ml of purified water was added to reduce the ethanol concentration to 5%. The previous washing process was repeated three times to thoroughly remove the ethanol.

[0204] To protect ALDH and ADH from the degradative effects of proteases present in yeast cells, 10 ml of 1X PBS was prepared by dissolving two protease inhibitor tablets (Pierce protease inhibitor mini-tablets, EDTA-free, Thermo Scientific). The above solution was added to the washed yeast pellet and allowed to fully release.

[0205] To prepare the lysates of the mutant strains prepared in this invention, 4 g of glass beads were added and stirred to disrupt the yeast cell walls. To prevent enzyme denaturation due to the heat generated during the yeast disruption process, vortexing for 30 seconds and incubating for 30 seconds were repeated six times.

[0206] After the yeast cell wall was completely broken down, 10 ml of 100 mM potassium phosphate buffer was added and mixed by vortexing for 3-5 seconds. The mixture was centrifuged 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® Syringe Filter, Sartorius, Goettingen, Germany) to prepare the KARC composition.

[0207] To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysates, intracellular proteases were removed and inhibited, and cell debris such as cell walls were removed. KARC compositions were prepared at free ratios with lysates selected from six mutant strains (KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02) or their mixtures (Table 6).

[0208] KARC1 was produced from KwonP-1. The enzyme activities of ADH and ALDH of KARC1 were 461.4 units / g and 28.6 units / g, respectively. 合計 and NADP 合計 The coenzyme contents of KARC1 and KARC2 were 176.2 nmole / g and 5.1 nmole / g, respectively. The GSH content of KARC1 was 0.98 wt%.

[0209] KARC2 was produced from KwonP-2. The enzyme activities of ADH and ALDH of KARC2 were 482.1 units / g and 29.8 units / g, respectively. 合計 and NADP 合計 The coenzyme contents of KARC2 and KARC3 were 175.4 nmole / g and 5.2 nmole / g, respectively. The GSH content of KARC2 was 0.96 wt%. KARC3 was produced from KwonP-3. The enzyme activities of ADH and ALDH in KARC2 were 477.5 units / g and 28.1 units / g, respectively. 合計 and NADP 合計 The coenzyme contents of KARC3 and KARC4 were 177.2 nmole / g and 5.1 nmole / g, respectively. The GSH content of KARC3 was 1.00 wt%.

[0210] KARC4 was produced from PicoYP. The enzyme activities of ADH and ALDH of KARC2 were 586.8 units / g and 33.8 units / g, respectively. 合計 and NADP 合計The coenzyme contents of KARC4 and KARC5 were 184.3 nmole / g and 5.7 nmole / g, respectively. The GSH content of KARC4 was 0.84 wt%.

[0211] KARC5 was produced from PicoYP-01. The enzyme activities of ADH and ALDH of KARC5 were 621.6 units / g and 38.2 units / g, respectively. 合計 and NADP 合計 The coenzyme contents of KARC5 and KARC6 were 186.9 nmole / g and 5.6 nmole / g, respectively. The GSH content of KARC5 was 0.84 wt%.

[0212] KARC6 was produced from PicoYP-02. The enzyme activities of ADH and ALDH in KARC5 were 664.1 units / g and 41.6 units / g, respectively. 合計 and NADP 合計 The coenzyme contents of the two were 195.0 nmole / g and 5.8 nmole / g, respectively. The GSH content of KARC6 was 0.88 wt%.

[0213] KARC was produced by free mixing of dry powder with lysates prepared from the six deposited strains. The mean enzyme activities of ADH and ALDH in the KARC composition were 547.6 units / g and 33.1 units / g, respectively. The coenzyme NAD in the KARC composition 合計 and the coenzyme NADP 合計 The average contents of glutathione in the KARC compositions were 180.4 nmole / g and 5.4 nmole / g, respectively. The average content of glutathione in the KARC compositions was 0.84 wt%.

[0214] The aldehyde degradation ability of KARC continued throughout the lysate production process. KARC demonstrated the ability to remove endogenous aldehydes such as HNE, MDA, and 3,4-dihydroxyphenylacetaldehyde (DOPAL). [Table 6]

[0215] [Example 8] Analysis of ALDH sequences contained in mutant strains Differences between the ALD (yeast aldehyde dehydrogenase) of both mutant and parent strains were investigated. Whole genome sequencing was performed on the parent strain and mutant strains KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02. Mutant cells were obtained by culturing pure strains on solid medium. The genome sequences of the obtained mutant strains were analyzed.

[0216] Among the ALDs (yeast aldehyde dehydrogenases) in the novel mutant strain, ALD2 (SEQ ID NO: 3) was found to be condensed with ALD3 (SEQ ID NO: 4) on chromosome 13. A non-coding region of 689 nucleotides was located between the genes encoding ALD2 and ALD3. ALD2 and ALD3 existed contiguously in the same genome. ALD2 and ALD3 encoded aldehyde dehydrogenases, respectively. The gene encoding ALD2 consisted of 1,521 nucleotides and 506 amino acids and was nearly identical to ALD3, but had an 8.2% sequence difference. ALD2 and ALD3 were identified as distinct aldehyde dehydrogenases, differing from each other by 125 base pairs (8.2%).

[0217] In six mutant strains (KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02), the protein is synthesized continuously because there is no stop codon at the end of the ALD2 sequence. As a result, a new, larger ALDH enzyme is created by linking parts of ALD2 and ALD3 (SEQ ID NO: 1).

[0218] ALD2 (SEQ ID NO: 3) of the type strain (KCTC7296) consisted of a 30-nucleotide sequence (5-GTTCACATAAATCTCTCTTTGGACAACTAA-3) encoding nine amino acids (N-VHINLSLDN-C) at the terminal, excluding the stop codon.

[0219] The ALD2 of the six mutant strains consisted of a specific 42-nucleotide sequence (5-AGATATAGATTATACACATTTAGAAAATTAGCCAAAAGAAAA-3) [SEQ ID NO: 2] encoding 14 amino acids (N-RYRLYTFRKLAKRK-C) between the 5′ end of ALD2 and ALD3.

[0220] The deletion of ALD2 from nucleotide 1492 to nucleotide 647 of the non-coding region resulted in no termination codon at the end of the gene sequence encoding ALD2. Finally, the six deposited mutant strains contained a new mutant gene consisting of a total of 3,054 bases encoding a novel ALD [SEQ ID NO: 1].

[0221] [Example 9] In vivo acetaldehyde (Ach) and malondialdehyde (MDA) reduction effect by oral administration of KARC For the acetaldehyde and MDA animal experiments, 5-week-old male Sprague Dawley (SD) rats (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 the KARC injection.

[0222] After completion of administration, blood samples were collected from the tail vein at 0, 1, 3, 5, and 8 hours after KARC injection, and after centrifugation, the plasma was stored at −80°C [Figures 7, 8].

[0223] 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 triglyceride). Mice were intraperitoneally administered the rotenone solution (2.5 mg / kg) daily for 60 days.

[0224] 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 L-dopa was orally administered two weeks after rotenone to observe the therapeutic effect on Parkinson's disease. Brain tissue was isolated and stored in liquid nitrogen at -80°C to quantify dopamine. (Figures 9 and 10)

[0225] 9-1: Effect of oral administration of KARC on acetaldehyde reduction * The effect of oral administration of KARC on total acetaldehyde reduction 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. 80 μl of blank working reagent (75 μl assay buffer, 8 μl NAD / MTT, 1 μl enzyme B) was dispensed into the remaining well. After dispensing, the plate was gently mixed and incubated at room temperature for 30 minutes. Upon completion of the reaction, absorbance was measured at 565 nm (520-600 nm).

[0226] Acetaldehyde concentrations reached a maximum 1 hour after ethanol administration and tended to decrease in the KARC composition-treated group. In the KARC-treated group, acetaldehyde concentrations were significantly reduced compared to the control group (vehicle) at 1, 3, and 5 hours after ethanol administration. In the high-dose KARC treated 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).

[0227] 9-2: MDA reduction effect of oral administration of KARC Total malondialdehyde content in blood was analyzed using the OxiTec™ TBARS Assay Kit according to the manufacturer's protocol (ZeptoMetric, Buffalo, NY, USA). 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 incubated for 60 minutes in a constant-temperature water bath at 95°C. After completion of the reaction, the sample was centrifuged at 1,600 rpm for 10 minutes at 4°C and allowed to stabilize at room temperature for 30 minutes. 150 μl of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 530-540 nm.

[0228] In the control group (vehicle), blood MDA concentrations reached a maximum 3 hours after ethanol administration, whereas in the KARC-treated group, they reached a maximum 1 hour after ethanol administration. Blood MDA concentrations decreased and showed significant differences from the control group 3 and 5 hours after ethanol administration. Blood MDA concentrations in the high-dose KARC group (F) were 0.232 and 0.137 μM, respectively, representing decreases of 80.4% and 86.3% compared to the control group (Figure 8).

[0229] These results indicated that oral administration of KARC was effective in reducing various endogenous aldehydes, such as acetaldehyde and malondialdehyde, in the blood.

[0230] 9-3: Effect of oral administration of KARC on reduction of DOPAL To assess the effect of oral administration of KARC on the reduction of dopamine-derived DOPAL, the DOPAL content in rat striatum-nigra samples was measured by HPLC / MMS. Samples were dissolved in trichloroacetic acid (3.0 M / 100 μl), and isoproterenol (1 nmol / ml, 100 μl) was added. The samples were then centrifuged using a Toyopack SP carton (Toso, Tokyo, Japan).

[0231] To dissolve the adsorbed amine compounds, 0.6 M KCl-acetonitrile (1:1, 2 ml) was added, and the DPE reagent was added to the solution to induce fluorescence. The resulting final solution was injected into an HPLC to measure dopamine.

[0232] To measure the DOPAC and HVA contents in the samples, the samples were dissolved in HClO (300 μl), homogenized, and centrifuged (50,000 g, 4°C, 15 min) to obtain the supernatant. The supernatant was filtered, and the DOPAC and HVA contents were measured by HPLC / MMS. The DOPAC content was calculated as ng / g tissue.

[0233] To investigate changes in dopamine metabolism in the brains of PD model animals, rotenone, DA, DOPAL, DOPAC, and HVA were measured using HPLC [Figure 9].

[0234] The brain levels of DA, DOPAL, DOPAC, and HVA in the control group were 1542, 22, 620, and 970 ng / g tissue weight, respectively. In the rotenone-induced PD group, the levels of DA, DOPAC, and HVA decreased by 1021, 234, and 102 ng / g tissue weight, respectively, compared with the control group. However, the DOPAL level increased by 70 ng / g tissue weight.

[0235] In the group treated with the reference drug L-Dopa, brain DA and DOPAL levels increased by 1816 and 96 ng / g tissue weight, respectively, compared with the control group, whereas DOPAC and HVA levels decreased by 281 and 126 ng / g tissue weight, respectively, similar results to those observed in the rotenone-treated group.

[0236] On the other hand, in the Parkinson's disease-induced model, the brain tissue concentrations of DA, DOPAL, DOPAC, and HVA were 1290, 21, 510, and 790 ng / g tissue weight, respectively. DA, DOPAC, and HVA were increased compared with the rotenone-treated group, while DOPAL was relatively decreased.

[0237] In particular, in the group given KARC pre-administration for preventive purposes, DA and DOPAL increased by 1522 and 18 ng / g tissue weight, respectively, compared with the control group, and DOPAC and HVA also increased by 590 and 860 ng / g tissue weight, resulting in overall levels of DA, DOPAL, DOPAC, and HVA that were almost consistent with those in the control group.

[0238] The ratio of dopamine metabolism index ((DOPAC + HVA) / DA) was used to indirectly examine the amount of DOPAL that remains unmetabolized during DA metabolism, since DA is metabolized via DOPAL and DOPAC and ultimately to HVA [Figure 10].

[0239] The dopamine conversion index [(DOPAC+HVA) / DA] was calculated and found to be 103.1% in the control group, 100.8% in the pre-KARC group, and 95.3% in the post-KARC group, which means that dopamine metabolism in vivo was progressing well in the three groups.

[0240] On the other hand, compared with the three groups, there was a 32.9% decrease in the rotenone group and a 22.4% decrease in the L-dopa group, which means that dopamine metabolic dysfunction was abnormally caused by Parkinson's disease.

[0241] This means that the metabolic intermediate DOPAL accumulates in vivo. KARC administration inhibits the accumulation of the neurotoxin DOPAL, restores normal dopamine metabolism, and accelerates the increase in DOPAC and HVA, which are relatively less toxic than DOPAL. Consequently, KARC has the effect of preventing and treating PD by restoring DA metabolic function.

[0242] [Example 10] Effect of reducing oxidative stress. Drinking alcohol increases reactive oxygen species or oxidative stress due to excess acetaldehyde (Ach) produced by alcohol dehydrogenase (ADH). Aldehyde dehydrogenase (ALDH) converts acetaldehyde to acetic acid and excretes it from the body. Excess aldehydes, caused by aldehyde dehydrogenase gene mutations or excessive alcohol consumption, can lead to lipid peroxidation.

[0243] The resulting acetaldehyde and malondialdehyde exacerbate oxidative stress and disrupt mitochondrial energy metabolism, inducing endoplasmic reticulum stress through the accumulation of unfolded proteins in cells, leading to cell death.

[0244] Blood acetaldehyde concentrations were measured over time following alcohol consumption [Figure 11]. The area under the curve (AUC) for blood acetaldehyde (Ach) was 13.02 ± 1.18 mg h / dL following alcohol consumption alone. When a 10 unit / kg KARC dose was administered, the area under the curve (AUC) for blood acetaldehyde (Ach) was measured at 9.39 ± 1.07 mg h / dL, a significant decrease of 26.13% compared to alcohol consumption alone (P = 0.005).

[0245] At a dose of 20 units / kg, the AUC of blood acetaldehyde (Ach) was significantly reduced by 55.71% compared with alcohol consumption alone, measuring 5.22 ± 0.99 mg·h / dL (P < 0.001). When comparing the KARC 10 units / kg and KARC 20 units / kg groups, blood acetaldehyde (Ach) was significantly reduced in the KARC 20 units / kg group (P = 0.034). KARC demonstrated a dose-dependent reduction in total blood acetaldehyde (Ach) over time.

[0246] The reduction in blood acetaldehyde (Ach) levels resulting from KARC administration has a positive impact on reducing oxidative stress and promoting health.

[0247] The blood malondialdehyde (MDA) concentration was measured during chemotherapy (Figure 12). The blood MDA concentration in the control group was 0.607 ± 0.161 μM. The group treated with KARC showed a significant 63.3% reduction in blood MDA concentration compared to the control group, measuring 0.223 ± 0.033 μM (P < 0.001).

[0248] In the control group, blood MDA concentrations ranged from 0.427 μM to 0.885 μM, with substantial variability. In the KARC-treated group, this range was significantly reduced, with values ​​ranging from 0.158 μM to 0.269 μM. This confirmed the effect of not only reducing but also stabilizing blood MDA concentrations, as demonstrated in Figure 13.

[0249] Various factors, such as drug intake, stress, and strenuous physical exercise, lead to an increase in intracellular reactive oxygen species, which induce lipid peroxidation and oxidation processes in endogenous amines such as dopamine, norepinephrine, serotonin, and histamine. Reactive aldehyde compounds, including 4-hydroxynonenal (HNE), malondialdehyde (MDA), acetaldehyde (Ac), and dopamine-derived aldehydes, accumulate intracellularly and exacerbate oxidative stress.

[0250] 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 exerts toxic effects on various cells and further enhances oxidative stress.

[0251] This cumulative oxidative stress disrupts intracellular mitochondrial energy metabolism, leading to the accumulation of aldehyde intermediates in aldehyde-based sugar metabolism, including methylglyoxal (MG) and glyceraldehyde-3-phosphate (GA3P). Chain reactions involving aldehydes result in the accumulation of stable end-glycation 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 cell apoptosis in neuronal cells.

[0252] 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, reinforcing and amplifying each other, is known to lead to increased endoplasmic reticulum stress (ER stress).

[0253] KARC administration effectively modulated malondialdehyde, a marker of reactive oxygen species and oxidative stress, demonstrating its potential to reduce oxidative stress and improve endoplasmic reticulum (ER) stress stability. KARC significantly reduced malondialdehyde levels in the bloodstream, demonstrating its ability to reduce reactive oxygen species and oxidative stress.

[0254] By reducing the levels of acetaldehyde and malondialdehyde in human blood, KARC demonstrated its potential to prevent and cure ER stress through the reduction of reactive oxygen species and oxidative stress. This suggests that by regulating intracellular reactive oxygen species and oxidative stress, KARC inhibits neuronal apoptosis, thereby suppressing and preventing Parkinson's disease. This leads to improvements in behavior and motor function.

[0255] [Example 11] Acute oral administration test 11-1. Preparation of experimental animals The experimental animals were female and male ICR mice (7 weeks old). The received ICR mice were acclimated for 7 days. The general symptoms of the adopted mice were observed during the acclimation period, and only healthy animals were used for the short-term administration toxicity test. Food and water were available ad libitum. Based on the average body weight of approximately 20 g on the day before oral administration, the groups were divided into 10 groups, 5 mice per group, and 5 mice per group.

[0256] 11-2. Administration of test substance The test substances were prepared by dissolving the mutant yeast lysate KARC of the present invention in physiological saline so that the doses administered to the 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.

[0257] The dosage standards for administration were in accordance with the Korean National Toxicology Program (KNTP) Toxicity Test Manual of the Ministry of Food and Drug Safety. The maximum applicable dose of 5,000 mg / kg as recommended by the KNTP manual was set as the maximum concentration for this experiment. The samples prepared for each group were orally administered once to each test animal. Normal group (G1) was administered saline.

[0258] 11-3. Observation and autopsy All animals in the test groups were observed for symptoms at least once a day from the day of acquisition until the day of necropsy. Symptoms were observed for 7 days after oral administration. After observing the symptoms of the rats, necropsies were performed. During the necropsy, changes in each organ were observed macroscopically.

[0259] A single-dose toxicity test of the ALDH-containing KARC composition of the present invention was conducted using mice. As a result, no mouse deaths were observed for 7 days at concentrations of up to 5,000 mg / kg of mutant yeast KARC. No abnormal characteristics such as changes in weight gain or food intake were found in the mice. No abnormal findings were found in the autopsy performed after the end of the observation period.

[0260] [Example 12] Observation of in vitro metabolism of various aldehydes by KARC The present invention confirmed the effectiveness of KARC in reducing exogenous and endogenous aldehydes. KARC (300 mg / ml) was reacted with various aldehydes (1 mM) at 37°C for 3 hours, resulting in a 24.4% reduction in 3,4-dihydroxyphenylacetaldehyde (DOPAL), a 74.9% reduction in succinic semialdehyde (SSA), a 23.8% reduction in glyoxal, a 99.6% reduction in cinnamaldehyde, and a 97.1% reduction in benzaldehyde. In the case of acetaldehyde, a 100.0% reduction was observed even after 1 hour of reaction at 30°C. [Figures 26-53]

[0261] 12-1: Reaction of KARC with various aldehydes Potassium chloride (KCl) was dissolved in 50 mM HEPES buffer (pH 7.5) to 200 mM. For experiments using acetaldehyde, glyoxal, DOPAL, cinnamaldehyde, and benzaldehyde, 935 μl of buffer, 15 μl of 100 mM EDTA in water, 30 μl of 100 mM NADP+ in water, 10 μl of 100 mM aldehyde in demineralized water (DW) or acetonitrile, and 10 μl of 300 mg / mL KARC were dispensed into a microtube. For negative controls, 935 μl of buffer, 15 μl of 100 mM EDTA in water, 30 μl of 100 mM NADP+ in water, 10 μl of 100 mM aldehyde in DW or acetonitrile, and 10 μl of DW were dispensed into a microtube.

[0262] For the SSA experiment, 845 μl of buffer solution, 15 μl of 100 mM EDTA solution, 30 μl of 100 mM NADP+ solution, 10 μl of 10 mM SSA in acetonitrile, and 10 μl of 300 mg / mL KARC were dispensed into a microtube. For the negative control, 845 μl of buffer solution, 15 μl of 100 mM EDTA solution, 30 μl of 100 mM NADP+ solution, 100 μl of 10 mM SSA in acetonitrile, and 10 μl of DW were dispensed into a microtube.

[0263] The reactions were shaken using a thermoshaker at 30°C or 37°C for 1 or 3 hours.

[0264] 12-2: Pretreatment before HPLC analysis For experiments using representative aliphatic aldehydes: SSA, acetaldehyde, and glyoxal, 500 μl of each reaction was dispensed 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 6 N HCl were further 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 6 N HCl were added and heated at 70°C for 40 minutes. After cooling, 10 μl of the heated solution was quantified and injected into the HPLC for analysis.

[0265] For experiments with DOPAL, cinnamaldehyde, and benzaldehyde, which represent aromatic aldehydes, 10 μl of the solution was reacted with KARC without heating with DNPH or DHBA, aliquoted, and injected into the HPLC for analysis.

[0266] 12-3: HPLC analysis An HPLC system (Waters Alliance 2690 / 2695 HPLC with a Waters 2996 PDA detector) was used for the analysis. The analytical column was a 150 mm ∘ 4.6 mm id (Shimadzu Corporation, Kyoto, Japan) packed with C18 with a particle size of 5 μm.

[0267] The gradient started with 80% water (1% v / v trifluoroacetic acid) and progressed to 20% after 15 minutes using reverse phase. Absorbance was analyzed at wavelengths of 254, 310, or 360 nm using a UV detector.

[0268] The results were confirmed by the progress of the reaction in which aldehyde was consumed via the reduction of DNPH-aldehyde conjugate or DHBA-aldehyde conjugate in the experimental group compared to the negative control group.

[0269] [Example 13] Preparation of food and pharmaceutical compositions for preventing and reversing auto-brewing symptoms by decomposing endogenous ethanol in vivo. Food and pharmaceutical compositions containing KARC as the active ingredient for suppressing auto-brewing symptoms were prepared. It is possible to prepare food or pharmaceutical compositions containing KARC powder with various composition ratios. For example, the powder composition of the present invention has the function of suppressing auto-brewing symptoms and oxidative stress through oral administration of 13 g of the composition twice daily. The weight ratios of the ingredients and phases of food or pharmaceutical compositions containing the powder composition are shown in Table 7.

[0270] [Table 7] The food and pharmaceutical compositions contain KARC dry powder, excipients, and natural sweeteners such as fructooligosaccharides, enzyme-treated stevia (stevia), citric acid anhydrous, isomaltodextrin (isomalt), xylitol, citrus juice powder, and citrus flavor powder. The processing and testing of the raw materials and final products of the food or pharmaceutical compositions were conducted in accordance with the general testing methods described in the Korean Food Code and the Health Functional Food Act. KARC-containing food or pharmaceutical compositions exhibit the effects of decomposing endogenous aldehydes and suppressing autogenous fermentation symptoms and oxidative stress. KARC-containing food or pharmaceutical compositions can prevent or improve irritable bowel syndrome.

[0271] The above examples provide a detailed description of the mutant yeast composition KARC containing aldehyde dehydrogenase, including its manufacturing method, pharmacological effects, administration methods, therapeutically effective doses for disease models, acute toxicity after short-term administration, and representative examples of food or pharmaceutical compositions. The effectiveness of KARC is described in detail in the above examples, but these are merely examples of the present invention.

[0272] 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.

[0273] Any food or therapeutic product containing a modified form of aldehyde dehydrogenase that embodies the technical idea of ​​the present invention as set forth in the claims is also within the scope of legal protection of the present invention.

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Claims

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

1.

2. A composition for promoting the degradation of endogenous aldehydes according to claim 1, characterized in that it contains an aldehyde dehydrogenase enzyme encoded by the gene of SEQ ID NO: 1, which includes SEQ ID NO:

2.

3. The composition for promoting the decomposition of endogenous aldehydes according to claim 1 or 2, wherein the endogenous aldehyde is an endogenous aldehyde produced by oxidation of an alcohol or an endogenous amine compound.

4. 4. The composition for promoting the degradation 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. 4. The composition for promoting the degradation 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-nonena, malondialdehyde (MDA), propionaldehyde, hexanal, palmitic aldehyde, succinic aldehyde, and acrylaldehyde.

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

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

8. A food composition for suppressing oxidative stress, comprising any one or a mixture of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP.

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

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

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

12. Mutant Saccharomyces cerevisiae KCTC14983BP.

13. Mutant Saccharomyces cerevisiae KCTC14984BP.

14. Mutant Saccharomyces cerevisiae KCTC14985BP.

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