Food and pharmaceutical compositions for inhibiting tremor or dyskinesia comprising novel aldehyde dehydrogenase

Abstract

The present invention relates to food and pharmaceutical compositions for inhibiting tremor or dyskinesia comprising a novel aldehyde dehydrogenase encoded by a gene having more than 98% homology with the gene of SEQ ID NO: 1. Furthermore, the present invention relates to a food composition and a pharmaceutical composition for inhibiting tremor or dyskinesia, said composition comprising a lysate of any one selected from the group consisting of KCTC 13925BP, KCTC 14122BP, KCTC 14123BP, KCTC 14983BP, KCTC 14984BP and KCTC 14985BP, or a mixture thereof.

Description

Technical Field

[0001] The present invention relates to a food and pharmaceutical composition for suppressing tremor or movement disorder, comprising a novel 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 a food and pharmaceutical composition comprising an aldehyde dehydrogenase encoded by the gene of SEQ ID NO: 1 including SEQ ID NO: 2.

[0002] In addition, the present invention relates to a food composition and a pharmaceutical composition for suppressing tremor or movement disorder, wherein the composition comprises a lysate of any one selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof. Background Art

[0003] Tremor or movement disorder (hereafter referred to as tremor) is a symptom that occurs not only in the hands, but also in the head, neck, jaw, tongue and voice. Although these tremor symptoms are rarely diagnosed, they can occur throughout the body, including the legs and feet.

[0004] Tremor symptoms are divided into essential tremor, rest tremor, Parkinson's disease tremor, psychogenic tremor, cerebellar tremor, intention tremor, ethanol withdrawal tremor and orthostatic tremor.

[0005] The cause of essential tremor has not been determined. More than 50% of people with essential tremor have a family history. Essential tremor occurs in all age groups. Essential tremor that occurs in people over 65 is called presbycusis.

[0006] Resting tremor and Parkinsonian tremor occur in people with Parkinson's disease. This tremor occurs mainly when the person is at rest and weakens or disappears when the person performs a desired movement.

[0007] Factitious or psychogenic tremor, also called hysterical tremor, may disappear when the person's attention is diverted away from the area where the tremor occurs.

[0008] When abnormal movements persist or when detailed motor control is required, a temporary, irregular tremor may occur. This is called a cerebellar tremor or intention tremor. This type of tremor symptom is usually caused by abnormalities in the cerebellum or abnormalities in the connections to the cerebellum. People with cerebellar dysfunction experience abnormal movement symptoms (for example, ataxia and difficulty controlling movement).

[0009] When the amplitude of the tremor increases for some reason (for example, anxiety, tension, stage fright, or an important game), the tremor observed with the naked eye is called enhanced physiologic tremor.

[0010] In addition to these tremor symptoms, there are also ethanol withdrawal tremors and orthostatic tremors.

[0011] In addition, acetaldehyde causes hangovers caused by excessive drinking and can also cause symptoms such as tremors or movement disorders. During intense exercise (e.g., football or basketball), malondialdehyde levels in athletes increase rapidly, causing symptoms such as tremors or movement disorders (e.g., painful muscle cramps).

[0012] Generally speaking, hand tremor refers to the presence of tremor symptoms (e.g., hand tremors). These hand tremors include resting tremor, postural tremor, action tremor, task-specific tremor, and intention tremor.

[0013] Hand tremor is one of the most common movement disorders caused by abnormal neurological function. Approximately 1% of the global population experiences hand tremor, and its incidence increases with age.

[0014] The main symptom of tremor is activity tremor, which occurs during voluntary activities. This type of tremor develops gradually with age and eventually makes daily activities, such as eating and driving, difficult, causing problems that affect quality of life.

[0015] In addition to a basic neurological examination, several methods are used to examine patients with tremor and objectively assess their symptoms. For academic purposes, the Fahn-Tolosa-Marin (FTM) scale is widely used. Disturbance in daily life is also assessed.

[0016] Motor tremors can be objectively assessed by having the patient draw an Archimedean spiral or by observing the patient's handwriting and handwriting style. Tremor symptoms can be objectively measured using accelerometers. In most patients, detailed visual observation of tremor symptoms is important for diagnosis.

[0017] The term "tremor" in this specification is intended to include all of the various tremor or movement disorder symptoms listed above.

[0018] When treating essential tremor (a tremor), medications are used to improve symptoms. The most widely used medications are propranolol and primidone. Propranolol is a non-selective beta-adrenergic antagonist, and primidone is a drug developed as an anti-epileptic drug. They have a suppressive effect on essential tremor.

[0019] When primidone is used in combination with propranolol, it has shown superior therapeutic efficacy compared to monotherapy. Clonazepam is a tremor treatment agent that is indicated for patients with diabetes and asthma, or for those who have difficulty taking primidone.

[0020] Essential tremor is a common symptom that can be improved with treatment. However, many patients do not receive treatment for essential tremor because they believe it is a symptom caused by aging. Because essential tremor can have a variety of causes, it is important to accurately diagnose the cause of the tremor.

[0021] The exact cause of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD), including essential tremor, has not yet been accurately determined. However, recent studies suggest that the main factor underlying neurodegenerative diseases is increased oxidative stress in nerve cells.

[0022] Oxidative stress is caused by the accumulation of reactive aldehydes within cells, including endogenous aldehydes (e.g., succinic semialdehyde (SSA) and glutamate semialdehyde (GSA) derived from glutamine) or acetaldehyde produced from ethanol.

[0023] These reactive aldehydes increase oxidative stress and trigger inflammation by binding to key proteins, DNA, and lipids in the body. Oxidative stress and chronic inflammation in the body accelerate aging. As people age, they may develop neurodegenerative diseases that cause tremors or abnormal movements.

[0024] There are approximately 19 types of ALDH (aldehyde dehydrogenase) in the human body that can break down these endogenous aldehydes. Among these ALDHs, mitochondrial ALDH2 plays an important role in the degradation of endogenous aldehydes.

[0025] ALDH is responsible for removing these endogenous aldehydes and protecting cells from oxidative stress. By acting as a free radical scavenger, ALDH stabilizes cells and prevents the accumulation of reactive aldehydes in the body. Thus, ALDH prevents neuronal death and inhibits the development of tremors or abnormal movements caused by neurodegeneration.

[0026] *Succinic semialdehyde (SSA), derived from endogenous acetaldehyde, malondialdehyde, and the neurotransmitter gamma-aminobutyric acid (GABA), has been implicated as a cause of tremor symptoms. These substances are the cause of the diseases that the food and pharmaceutical compositions of the present invention are intended to prevent or treat.

[0027] GABA is produced in the body from its precursor, glutamate. It is converted into GABA aldehyde (succinic semialdehyde) by the action of MAOB (monoamine oxidase B). GABA aldehyde (succinic semialdehyde, hereinafter referred to as SSA) is oxidized to succinic acid in the human body through the action of ALDH.

[0028] At the same time, the tremor symptoms that occur during spastic paraplegia are related to the glutamate metabolite glutamyl semialdehyde (GSA, glutamate-5-semialdehyde).

[0029] When aldehyde dehydrogenase (ALDH) expression is reduced or mutated in humans, the breakdown of acetaldehyde, malondialdehyde, glutamate semialdehyde (GSA), and succinate semialdehyde (SSA) in the body is delayed. Consequently, oxidative stress increases in the body and causes motor neurological abnormalities, leading to symptoms such as tremors or movement disorders.

[0030] [Prior art literature]

[0031] [Patent Document]

[0032] U.S. Patent Application Publication No. US2021-0254023-A1, dated August 21, 2021

[0033] [Non-patent literature]

[0034] 1. Haubenberger, D., & Hallett, M. (2018). Essential Tremor. New England Journal of Medicine, 378(19), 1802-1810. doi: 10.1056 / nejmcp1707928

[0035] 2. Louis, ED (2005). Essential tremor. The Lancet Neurology, 4(2), 100-110. doi: 10.1016 / s1474-4422(05)00991-9

[0036] 3. Abboud, H.; Ahmed, A.; Femandez, HH, Essential tremor: choosing the right management plan for your patient. Cleve Clin J Med 2011, 78, (12), 821-8.

[0037] 4.Vilarino-Gell,C.;Ross,O.A.;Wider,C.;Jasinska-Myga,B.;Cobb,S.A.;Soto-Ortolaza.A.I.;Kachergus,J.M.;Keeling,B.H.;Dachsel,J.C.;Melrose,H.L.,LINGO 1rs9652490 is associated with essential tremor and Parkinsondisease.Parkinsonism&related disorders 2010,16,(2),109-111.

[0038] 5.Louis,E.D.;Ferreira,J.J.,How common is the most common adultmovement disorder Update on the worldwide prevalence of essentialtremor.Movement Disorders 2010,25,(5),534-541.

[0039] 6.Lorenz,D.;Poremba,C.;Papengut,F.;Schreiber.S.;Deuschl,G.,The psy-chosocial burden of essential tremor in an outpatient and a community-basedcohort.European Journal of Neurology 2011,18,(7),972-979.

[0040] 7.Dogu,O.;Louis,E.D.;Sevim,S.;Kaleagasi,H.;Aral,M.,Clinicalcharacteristics of essential tremor in Mersin,Turkey:A Population-based door-to-door study.Journal of neurology 2005,252,570-574.

[0041] 8.Louis,E.D.;Barnes,L.;Albert,S.M.;Cote,L.;Schneier,F.R.;Pullman,S.L.;Yu,Q.,Correlates of functional disability in essential tremor.Mov Disord2001,16,(5),914-20.

[0042] 9.Fahn,S.;Tolosa,E.;Marln,C.,Clinical rating scale fortremor.Parkinson′s disease and movement disorders 1993,2,271-280.

[0043] 10.Deuschl,G.;Raethjen,J.;Hellriegel,H.;Elble,R.,Treatment ofpatients with essential tremor.Lancet Neurol 2011,10,(2),148-61.

[0044] 11.Zesiewicz,T.A.;Elble,R.J.;Louis,E.D.;Gronseth,G.S.;Ondo,W.G.;Dewey,R.B.,Jr.;Okun.M.S.;Sullivan,K.L.;Weiner,W.J.,Evidence-based guidelineupdate:treatment of essential tremor:report of the Quality Standards sub-committee of the American Academy of Neurology.Neurology 2011,77,(19),1752-5.

[0045] 12.Young,R.R.;Growdon,J.H.;Shahani,B.T.,Beta-adrenergic mcchanisms inaction tremor.N Engl J Med 1975,293,(19),950-3.

[0046] 13.Heo,J.Y.;Nam,M.H.;Yoon,H.H.;Kim,J.;Hwang,Y.J.;Won,W.;Woo,D.H.;Lee,J.A.;Park,H.J.;Jo,S.;Lee,M.J.;Kim,S.;Shim,J.E.;Jang,D.P.;Kim,K.I.;Huh,S.H.;Jeong,J.Y.;Kowall,N.W.;Lee,J.;Im,H.;Park,J.H.;Jang,B.K.;Park,K.D.;Lee,H.J.;Shin,H.;Cho,I.J.;Hwang,E.M.;Kim,Y.;Kim,H.Y.;Oh,S.J.;Lee,S.E.;Paek,S.H.;Yoon,J.H.;Jin,B.K.;Kweon,G.R.;Shim,I.;Hwang,O.;Ryu,H.;Jeon,S.R.;Lee,C.J.,AbcrrantTonic Inhibition of Dopaminergic Neuronal Activity Causes Motor Symptoms inAnimal Models of Parkinson′s Disease.Curr Biol 2020,30,(2),276-291e9.

[0047] 14.Raethjen,J.;Deuschl,G.,Tremor.Curr Opin Neurol 2009,22,(4),400-5.

[0048] 15.Abboud,H.;Ahmed,A.;Fernandez,H.H.,Essential tremor:choosing theright management plan for your patient.Cleve Clin J Med 2011,78,(12),821-8.

[0049] 16.Jo,S.;Yarishkin,O.;Hwang,Y.J.;Chun,Y.E.;Park,M.;Woo,D.H.;Bae,J.Y.;Kim,T.;Lee,J.;Chun,H.;Park,H.J.;Lee,D.Y.;Hong,J.;Kim,H.Y.;Oh,S.J.;Park,S.J.;Lee,H.;Yoon,B.E.;Kim,Y.;Jeong,Y.;Shim,I.;Bae,Y.C.;Cho,J.;Kowall,N.W.;Ryu,H.;Hwang,E.;Kim,D.;Lee,C.J.,GABA from reactive as-trocytes impairs memory inmouse models of Alzheimer's disease.Nat Med 2014,20,(8),886-96.

[0050] 17.Nam,M.H.;Cho,J.;Kwon,D.H.;Park,J.Y.;Woo,J.;Lee,J.M.;Lee,S.;Ko,H.Y.;Won,W.;Kim,R.G.;Song,H.;Oh.S.J.;Choi,J.W.;Park,K.D.;Park,E.K.;Jung,H.;Kim,H.S.;Lee,M.C.;Yun,M.;Lee,C.J.;Kim,H.I.,Excessive Astrocytic GABA CausesCortical Hypometabolism and Impedes Functional Recovery after Sub-corticalStroke.Cell Rep 2020,32,(3),107975.

[0051] 18. Woo, J.; Min, J. O.; Kang, D. S.; Kim, Y. S.; Jung, G. H.; Park, H. J.; Kim, S.; An, H.; Kwon, J.; Kim, J.; Shim, I.; Kim, H. G.; Lee, C. J.; Yoon, B. E., Control of motor coordination by astrocytic tonic GABA release through modulation of ex - citation / inhibition balance in cerebellum. Proc Natl Acad Sci U S A 2018, 115, (19), 5004 - 5009.

[0052] 19. Ishibashi, M.; Egawa, K.; Fukuda, A., Diverse Actions of Astrocytes in GABAergic Signaling. Int J Mol Sci 2019, 20, (12).

[0053] 20. Yoon, B. E.; Lee, C. J., GABA as a rising gliotransmitter. Front Neural Circuits 2014, 8, 141.

[0054] 21. Edenberg, H. J., The genetics of alcohol metabolism: role of alcohol dehydrogenase and aldehyde dehydrogenase variants. Alcohol Res Health 2007, 30, (1), 5 - 13.

[0055] 23. Keung, W. M.; Vallee, B. L., Daidzin and its antidipsotropic analogs inhibit serotonin and dopamine metabolism in isolated mitochondria. Proc Natl Acad Sci USA 1998, 95, (5), 2198 - 203. Summary of the Invention Technical Problem

[0056] Through the above-mentioned various previous studies, it was found that as acetaldehyde, malondialdehyde, glutamate semialdehyde (GSA), or succinate semialdehyde (SSA) increases in the body, symptoms of tremor or movement disorder appear.

[0057] Therefore, the main object of the present invention is to provide food compositions and pharmaceutical compositions comprising aldehyde dehydrogenase that rapidly converts glutamate semialdehyde (GSA), endogenous aldehydes derived from acetaldehyde, malondialdehyde and glutamate, and succinate semialdehyde (SSA) into acidic compounds.

[0058] That is, an object of the present invention is to provide a food composition or a pharmaceutical composition that suppresses various tremor symptoms occurring due to abnormalities in the oxidation and excretion processes of acetaldehyde, malondialdehyde, succinic semialdehyde (SSA), and glutamate semialdehyde (GSA) caused by incomplete function of acetaldehyde dehydrogenase (ALDH) in the human body.

[0059] Another object of the present invention is to provide a food composition or a pharmaceutical composition for suppressing tremor, the food composition or the pharmaceutical composition comprising aldehyde dehydrogenase (ALDH) encoded by the gene of SEQ ID NO: 1 including SEQ ID NO: 2.

[0060] Another object of the present invention is to provide a food composition or a pharmaceutical composition for suppressing tremor, comprising a dry powder of a lysate of any one selected from the group consisting of KCTC 13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof (hereinafter abbreviated as KARC).

[0061] Solution to the problem

[0062] The main object of the present invention as described above can be achieved by providing a food composition and a pharmaceutical composition comprising an aldehyde dehydrogenase contained in a lysate of any one selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

[0063] The object of the present invention as described above can be achieved by providing a food composition and a pharmaceutical composition, which can convert acetaldehyde, succinic semialdehyde (SSA) and glutamate semialdehyde (GSA) into acidic compounds, and the composition comprises an aldehyde dehydrogenase contained in the lysate of any one selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

[0064] Advantageous Effects of the Invention

[0065] The composition of the present invention comprising KARC exhibits an effect of promoting the dehydrogenation reaction of succinic semialdehyde (SSA), glutamic acid 5-semialdehyde (GSA), acetaldehyde or malondialdehyde, which are suspected to be the cause of tremor symptoms including hand tremor or abnormal movement symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] [ Figure 1 ]and[ Figure 2 ] is a chemical formula showing the production and decomposition process of endogenous aldehydes in the body.

[0067] Ethanol or ethanol derivatives (2-substituted alcohols, R-CH2CH2-OH) are reversibly converted into acetaldehyde derivatives (R-CH2-CHO) in the body by alcohol dehydrogenase (ADH). Acetaldehyde derivatives (highly toxic substances) are irreversibly converted into relatively non-toxic acetic acid derivatives (R-CH2-CO2H).

[0068] Endogenous monoamines (R-CH2CH2-NH2) are converted to aldehydes (R-CH2-CHO) by the enzyme monoamine oxidase (MAO), which are then detoxified to acetic acid (R-CH2-CO2H) through the reactions of aldehyde dehydrogenase and alcohol dehydrogenase. This is the same process as alcohol metabolism.

[0069] [ Figure 3 ] shows the breakdown of GABA in vivo.

[0070] The monoamine neurotransmitter GABA is oxidized by the enzyme monoamine oxidase (MAO) and converted into the endogenous aldehyde, succinic semialdehyde (SSA), which binds to and denatures surrounding proteins. As a result, the accumulation of denatured proteins in the endoplasmic reticulum acts as a cytotoxic agent, inducing cell death. Figure 3 ].

[0071] [ Figure 4 ] is a graph showing the ability of KARC of the present invention to decompose endogenous acetaldehyde. These results indicate that the composition of the present invention decomposes endogenous acetaldehyde and suppresses self-brewing symptoms.

[0072] [ Figure 5 ] is a graph showing the malondialdehyde decomposition ability of KARC.

[0073] In animal studies, ethanol intake increases the endogenous toxic aldehyde, malondialdehyde [ Figure 5 ].

[0074] [ Figure 7 ] is a graph showing the ability of KARC of the present invention to decompose acetaldehyde in the human body.

[0075] [ Figure 8 ] is a graph showing the ability of KARC to break down malondialdehyde in the human body.

[0076] In tests, in animals [ Figure 4 、 Figure 5 、 Figure 6 ] and in humans [ Figure 7 、 Figure 8 ] demonstrated a reduction in endogenous blood acetaldehyde and malondialdehyde, suggesting that tremor symptoms (including hand tremors) or movement disorders may be suppressed by reducing oxidative stress induced by alcohol consumption and fatigue.[ Figure 4 、 Figure 5 、 Figure 6 、 Figure 7 ].

[0077] exist[ Figure 8 ], under conditions where oxidative stress was increased due to medication, KARC administration reduced malondialdehyde, a biomarker of oxidative stress and reactive oxygen species. This confirms the role of KARC in alleviating tremor symptoms by regulating oxidative stress.

[0078] [ Figure 9 ] shows changes in enzyme activity when the KwonP-1 strain contained in the KARC of the present invention is orally administered.

[0079] [ Figure 10 ] shows changes in enzyme activity when the KwonP-2 strain contained in the KARC of the present invention is orally administered.

[0080] [ Figure 11 ] shows changes in enzyme activity when the KwonP-3 strain contained in the KARC of the present invention is orally administered.

[0081] [ Figure 12 ] shows changes in enzyme activity when the PicoYP strain contained in the KARC of the present invention is orally administered.

[0082] [ Figure 13 ] shows changes in enzyme activity when the PicoYP-01 strain contained in the KARC of the present invention is orally administered.

[0083] Figure 14 Shows the change in enzyme activity when the PicoYP-02 strain contained in the KARC of the present invention is orally administered.

[0084] Figure 9 、 Figure 10 、 Figure 11 、 Figure 12 、 Figure 13 、 Figure 14 Show that KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02 are orally administered for 90 minutes under conditions similar to the digestive process of the human stomach (1 < pH < 5). The change in ALDH enzyme activity is measured.

[0085] At pH = 5 (similar to the conditions observed during food intake), the ALDH enzyme activity remains at a minimum of 37.29 units / g and a maximum of 52.24%. It is confirmed that the enzyme activity is maintained when KARC is orally administered.

[0086] Figure 15 Shows the growth curve and enzyme activity of the KwonP-1 strain cultured in a 5 L fermenter.

[0087] Figure 16 Shows the growth curve and enzyme activity of the KwonP-2 strain cultured in a 5 L fermenter.

[0088] Figure 17 Shows the growth curve and enzyme activity of the KwonP-3 strain cultured in a 5 L fermenter.

[0089] Figure 18 Shows the growth curve and enzyme activity of the PicoYP strain cultured in a 5 L fermenter.

[0090] Figure 19 Shows the growth curve and enzyme activity of the PicoYP-01 strain cultured in a 5 L fermenter.

[0091] * Figure 20 Shows the growth curve and enzyme activity of the PicoYP-02 strain cultured in a 5 L fermenter.

[0092] At Figure 15 、 Figure 16 、 Figure 17 、 Figure 18 、 Figure 19 、 Figure 20 ​​​​​​​], the new mutants KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-01 were cultured in 5 L fermentors using YPD medium under the same conditions at 30°C and 200 rpm for 48 hours.

[0093] When the growth curves (OD660nm) and ALDH enzyme activities of each strain were compared with those of the model strain, the ALDH enzyme activities were at least 10.5 times and at most 18.75 times higher. PicoYP-01 had the highest ALDH activity at 52.68 units / g, and KwonP-3 had the lowest value at 29.5 units / g.

[0094] [ Figure 21 ] is the HPLC spectrum of a mixture of distilled water and SSA.

[0095] [ Figure 22 ] is an HPLC spectrum of a mixture of KARC and SSA of the present invention kept at 37°C for 1 hour.

[0096] [ Figure 23 ] is an HPLC spectrum of a mixture of KARC and SSA of the present invention kept at 37°C for 3 hours.

[0097] exist[ Figure 21 、 Figure 22 、 Figure 23 In the study, when KARC was treated at 37°C, it decreased by 55% within 1 hour and by 74.9% within 3 hours. KARC oxidizes SSA, a metabolite of GABA. DETAILED DESCRIPTION

[0098] *Hereinafter, the method for producing the KARC dry powder of the present invention, i.e., the lysate of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, KCTC14985BP, will be described in more detail.

[0099] These examples are only for the purpose of illustrating compositions that can achieve the objects of the present invention, and therefore, the scope of the present invention is not limited to the compositions described in the following examples.

[0100] Example

[0101] [Example 1] Screening of wild yeast parent strains for mutation.

[0102] In the present invention, each Makgeolli (Korean traditional liquor) suspension was prepared by mixing various types of Makgeolli with a 0.9% NaCl solution. The Makgeolli suspension was stirred at 200 rpm for 1 hour. The supernatant containing the wild yeast strain was diluted with YPD (yeast extract peptone dextrose broth) medium. The diluted solution was prepared to be 10% of the original solution. -6 times.

[0103] The diluted solution was spread onto YPD agar medium. The agar medium was incubated statically at 30°C under aerobic conditions for one week. Preliminary screening of Saccharomyces cerevisiae was performed based on colony morphology, growth characteristics in YPD agar, and microscopic observation.

[0104] The ALDH activity and glutathione content of the screened Saccharomyces cerevisiae were measured. Parent strains were selected based on ALDH activity and glutathione production.

[0105] 1-1: Measurement of aldehyde dehydrogenase

[0106] Acetaldehyde reacts with dinitrophenylhydrazine (DNPH) to form an acetaldehyde-hydrazone (Ach-DNPH) compound. The Ach-DNPH compound is detected by HPLC equipped with a C18 column at 360 nm. The amount of Ach-DNPH compound detected quantifies the amount of aldehyde reduced by the decomposition reaction by aldehyde dehydrogenase (ALDH).

[0107] The enzyme reaction was performed at 30°C by adding 10 μL of yeast lysate to 990 μL of a reaction mixture [50 mM potassium phosphate buffer (pH 8.0), 1.5 mM acetaldehyde, and 3 mM NADP+]. After the enzyme reaction was complete, 50 μL of 10 mM DNPH was added to induce the formation of Ach-DNPH. Ach-DNPH formation was performed at 22°C for 1 hour.

[0108] The formation of Ach-DNPH was terminated by adding 3M sodium acetate (pH 9). The formed Ach-DNPH compound was isolated by adding two volumes of acetonitrile. The isolated Ach-DNPH compound (in ACN) was analyzed by injection HPLC.

[0109] The concentration of Ach-DNPH compounds was analyzed by HPLC at a wavelength of 360 nm using a mobile phase (acetonitrile, water) developed on a C18 column at a rate of 1 mL / min. The area values ​​of the chromatograms obtained from the HPLC results were converted using a standard curve for aldehyde-DNPH (Sigma-Aldrich) to quantify the concentration of Ach-DNPH compounds. A decrease of 1 mM in Ach-DNPH concentration per minute was calculated as 1 unit of ALDH. ALDH activity was normalized to units per mg of protein.

[0110] 1-2: Glutathione measurement

[0111] Yeast cells were harvested by centrifuging 1 mL of Saccharomyces cerevisiae culture medium. A suspension was prepared by adding 1 mL of water to the harvested yeast cells. Glutathione was extracted by stirring the suspension at 85°C and 1,000 rpm for 2 hours. The suspension was centrifuged to remove the yeast cells, and the supernatant was filtered through a 0.22 μm filter to obtain a glutathione-containing sample.

[0112] The glutathione concentration in the sample was analyzed by HPLC (Shimazu LC-20AD) equipped with a C18 column. Glutathione concentration was analyzed at a wavelength of 210 nm under conditions of developing a mobile phase (2.02 g / L sodium 1-heptanesulfonate monohydrate, 6.8 g / L potassium dihydrogen phosphate, pH 3.0, methanol mixture) at a rate of 1 mL / min. The area value of the chromatogram obtained as the HPLC result was analyzed using a standard curve analysis of glutathione.

[0113] The ALDH activity and glutathione content of 200 different yeasts obtained from Korean Makgeolli were analyzed. The 10 yeasts listed in [Table 1] showed higher ALDH activity or glutathione production capacity than other yeasts.

[0114] Yeast #97 had an ALDH activity of 0.10 units / mg protein, the second highest overall. Yeast #97 also had a glutathione content of 0.42%, the highest of all strains. Yeast #97 was selected as the parent strain and subjected to the mutation induction procedure.

[0115] Table 1

[0116] [Example 2] Identification of parent strains used in the mutagenesis process

[0117] Identification was performed to confirm the exact species of the wild-type parent strain (yeast #97, wild-type yeast). To ensure sufficient yeast cells for DNA extraction, only a single yeast colony was plated on YPD agar medium. DNA was extracted using a genomic DNA preparation kit (HiGene™, BIOFACT Co., Ltd., Daejeon, South Korea) according to the manufacturer's instructions.

[0118] To amplify the rRNA gene in the yeast ITS region, polymerase chain reaction (PCR) was performed on yeast chromosomal DNA using ITS5 (forward) and ITS4 (reverse) primers. PCR results were analyzed by DNA sequencing.

[0119] The DNA sequence of the parent strain was isolated using the Bioedit program. The reverse strand of the PCR result was converted to a paired base sequence using the reverse completion process.

[0120] The forward strand sequence matched its reverse strand counterpart using the Cluster X program. Using the BLAST database provided by the National Center for Biotechnology Information (NCBI), the parent strain was identified as matching the sequence identified in the experimental procedure. The rRNA in the parent strain's ITS was found to be 100% identical to that of Saccharomyces cerevisiae.

[0121] [Example 3] Selection of mutants with improved aldehyde dehydrogenase production

[0122] The mutagenesis procedure for the wild-type S. cerevisiae parent strain was performed according to the methods described in US Patent Application No. 17 / 176,365.

[0123] To induce mutations in the yeast parent strain, wild yeast strains that produce both ALDH and glutathione were treated with ethyl methanesulfonate (EMS) or nitrosoguanidine (NGD). The induced mutant yeast strains were exposed to varying concentrations of methylglyoxal. Mutants with excellent adaptability to methylglyoxal were screened. The selected yeast strains were exposed to varying concentrations of lysine. Mutants with excellent adaptability to lysine were screened. 30 mutants with excellent adaptability to both methylglyoxal and lysine were obtained. Each of the 30 yeast strains was evaluated for five properties: growth curve, ALDH activity, ADH activity, coenzyme content, and glutathione content.

[0124] 3-1: Growth characteristics

[0125] Saccharomyces cerevisiae is a Crabtree-positive microorganism that produces ethanol while growing under aerobic conditions. Cultivating high-yield yeast requires Saccharomyces cerevisiae with high ethanol tolerance.

[0126] YPD medium with different ethanol concentrations (no ethanol, 5%, 7% and 10%) was prepared. Saccharomyces cerevisiae (yeast) medium adjusted to OD=1 at 660nm was prepared. Each mixture of the prepared YPD medium and yeast medium was diluted in a ratio of 99:1. Finally, YPD medium containing yeast with four different alcohol concentrations was prepared. Each YPD medium mixed with yeast was shaken at 30°C and 200rpm. The growth curve of the mutant strain was measured every 3 hours for 48 hours. The growth curve of each mutant strain was evaluated by three characteristics: the time (or period) of the lag phase, the specific growth rate (OD660nm / hr) in the exponential phase and the maximum density (OD660nm).

[0127] The higher the ethanol concentration in YPD medium, the longer the lag phase. The maximum density and specific growth rate decreased. Comparing the maximum density of mutant strains at low (5%) and high (10%) ethanol concentrations revealed that, for nine mutant strains, the high concentration maintained 50% growth compared to the low concentration. The nine mutant strains are distinguished from other strains by their short lag phase and high specific growth rate.

[0128] [Table 2]

[0129] 3-2: Activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH)

[0130] The activity of alcohol dehydrogenase (ADH) was measured by adding 10 μL of yeast lysate to 990 μL of a reaction mixture consisting of 50 mM potassium phosphate buffer (pH 8.0), 2 mM NAD+, and 1% ethanol. The activity of aldehyde dehydrogenase (ALDH) was measured by adding 10 μL of yeast lysate to 990 μL of a reaction mixture consisting of 50 mM potassium phosphate buffer (pH 8.0), 3 mM NAD+, and 1.5 mM acetaldehyde. The enzyme reaction of ADH and ALDH was carried out at 30°C for 5 minutes, and the concentration of NAD(P)H generated by the enzyme reaction was measured by absorbance at 340 nm.

[0131] The enzyme activities of the nine mutant strains (K-1 to K-9) selected in the present invention were measured. The ADH activity of the mutant strains was as low as 382.69 units / g and as high as 975.29 units / g. Compared with the model strain (reference yeast, Saccharomyces cerevisiae KCTC7296), the ADH activity of the mutant strains increased by at least 5.1 times and as much as 13.1 times. The ALDH activity of the mutant strains was as low as 15.23 units / g and as high as 72.16 units / g. Compared with the enzyme activity of the model strain, the ALDH activity of the mutant strains increased by at least 5.3 times and as much as 24.9 times.

[0132] Six mutant strains (K-1, K-4, K-6, K-7, K-8, and K-9) showed similar rates of increase in ADH and ALDH enzyme activities compared to the model strain. The ALDH enzyme activities in three mutant strains (K-2, K-3, and K-5) were 18.3-, 23.2-, and 24.9-fold higher than those in the model strain, respectively. The ADH enzyme activities in three mutant strains (K-2, K-3, and K-5) were 9.7-, 11.6-, and 13.1-fold higher than those in the model strain, respectively. The rate of increase in ALDH enzyme activity in the three mutant strains (K-2, K-3, and K-5) was twice that of ADH.

[0133] The present inventors named three new mutants (K-2, K-3, and K-5) adapted to increase aldehyde dehydrogenase (ALDH) activity PicoYP, PicoYP-01, and PicoYP-02, respectively. These three new mutants were deposited with the Bioresource Center of the Korea Institute of Bioscience and Biotechnology under the accession numbers KCTC14983BP, KCTC14984BP, and KCTC14985BP, respectively.

[0134] 3-3: Content of coenzymes (NAD and NADP)

[0135] NAD total and NADP total were measured in lysates extracted from the mutant strain using the NADH / NAD+ assay kit and the NADPH / NADP+ assay kit, respectively. NAD(P) in the sample was converted to NAD(P)H using an NAD(P) cycling buffer and an NAD(P) cycling enzyme mix. A chromogenic reaction was induced with an NAD(P) chromogen, and the absorbance at 450 nm was measured. The chromogenic reaction was measured by absorbance at 450 nm. The sample absorbance was substituted into the equation corresponding to the standard curve to calculate the NAD(P) total in the yeast lysate.

[0136] The coenzyme content of the nine mutant strains (K-1 to K-9) selected in the present invention was measured. The NADtotal of the mutant strains was as low as 126 nmol / g and as high as 195 nmol / g. Compared with the model strain, the NADtotal of the mutant strain increased by at least 7.3 times and as much as 10.8 times. The NADPtotal content of the mutant strain was as low as 2.4 nmol / g and as high as 5.8 nmol / g. Compared with the model strain, the NADPtotal content of the mutant strain increased by at least 11.4 times and as much as 27.6 times.

[0137] In the six mutants (K-1, K-4, K-6, K-7, K-8, and K-9), the rate of increase in NADPtotal was less than twice the rate of increase in NADtotal. The rates of increase in total NADP content in the three new mutants (PicoYP, PicoYP-01, and PicoYP-02) were 25.7-fold, 22.9-fold, and 27.6-fold, respectively. The rates of increase in total NAD content in the three new mutants were 10.8-fold, 9.9-fold, and 11.3-fold, respectively. The rate of increase in NADPtotal in the three new mutants was greater than twice the rate of increase in NADtotal.

[0138] 3-4: Glutathione (GSH) content

[0139] Glutathione content was measured in nine mutant strains using the same method as in Examples 1-2. Glutathione content ranged from a low of 0.85% to a high of 1.05%. Compared to the model strain, glutathione content increased by at least 2.7 times and as much as 3.3 times in the mutants. Three of the new mutants (PicoYP, PicoYP-01, and PicoYP-02) showed higher increases in ALDH activity and coenzyme content than in the other strains.

[0140] The three new mutant yeasts (PicoYP, PicoYP-01, and PicoYP-02) have similar glutathione production capabilities to existing deposited strains (Kwon P-1, Kwon P-2, and Kwon P-3). Compared to existing deposited strains, the three new mutant yeasts have significantly increased ADH and ALDH enzyme activities and coenzyme content.

[0141] [Table 3]

[0142] [Table 4]

[0143] [Example 4] Comparison of carbon source preferences

[0144] The carbon source preferences of three mutants (KwonP-1, KwonP-2, and KwonP-3) with elevated ALDH and glutathione levels were investigated, for which a domestic patent application was filed on February 18, 2020. The carbon source preferences of a reference yeast strain (KCTC7296) were measured. To determine the maximum capacity for ALDH production, the carbon source preferences of three new mutants (PicoYP, PicoYP-01, and PicoYP-02) were investigated.

[0145] The carbon source preference of the strains was analyzed for specificity and novelty using the API 50CHL kit (API Systems, BIOMERIEUX, SA, France).

[0146] Prepare 15 mL conical tubes containing 8 mL of YPD medium. Inoculate each of the seven mutant strains into the prepared conical tubes.

[0147] After incubating the inoculated conical tubes at 30°C and 200 rpm for 24 hours, each of the seven mutant strains was isolated from the exponential growth phase. To eliminate the effects of residual carbon source in the YPD medium, the yeast was washed three times using a centrifuge. A yeast suspension at a 2 McFarland concentration was prepared using API 50CHL medium. The prepared yeast suspension was placed in a test strip tube. The test strips, upon which the suspension was dispensed, were incubated at 30°C for 24 hours.

[0148] API 50CHL medium, used for API testing, is purple. As acids are produced through energy metabolism, API 50CHL medium turns blue, then green, and finally yellow. The carbon source used by the mutant is recorded based on the color change, such as purple x, blue +, green ++, and yellow +++.

[0149] All seven mutant strains tested used 19 carbon sources for energy production and growth: L-arabinose, ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, N-acetylglucosamine, arbutin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, raffinose, and gentiobiose.

[0150] Only three mutants used rhamnose: KwonP-1, PicoYP-01, and PicoYP-02. Four mutants used sorbitol: KwonP-1, KwonP-3, PicoYP-01, and PicoYP-02. Four mutants used α-methyl-D-mannoside: the model strain, KwonP-1, KwonP-2, and PicoYP-02. Six mutants used amygdalin: KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02. Four mutants used D-turanose: the model strain, KwonP-1, KwonP-3, and PicoYP-02. Three mutants used D-tagatose: the model strain, KwonP-3, and PicoYP-3. Only the model strain used gluconate.

[0151] Mannitol and sorbitol, corresponding to alcoholic carbon sources, had a significant effect on yeast growth. The three new mutants differed from the other four yeast strains in the types of sugars they utilized for growth. The three new mutants (PicoYP, PicoYP-01, and PicoYP-02) showed slight differences in their preferred alcoholic carbon sources [Table 5].

[0152] [Table 5]

[0153] [Example 5] Changes in ALDH activity of mutant strains in gastric juice

[0154] When KARC is administered orally, in order to maintain enzyme activity in the intestine, the enzyme activity must pass safely without being destroyed by gastric acid, which secretes strong proteolytic enzymes such as pepsin.

[0155] NaOH solution was added to artificial gastric fluid at pH 1.17 to create two simulated solutions at pH 3 and pH 5, similar to the human gastric environment during food digestion. 1g of KARC was added to 7mL of artificial gastric fluid and 7mL of each simulated solution and mixed at 36.5°C for 5, 30, 60, and 90 minutes, respectively. NaOH solution was added to each reaction mixture to adjust the acidity to pH 7. 10mL samples were collected from each of the adjusted pH 7 solutions for analysis. ALDH activity was assayed for each sample.

[0156] At pH 1.17, ALDH activity in samples decreased by over 92.88% compared to the control within 5 minutes of reaction. At pH 1.17, ALDH activity in samples decreased by an average of 98.89% over 90 minutes. At pH 3, ALDH activity decreased by an average of 96.66% after 90 minutes, and at pH 5, it decreased by 56.83%. Finally, ALDH activity at pH 3 and 5 remained relatively higher than at pH 1.17 during the 90-minute reaction.

[0157] Specifically, at pH 1.17, the ALDH activity of KwonP-1 (KCTC13925BP) decreased by 90.94% to 5.57 units / g after 5 minutes of reaction compared to the control group. The ALDH activity of KwonP-1 decreased by 98.57% to 0.88 units / g after 90 minutes of reaction. Figure 14 The enzyme activity at pH = 3 and pH = 5 remained relatively higher than that at pH = 1.17. After 90 minutes of reaction, the ALDH activity of KwonP-1 decreased by 96.66% to 5.57 units / g at pH = 3, and decreased by 98.57% to 0.88 units / g at pH = 5.

[0158] At pH = 1.17, the ALDH activity of KwonP-2 (KCTC14122BP) decreased by 91.18% to 5.43 units / g after 5 minutes of reaction. The ALDH activity of KwonP-2 decreased by 98.81% to 0.73 units / g after 90 minutes of reaction. Figure 15 At pH 3 and pH 5, higher enzyme activity was maintained than at pH 1.17. After 90 minutes of reaction, ALDH activity decreased by 97.62% to 1.47 units / g at pH 3, and decreased by 56.11% to 26.99 units / g at pH 5.

[0159] At pH = 1.17, the ALDH activity of KwonP-3 (KCTC14123BP) decreased by 89.99% to 6.16 units / g after 5 minutes of reaction. The ALDH activity of KwonP-3 decreased by 97.85% to 1.32 units / g after 90 minutes of reaction. Figure 16 At pH 3 and pH 5, higher enzyme activity was maintained than at pH 1.17. After 90 minutes of reaction, ALDH activity decreased by 92.61% to 4.55 units / g at pH 3, and decreased by 62.31% to 22.18 units / g at pH 5.

[0160] At pH = 1.17, the ALDH activity of PicoYP (KCTC14983BP) decreased by 92.84% to 4.40 units / g after 5 minutes of reaction. The ALDH activity of PicoYP decreased by 98.33% to 1.03 units / g after 90 minutes of reaction. Figure 17 High enzyme activity was maintained at both pH 3 and pH 5. After 90 minutes of reaction, ALDH activity decreased by 96.66% at pH 3 to 2.05 units / g, and decreased by 53.97% at pH 5 to 28.31 units / g.

[0161] At pH = 1.17, the ALDH activity of PicoYP-01 (KCTC14984BP) decreased by 95.71% to 2.64 units / g after 5 minutes of reaction. The ALDH activity of PicoYP-01 decreased by 99.76% to 0.15 units / g after 90 minutes of reaction. Figure 18 At pH 3 and pH 5, higher enzyme activity was maintained than in gastric juice. After 90 minutes of reaction, ALDH activity decreased by 98.21% to 1.10 units / g at pH 3, and decreased by 58.74% to 25.38 units / g at pH 5.

[0162] At pH = 1.17, the ALDH activity of PicoYP-02 (KCTC14985BP) decreased by 96.66% to 2.05 units / g after 5 minutes of reaction. The ALDH activity of PicoYP-02 decreased by 99.76% to 0.15 units / g after 90 minutes of reaction. Figure 19 At pH 3 and pH 5, higher enzyme activity was maintained than in gastric juice. After 90 minutes of reaction, at pH = 3, ALDH activity decreased by 98.21% to 1.10 units / g, and at pH = 5, ALDH activity decreased by 62.08% to 23.32 units / g.

[0163] The pH of secreted gastric juice is 1.17. When food is consumed, the pH of the original gastric juice and food mix in the stomach, raising it from 3 to 5, making it unlikely to reach a pH of 1.17. Despite this, ALDH activity in the mutant strain is maintained even under this extreme pH condition.

[0164] Finally, the ALDH enzyme activity of the new mutants (PicoYP, PicoYP-01, PicoYP-02) remained at 2 to 5 units / g, despite a 92% to 97% decrease under the strongly acidic conditions of pH 1.17. Maintaining 2-5 units of enzyme activity is sufficient to function in the intestine. Compared to pH 1.17, it was even higher at pH 3 and pH 5. This leads to the conclusion that the new mutants (PicoYP, PicoYP-01, PicoYP-02) can be administered orally.

[0165] [Example 6] Growth characteristics of 5L fermenter culture

[0166] Be inoculated in YPD substratum (2% peptone, 1% yeast extract, 2% glucose) respectively, and carry out primary seed culture 18 hours at 30 ℃ and 200rpm.The seed of 20mL culture is inoculated in 1980mL YPD substratum, and in 5L, cultivate again.The cultivation in 5L culture tank was carried out 48 hours at 30 ℃ and 200rpm.Use the 10mL sample analysis OD660nm growth curve and the enzyme activity under collecting from secondary culture.

[0167] The maximum density (OD660nm) of KwonP-1 (KCTC13925BP) was 134.4. The maximum density of KwonP-1 was 4.35% higher than that of the model strain (KCTC7296). The growth curve characteristics and specific growth rate (OD660nm / hr) of KwonP-1 were similar to those of the model strain. The ALDH activity of KwonP-1 was 33.6 units / g. The ALDH activity of KwonP-1 was 11.96 times higher than that of the model strain. Figure 20 ].

[0168] The maximum density (OD660nm) of KwonP-2 (KCTC14122BP) was 133.8. The maximum density of KwonP-2 was 3.88% higher than that of the model strain. The growth of KwonP-2 ended earlier than that of the model strain. The specific growth rate (OD660nm / hr) of KwonP-2 was 14.8% higher than that of the model strain. The ALDH activity of KwonP-2 was 31.5 units / g. The ALDH activity of KwonP-2 was 11.21 times higher than that of the model strain. Figure 21 ].

[0169] The maximum density (OD660nm) of KwonP-3 (KCTC14123BP) was 134.1. The maximum density of KwonP-3 was 4.12% higher than that of the model strain. The growth of KwonP-3 ended earlier than that of the model strain. The specific growth rate (OD660nm / hr) of KwonP-3 was 6.08% higher than that of the model strain. The ALDH activity of KwonP-3 was 29.5 units / g. The ALDH activity of KwonP-3 was 10.5 times higher than that of the model strain. Figure 22 ].

[0170] The maximum density (OD660nm) of PicoYP (KCTC14983BP) was 123.8. The maximum density of PicoYP was 3.88% higher than that of the model strain. The growth curve characteristics of PicoYP were similar to those of the model strain. The specific growth rate (OD660nm / hr) of PicoYP was 6.22% higher than that of the model strain. The ALDH activity of PicoYP was 44.2 units / g. The ALDH activity of PicoYP was 15.73 times higher than that of the model strain. Figure 23 ].

[0171] The maximum density (OD660nm) of PicoYP-01 (KCTC14984BP) was 126.9. This was 1.47% higher than that of the model strain. The growth curve characteristics of PicoYP-01 were similar to those of the model strain. The specific growth rate (OD660nm / hr) of PicoYP-01 was 2.14% higher than that of the model strain. The ALDH activity of PicoYP-01 was 47.1 units / g. This ALDH activity was 16.76-fold higher than that of the model strain [Figure 24].

[0172] The maximum density (OD660nm) of PicoYP-02 (KCTC14985BP) was 148.1. This was 14.99% higher than that of the model strain. The growth curve of PicoYP-02 was at the top compared to the model strain. The specific growth rate (OD660nm / hr) of PicoYP-02 was 9.64% lower than that of the model strain. The ALDH activity of PicoYP-02 was 52.68 units / g. This ALDH activity of PicoYP-02 was 18.75-fold higher than that of the model strain [Figure 25].

[0173] [Example 7] Preparation of mutant strain lysate (KARC)

[0174] To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysate, proteases are removed and inhibited. To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysate, cell debris is removed. The dried products or lysates of the mutant strains are mixed to prepare a KARC composition.

[0175] The mutant strain and the culture medium containing it contain a variety of substances, such as yeast metabolites and proteolytic enzymes secreted by the yeast. To extract and preserve the ALDH, coenzymes, and glutathione present in the yeast, it is necessary to fully remove substances outside the yeast. To this end, a washing procedure was performed. Washing of the mutant strain was performed by dispensing 40 mL of culture medium into a 50 mL conical tube, centrifuging at 13,000 rpm for 15 minutes, and removing the supernatant.

[0176] As a result of centrifugation, the yeast cells aggregated into a pellet containing residual culture medium. Add 30 mL of purified water and vortex to loosen the pellet. Repeat this procedure three times to fully remove the residual culture medium.

[0177] Yeast is known to have an ethanol tolerance of up to 13%, and yeast will die when exposed to high ethanol concentrations. Dissolve the washed pellet thoroughly in 10 mL of 20% ethanol solution to induce yeast death. Stir the pellet in ethanol at 100 rpm for 30 minutes to induce yeast death. At the end of the reaction time, add 30 mL of purified water to bring the ethanol concentration to 5%. Repeat this washing procedure three times to fully remove the ethanol.

[0178] To prevent the degradation of ALDH and ADH by proteases present in yeast cells, 10 mL of 1X PBS was prepared by dissolving two protease inhibitor tablets (Pierce Protease Inhibitor Tablets, EDTA-free, Thermo Scientific). The solution was added to the washed yeast pellet and distributed thoroughly.

[0179] To prepare the lysate of the mutant strain prepared in the present invention, 4 g of glass beads were added and stirred to disrupt the yeast cell walls. To prevent heat generated during yeast disruption from denaturing the enzyme, vortexing for 30 seconds and incubating on ice for 30 seconds were repeated 6 times.

[0180] After the yeast cell wall was broken, 10 mL of 100 mM potassium phosphate buffer was added and mixed by vortexing for 3-5 seconds. Centrifugation was performed at 13,000 rpm for 15 minutes to remove cell structures such as yeast cell walls and glass beads. The supernatant was filtered through a 0.2 μm filter (Minisart The KARC composition was prepared by filtration through a syringe filter (Sartorius, Goettingen, Germany).

[0181] To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysates, remove and inhibit intracellular proteases, and remove cellular debris such as cell walls, KARC compositions were prepared using lysates of six mutant strains (KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, PicoYP-02) or mixtures thereof in a free ratio [Table 6].

[0182] KARC 1 is made from Kwon P-1. KARC 1's ADH and ALDH enzyme activities are 461.4 units / g and 28.6 units / g, respectively. The NAD and NADP total coenzyme contents in KARC 1 are 176.2 nanomoles / g and 5.1 nanomoles / g, respectively. KARC 1 also contains 0.98 wt% GSH.

[0183] KARC 2 is made from Kwon P-2. KARC 2's ADH and ALDH enzyme activities are 482.1 units / g and 29.8 units / g, respectively. KARC 2's NAD total and NADP total coenzyme contents are 175.4 nanomoles / g and 5.2 nanomoles / g, respectively. KARC 2 also contains 0.96 wt% GSH.

[0184] KARC 3 is made from Kwon P-3. KARC 2 has ADH and ALDH enzyme activities of 477.5 units / g and 28.1 units / g, respectively. KARC 3 has NAD total and NADP total coenzyme contents of 177.2 nanomoles / g and 5.1 nanomoles / g, respectively. KARC 3 also has a GSH content of 1.00 wt%.

[0185] KARC 4 is made from PicoYP. KARC 2 has ADH and ALDH enzyme activities of 586.8 units / g and 33.8 units / g, respectively. KARC 4 has NAD total and NADP total coenzyme contents of 184.3 nanomoles / g and 5.7 nanomoles / g, respectively. KARC 4 also has a GSH content of 0.84 wt%.

[0186] KARC 5 is produced from PicoYP-01. KARC 5 exhibits ADH and ALDH enzyme activities of 621.6 units / g and 38.2 units / g, respectively. The NAD and NADP total coenzyme contents in KARC 5 are 186.9 nanomoles / g and 5.6 nanomoles / g, respectively. KARC 5 also contains 0.84 wt% GSH.

[0187] KARC 6 is made from PicoYP-02. KARC 5 has ADH and ALDH enzyme activities of 664.1 units / g and 41.6 units / g, respectively. KARC 6 has NAD total and NADP total coenzyme contents of 195.0 nanomoles / g and 5.8 nanomoles / g, respectively. KARC 6 also has a GSH content of 0.88 wt%.

[0188] KARC was prepared by freely mixing dry powders and lysates prepared from six deposited strains. The average enzyme activities of ADH and ALDH in the KARC composition were 547.6 units / g and 33.1 units / g, respectively. The average contents of the coenzyme NAD total and coenzyme NADP total in the KARC composition were 180.4 nanomoles / g and 5.4 nanomoles / g, respectively. The average glutathione content in the KARC composition was 0.84 wt%.

[0189] The aldehyde decomposition ability of KARC was maintained during the lysate production process. KARC exhibited the ability to remove endogenous aldehydes such as HNE, MDA, and 3,4-dihydroxyphenylacetaldehyde (DOPAL).

[0190] [Table 6]

[0191] [Example 8] Sequence analysis of ALDH contained in mutant strains. The differences in ALD (yeast aldehyde dehydrogenase) between the mutants and the parent strain were investigated. Whole-genome sequencing was performed on the parent strain and mutants KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02. Mutant cells were isolated by culturing pure strains on solid culture medium. The resulting mutant genome sequences were then analyzed.

[0192] In the ALD (yeast aldehyde dehydrogenase) of the new mutant strain, ALD2 (SEQ ID NO: 3) and ALD3 (SEQ ID NO: 4) were found to be densely distributed on chromosome 13. A 689-nucleotide noncoding region was located between the ALD2 and ALD3 encoding genes.

[0193] ALD2 and ALD3 coexist in the same genome. ALD2 and ALD3 encode separate aldehyde dehydrogenases. The ALD2 gene is nearly identical to ALD3, consisting of 1,521 nucleotides and 506 amino acids, but differs by 8.2% in sequence. ALD2 and ALD3 were identified as separate aldehyde dehydrogenases, differing by 125 bases (8.2%).

[0194] In six mutant strains (KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02), the ALD2 sequence lacks a stop codon, allowing protein synthesis to proceed. Consequently, a new, larger ALDH enzyme was created by linking a portion of ALD2 to ALD3 [SEQ ID NO: 1].

[0195] ALD2 [SEQ ID NO. 3] of the model strain (KCTC7296) consists of a 30-nucleotide sequence (5'-GTTCACATAAATCTCTCTTTGGACAACTAA-3') encoding 9 amino acids (N-VHINLSLDN-C) at the terminal, excluding the stop codon.

[0196] ALD2 of the six mutants consists of a specific 42-nucleotide sequence (5'-AGATATAGATTATACACATTTAGAAAATTAGCCAAAAGAAAA-3') encoding 14 amino acids (N-RYRLYTFRKLAKRK-C) between the 5' end of ALD2 and ALD3 [SEQ ID NO. 2].

[0197] The ALD2 gene encoding ALD2 is deleted from nucleotide 1492 to nucleotide 647 in the noncoding region, eliminating the termination codon. Ultimately, the six deposited mutants contain a novel mutant gene consisting of 3,054 bases encoding a novel ALD2 gene [SEQ ID NO: 1].

[0198] [Example 9] Observation of in vitro decomposition of succinic semialdehyde (SSA) by KARC

[0199] The present invention demonstrates the effect of KARC in reducing succinic semialdehyde (SSA).

[0200] 9-1: Reaction of succinic semialdehyde (SSA)

[0201] Potassium chloride (KCl) was dissolved in 50 mM HEPES buffer solution at pH 7.5 to 200 mM for buffering.

[0202] For the SSA experiment, 845 μL of this buffer, 15 μL of 100 mM EDTA in water, 30 μL of 100 mM NADP+ in water, 10 μL of 10 mM SSA in acetonitrile, and 10 μL of 300 mg / mL KARC were dispensed into a microtube. As a negative control, 845 μL of the buffer solution, 15 μL of 100 mM EDTA in water, 30 μL of 100 mM NADP+ in water, 100 μL of 10 mM SSA in acetonitrile, and 10 μL of DW were dispensed into a microtube. The reaction was shaken at 30°C or 37°C for 1 or 3 hours.

[0203] 9-2: Pretreatment before HPLC analysis

[0204] For experiments using representative fatty aldehydes (SSA and acetaldehyde), 500 μL of each reaction was aliquoted into a microtube at the end of the reaction. 470 μL of methanol, 20 μL of 50 mM DNPH in acetonitrile, and 10 μL of 6N HCl were additionally dispensed into the microtube containing the reaction solution and heated at 70°C for 40 minutes. Alternatively, 480 μL of methanol, 10 μL of 100 mM DHBA in acetonitrile, and 10 μL of 6N HCl were added and heated at 70°C for 40 minutes. After cooling the heated solution, 10 μL was aliquoted and injected into the HPLC for analysis.

[0205] 9-3: HPLC analysis

[0206] Analyses were performed using an HPLC system (Waters Alliance 2690 / 2695 HPLC with a Waters 2996 PDA detector). The analytical column was 150 mm x 4.6 mm id, packed with C18, 5 μm particle size (Shimadzu Scientific Instruments, Kyoto, Japan).

[0207] The gradient started from 80% water (1 v / v% trifluoroacetic acid) and after 15 minutes was adjusted to 20% in reverse phase.The absorbance was analyzed with a UV detector at a wavelength of 254 nm, 310 nm or 360 nm.

[0208] Compared with the negative control group, the experimental group confirmed this result by the reduction and consumption of aldehyde by DNPH-aldehyde conjugate or DHBA-aldehyde conjugate. Figure 21 、 Figure 22 、 Figure 23 ].

[0209] [Example 10] Effect of Oral Administration of KARC on Reduction of Acetaldehyde (Ach) and Malondialdehyde (MDA) in the Body

[0210] For the acetaldehyde and MDA animal experiments, 5-week-old male Sprague Dawley (SD) rats were used. The KARC composition was orally administered to the rats at a dose of 10 units / kg or 20 units / kg, and ethanol (3 g / kg) was orally administered to the rats 30 minutes after KARC injection.

[0211] After administration, blood samples were collected from the tail vein at 0 h, 1 h, 3 h, 5 h, and 8 h after KARC injection, and the plasma was stored at −80 °C after centrifugation. Figure 7 、 Figure 8 ].

[0212] Seven-week-old male Wistar rats (7 weeks old, 250 g, n=8-10) were used. Rotenone solution (2.5 mg rotenone / mL, 20 μL DMSO / mL) was prepared using natural oil (medium-chain triglycerides). Mice were administered rotenone solution (2.5 mg / kg) by intraperitoneal injection daily for 60 days.

[0213] Two administration methods were used to confirm the preventive and therapeutic effects of KARC on Parkinson's disease. KARC (20 units / kg) was orally administered at the same time as rotenone to observe the preventive effect on Parkinson's disease. KARC (20 units / kg) or levodopa was orally administered two weeks after rotenone administration to observe the therapeutic effect on Parkinson's disease. For the quantification of dopamine, brain tissue was isolated and stored in liquid nitrogen at -80°C. Figure 9 、 Figure 10 ].

[0214] 10-1: Acetaldehyde-reducing effect by oral administration of KARC

[0215] The total acetaldehyde reduction effect of oral administration of KARC was evaluated using an acetaldehyde assay kit (LSBio, Seattle, WA, USA). 20 μL of each sample was distributed to 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 distributed to one well. In the remaining wells, 80 μL of blank working reagent (75 μL assay buffer, 8 μL NAD / MTT, 1 μL enzyme B) was distributed. The distributed plate was gently mixed and reacted at room temperature for 30 minutes. After the reaction was complete, the absorbance was measured at 565 nm (520-600 nm).

[0216] In the KARC composition-administered group, acetaldehyde concentrations reached their maximum 1 hour after ethanol administration and then showed a downward trend. In the KARC-administered group, acetaldehyde concentrations were significantly lower 1, 3, and 5 hours after ethanol administration compared to the control group (vehicle). In the high-dose KARC-administered group (F), blood acetaldehyde concentrations were 0.356 mM, 0.224 mM, and 0.091 mM, respectively, representing decreases of 39.2%, 58.4%, and 72.1% compared to the control group. Figure 7 ].

[0217] 10-2: MDA-reducing effect by oral administration of KARC

[0218] According to the manufacturer's protocol (ZeptoMetric, Buffalo, NY, USA), the total malondialdehyde content in the blood was analyzed using the OxiTecTM TBARS assay kit. 100 μL of sample, 100 μL of 8.1% SDS solution and 4 mL of color indicator (TBA, 10% NaOH solution, 20% acetic acid) were added to a conical tube and then reacted in a 95°C thermostatic water bath for 60 minutes. After the reaction was complete, the sample was centrifuged at 4°C and 1,600 rpm for 10 minutes and stabilized at room temperature for 30 minutes. 150 μL of supernatant was transferred to a 96-well plate and the absorbance was measured at 530-540 nm.

[0219] In the control group (vehicle), the MDA concentration in the blood reached its maximum 3 hours after ethanol administration, while in the KARC administration group, it reached its maximum 1 hour after ethanol administration. The MDA concentration in the blood decreased 3 and 5 hours after ethanol administration, showing a significant difference from the control group. The blood MDA concentrations in the high-dose KARC administration group (F) were 0.232 μM and 0.137 μM, respectively, which were decreased by 80.4% and 86.3% compared to the control group. Figure 8 ].

[0220] These results indicate that oral administration of KARC is effective in reducing multiple endogenous aldehydes such as acetaldehyde and malondialdehyde in the blood.

[0221] [Example 11] Effect of reducing oxidative stress.

[0222] When drinking alcohol, excessive acetaldehyde (ACh) is produced by alcohol dehydrogenase (ADH), leading to increased reactive oxygen species and oxidative stress. Aldehyde dehydrogenase (ALDH) converts ACh into acetic acid and excretes it from the body. Excessive aldehyde production due to mutations in the ALDH gene or from excessive alcohol consumption can lead to lipid peroxidation.

[0223] The resulting acetaldehyde and malondialdehyde exacerbate oxidative stress and interfere with mitochondrial energy metabolism. Endoplasmic reticulum stress is caused by the accumulation of denatured proteins in cells, leading to cell death.

[0224] Blood acetaldehyde concentration was measured over time after drinking. Figure 11 When drinking alone, the area under the curve (AUC) of blood acetaldehyde (Ach) was 13.02±1.18 mg·h / dL. When administered at a dose of 10 units / kg of KARC, the area under the curve (AUC) of blood acetaldehyde (Ach) decreased significantly by 26.13% compared to drinking alone, measuring 9.39±1.07 mg·h / dL (P=0.005).

[0225] When administered at a dose of 20 units / kg, the AUC of blood acetaldehyde (ACh) significantly decreased by 55.71% compared to alcohol consumption alone, reaching 5.22 ± 0.99 mg·h / dL (P < 0.001). Compared to the group administered 10 units / kg of KARC, the 20 units / kg group showed a significant decrease in blood acetaldehyde (ACh) (P = 0.034). KARC demonstrated a dose-dependent reduction in total blood acetaldehyde (ACh) over time.

[0226] The reduction in blood acetaldehyde (Ach) concentration due to KARC administration has a positive impact on reducing oxidative stress and promoting health.

[0227] Measurement of blood malondialdehyde (MDA) concentration during chemotherapy Figure 12 The blood MDA concentration in the control group was 0.607±0.161 μM. The group receiving KARC treatment showed a significant 63.3% decrease in blood MDA concentration compared to the control group, measuring 0.223±0.033 μM (P<0.001).

[0228] In the control group, blood MDA concentration ranged from 0.427 μM to 0.885 μM, showing a wide variation. In the KARC-administered group, the range was significantly narrowed, with values ​​ranging from 0.158 μM to 0.269 μM. This confirms not only the effect of lowering blood MDA concentration but also stabilizing it, as shown in [ Figure 13 ] confirmed.

[0229] Various factors, including drug ingestion, stress, and intense physical exercise, lead to an increase in intracellular reactive oxygen species (ROS). This triggers lipid peroxidation and the oxidation of endogenous amines (such as dopamine, norepinephrine, serotonin, and histamine). Reactive aldehyde compounds, including 4-hydroxynonenal (HNE), malondialdehyde (MDA), acetaldehyde (ACh), and dopamine-induced aldehydes, accumulate within cells, exacerbating oxidative stress.

[0230] 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 (ALPEs). The accumulation of these products has toxic effects on various cells, further exacerbating oxidative stress.

[0231] This cumulative oxidative stress disrupts mitochondrial energy metabolism within the cell and leads to the accumulation of aldehyde intermediates in aldose sugar metabolism, including methylglyoxal (MG) and glyceraldehyde-3-phosphate (GA3P). Chain reactions involving aldehydes lead to the accumulation of stable final sugar oxidation products, known as advanced glycation end products (AGEs), which weaken intracellular antioxidant defense systems such as glutathione (GSH). These processes increase endoplasmic reticulum (ER) stress, leading to increased apoptosis in neuronal cells.

[0232] Increased reactive oxygen species and oxidative stress are associated with elevated levels of reactive aldehydes, such as HNE and MDA, and modified proteins, such as advanced glycation end products (AGEs) and advanced lipid peroxidation end products (ALEs). This series of events is known to reinforce and amplify each other, leading to increased endoplasmic reticulum stress (ER stress).

[0233] KARC administration effectively regulated malondialdehyde, a marker of reactive oxygen species and oxidative stress, demonstrating the potential to reduce oxidative stress and improve endoplasmic reticulum (ER) stress stability. KARC significantly reduced malondialdehyde concentrations in the bloodstream, demonstrating its ability to reduce reactive oxygen species and oxidative stress. By reducing acetaldehyde and malondialdehyde levels in human blood, KARC demonstrated the potential to prevent and correct ER stress by reducing reactive oxygen species and oxidative stress. This suggests that by modulating intracellular reactive oxygen species and oxidative stress, KARC inhibits neuronal apoptosis, thereby suppressing and preventing Parkinson's disease. This resulted in improved behavior and motor function.

[0234] [Example 12] Acute oral administration test

[0235] 12-1. Preparation of experimental animals

[0236] The experimental animals were female and male ICR mice (7 weeks old). The received ICR mice were adapted for 7 days. The general symptoms of the adopted mice during the adaptation period were observed, and only healthy animals were used for short-term toxicity testing. Feed and water were available freely. Based on an average body weight of about 20g the day before oral administration, the group was divided into 10 groups of 5 mice each.

[0237] 12-2. Administration of test substance

[0238] The test substances were prepared by dissolving in physiological saline so that the doses for experimental animals were 0 mg / kg, 750 mg / kg, 3,000 mg / kg, and 5,000 mg / kg, respectively, based on the content of the mutant yeast lysate KARC of the present invention.

[0239] The dosage standards were in accordance with the Korea National Toxicology Program (KNTP) Toxicity Test Manual of the Ministry of Food and Drug Safety. The maximum dose of 5,000 mg / kg, as specified in the KNTP Manual, was used as the maximum concentration for this experiment. Samples prepared for each group were administered orally once to each test animal. The normal group (G1) received normal saline.

[0240] 12-3. Observation and autopsy

[0241] For all test groups, mice were observed for symptoms at least once daily from the date of acquisition until the date of necropsy. Symptoms were observed for 7 days after oral administration. Necropsy was performed after observing the symptoms of the rats. During the rat necropsy, changes in various organs were observed visually.

[0242] Single-dose toxicity testing of the ALDH-containing KARC composition of the present invention was conducted in mice. Results showed that no mouse deaths were observed within 7 days at concentrations of up to 5,000 mg / kg of mutant yeast KARC. No abnormalities, such as weight gain or changes in feed intake, were observed in the mice. Post-observation autopsies revealed no abnormal findings.

[0243] [Example 13] Preparation of food and pharmaceutical compositions for alleviating tremor and oxidative stress by decomposing endogenous alcohols and aldehydes.

[0244] Food and pharmaceutical compositions containing KARC as an active ingredient for alleviating tremors and oxidative stress have been prepared. Food or pharmaceutical compositions containing KARC powder can be prepared in various composition ratios. For example, the powder composition according to the present invention can suppress tremors and oxidative stress by taking 13 g of the composition twice daily. The weight ratios of the components and phases of the food or pharmaceutical compositions containing the powder composition are shown in Table 7.

[0245] [Table 7] Industrial Applicability

[0246] KARC dry powder, excipients, and natural sweeteners such as oligofructose, enzyme-treated stevia, anhydrous citric acid, isomaltodextrin, and xylitol, as well as citrus juice powder and citrus flavoring powder, are added to food and pharmaceutical compositions. The processing and testing of the raw materials and final products of the food or pharmaceutical compositions are performed in accordance with general testing methods and the Health Functional Food Act as outlined in the Korean Food Code.

[0247] A food or pharmaceutical composition containing KARC decomposes endogenous aldehydes and exhibits an effect of suppressing tremor or movement disorder.

[0248] The above examples describe in detail the mutant yeast composition KARC, which contains aldehyde dehydrogenase, including its production methods, pharmacological effects, administration methods, therapeutically effective doses for disease models, acute toxicity after short-term administration, and representative examples of food or pharmaceutical compositions. While the above examples describe the efficacy of KARC in detail, they are merely examples of the present invention.

[0249] 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.

[0250] Even foods or therapeutic agents containing modified forms of aldehyde dehydrogenase that embody the technical gist of the present invention as described in the patent claims fall within the scope of legal protection of the present invention.

Claims

A food composition for suppressing or ameliorating the symptoms of tremor or movement disorder, comprising an aldehyde dehydrogenase encoded by a gene having more than 98% homology to the gene of SEQ ID NO:

1. 2 . The food composition for suppressing tremor or movement disorder according to claim 1 , comprising aldehyde dehydrogenase encoded by the gene of SEQ ID NO: 1 including SEQ ID NO:

2. 3 .

3. The food composition for suppressing tremor or movement disorder according to claim 1 or 2, wherein The aldehyde is an endogenous aldehyde.

4. The food composition for suppressing tremor or movement disorder according to claim 3, wherein The endogenous aldehyde is an endogenous aldehyde produced by oxidation of endogenous amine compounds or alcohols.

5. The food composition for suppressing tremor or movement disorder according to claim 3, wherein The endogenous aldehyde is selected from the group consisting of acetaldehyde, malondialdehyde (MDA), glutamate semialdehyde (GSA) and succinate semialdehyde (SSA).

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

7. A food composition for suppressing tremor or movement disorder, comprising any one or a mixture thereof selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP.

8. A pharmaceutical composition for suppressing tremor or movement disorder, comprising an aldehyde dehydrogenase encoded by a gene having more than 98% homology to the gene of SEQ ID NO:

1. 9 . The pharmaceutical composition for suppressing tremor or movement disorder according to claim 8 , comprising aldehyde dehydrogenase encoded by the gene of SEQ ID NO: 1 including SEQ ID NO:

2.

10. The pharmaceutical composition for suppressing tremor or movement disorder according to claim 8 or 9, wherein The aldehyde dehydrogenase is contained in any one selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

11. A pharmaceutical composition for inhibiting tremor or movement disorder, comprising a lysate of any one selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

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