Novel aldehyde dehydrogenase food and drug compositions for improving behavior and motor function

Abstract

The present invention is directed to a food or pharmaceutical composition for improving behavior and motor function, and a pharmaceutical composition for inhibiting or preventing Parkinson's disease, which contains an aldehyde dehydrogenase encoded by a gene having more than 98% homology to the gene of SEQ ID NO: 1, particularly encoded by the gene of SEQ ID NO: 1 including SEQ ID NO: 2, and contained in a lysate of any one or a mixture thereof selected from the group consisting of Saccharomyces cerevisiae, KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP.

Description

[Technical Field]

[0001] The present invention relates to foods and medicines containing a novel aldehyde dehydrogenase (ALDH) for improving behavior and motor function.

[0002] More specifically, the present invention relates to a food for improving behavior and motor function, and a pharmaceutical composition for inhibiting or preventing Parkinson's disease, which contains an aldehyde dehydrogenase encoded by a gene having more than 98% homology to the gene of SEQ ID NO: 1, particularly the gene of SEQ ID NO: 1, including SEQ ID NO: 2.

[0003] More specifically, the present invention is directed to a food or pharmaceutical composition for improving behavior and motor function, and a pharmaceutical composition for inhibiting or preventing Parkinson's disease, which contains an aldehyde dehydrogenase contained in a lysate of any one or a mixture of Saccharomyces cerevisiae, KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, which is encoded by a gene having more than 98% homology to the gene of SEQ ID NO: 1, particularly SEQ ID NO: 2. [Background technology]

[0004] Dopamine (DA) is a substance secreted by cells in the substantia nigra of the brain, and transmits signals by increasing or decreasing the electrical potential of adjacent neurons through synapses. Parkinson's disease occurs when dopamine-secreting neurons die in the substantia nigra of the brain, resulting in a decrease in dopamine secretion.

[0005] Generally, neurotransmitters such as dopamine reside within the synaptic endoplasmic reticulum of neurons. When a signal is transmitted to a neuron, calcium ion channels open, allowing calcium to flow into the neuron. As a result, synaptic vesicles migrate to the neuron's plasma membrane. At this time, membrane fusion occurs between the endoplasmic reticulum that has migrated to the plasma membrane and the plasma membrane, and the neurotransmitters present in the endoplasmic reticulum are released from the axon terminal into the synapse.

[0006] Dopamine is a precursor to adrenaline (epinephrine) or norepinephrine and is associated with various brain functions such as motivation, happiness, memory, and cognition. It acts as a motor neurotransmitter in the substantia nigra of the brain and plays a role in stabilizing muscle control.

[0007] While sufficient dopamine secretion gives people a sense of motivation and accomplishment, reduced dopamine secretion causes emotional anxiety such as depression as well as physical instability, including balance disorders. Excess dopamine secretion can cause hallucinations and delusions in humans.

[0008] Dopamine produces endogenous aldehydes, such as DOPAL, MOPAL, DOPEGAL, and MOPEGAL, via the action of monoamine oxidase (MAO) [Figures 1 and 2]. These endogenous aldehydes are highly reactive, leading to the accumulation of abnormally aggregated α-synuclein (α-Syn) and other denatured proteins in the endoplasmic reticulum of substantia nigra neurons. The accumulation of these denatured proteins can result in increased endoplasmic reticulum stress.

[0009] This increase in endoplasmic reticulum stress in nigral cells is known to rapidly induce neuronal cell death (apoptosis) in the substantia nigra, resulting in a decrease in dopamine secretion in the substantia nigra, and leading to the onset of Parkinson's disease.

[0010] In other words, denatured α-synuclein aggregates deposit within the endoplasmic reticulum of substantia nigra neurons, promoting the death of dopamine-secreting cells and thereby reducing the secretion of dopamine, which is required for normal brain function, resulting in various symptoms such as slowed movement and gait, resting hand tremors, muscle rigidity, and postural instability, as well as decreased motor skills, decreased cognitive function, and sleep disturbances.

[0011] To date, no effective drugs have been developed to increase dopamine-secreting neurons or to reduce neuronal death.

[0012] Currently available therapeutic drugs are used to alleviate various symptoms or slow the progression of Parkinson's disease. For example, drugs have been developed to temporarily activate dopamine in the brain by exogenously replenishing dopamine, whose secretion is reduced due to the death of dopamine cells in the substantia nigra. Dopamine precursors such as levodopa, dopamine agonists that activate dopamine receptors, or dopamine degrading enzyme inhibitors are currently used as drugs to alleviate the symptoms of Parkinson's disease.

[0013] Continued administration of the dopamine supplement levodopa can cause various side effects, including dyskinesia, motor fluctuations, and wearing-off effects.

[0014] Monoamine oxidase (MAO), which is used to inhibit the breakdown of dopamine in the brains of Parkinson's disease patients, is an enzyme involved in the oxidative deamination of the primary amine dopamine. MAO is present in particularly large amounts in the brain, liver, stomach, and myocardium, and has recently attracted attention as an indicator of fibrosis that progresses from hepatitis to cirrhosis. This is because monoamine oxidase is involved in the cross-linking reaction that converts soluble collagen into insoluble collagen.

[0015] Increased monoamine oxidase activity is manifested in collagen metabolism, particularly in conditions where collagen synthesis is enhanced, such as acromegaly and progressive scleroderma. In addition, monoamine oxidase is also involved in the metabolism of noradrenaline, serotonin, and other neurotransmitters.

[0016] The present inventor has long been researching the development of a drug that inhibits the accumulation of denatured proteins, including alpha-synuclein aggregates, in the endoplasmic reticulum, induces a reduction in oxidative stress and endoplasmic reticulum stress, and prevents apoptosis of neurons in the substantia nigra of the brain, by promoting the degradation and metabolism of DOPAL (dopamine derived from an aldehyde), a toxic aldehyde substance produced in the metabolic process of dopamine by monoamine oxidase (MAO) in the substantia nigra of the brain, and aldehyde compounds of other origins.

[0017] As a result of such investigations, the inventors have come to the conclusion that if aldehyde dehydrogenase derived from yeast (Saccharomyces cerevisiae), which is similar to human aldehyde dehydrogenase, can be administered to the human body to rapidly oxidize endogenous toxic aldehydes and convert them into organic acid forms, it may be possible to effectively inhibit and prevent the onset of Parkinson's disease by reducing the apoptosis of dopaminergic neurons through the reduction of endogenous toxic aldehydes in the substantia nigra. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] U.S. Patent Application Publication No. 2021-0254023A1, dated August 21, 2021 [Non-patent literature]

[0019] TIFF2025539868000001.tif37108TIFF2025539868000002.tif166109TIFF2025539868000003.tif99108 Summary of the Invention [Problem to be solved by the invention]

[0020] Disclosure of the Invention Technical issues Numerous previous studies, such as those mentioned above, have revealed that the strong reactivity of DOPANAL (a dopamine-derived aldehyde) increased by the action of monoamine oxidase in the substantia nigra leads to the accumulation of denatured proteins, such as alpha-synuclein aggregates, in the endoplasmic reticulum of nigral neurons, which can cause the death of nigral neurons.

[0021] Despite many previous studies such as those mentioned above, no food or pharmaceutical composition has yet been developed that can effectively inhibit or prevent the occurrence of Parkinson's disease by reducing the apoptosis of substantia nigra cells, thereby reducing the accumulation of degenerated proteins in neural tissues through the activation of aldehyde dehydrogenase, which rapidly oxidizes and detoxifies reactive aldehydes produced during dopamine metabolism.

[0022] The main object of the present invention is to provide food and pharmaceutical compositions that exhibit the ability to improve behavioral and motor functions or suppress and prevent Parkinson's disease, which contain aldehyde dehydrogenase, which promotes the acid conversion of endogenous aldehydes produced by the decomposition of alcohol or the oxidation of endogenous amine compounds such as dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid (GABA).

[0023] Another object of the present invention is to provide a food for improving behavior and motor function, and a pharmaceutical composition for inhibiting or preventing Parkinson's disease, which contain aldehyde dehydrogenase encoded by a gene having more than 98% homology to the gene of SEQ ID NO: 1, particularly encoded by the gene of SEQ ID NO: 1, including SEQ ID NO: 2.

[0024] Yet another object of the present invention is to provide a food or pharmaceutical composition for improving behavior and motor function, and a pharmaceutical composition for inhibiting or preventing Parkinson's disease, which contain aldehyde dehydrogenase contained in a lysate of any one selected from the group consisting of Saccharomyces cerevisiae, KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof. [Means for solving the problem]

[0025] Solutions to problems The above-mentioned objects of the present invention can be achieved by providing a food composition and a pharmaceutical composition containing a dry powder of a lysate of any one selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP or a mixture thereof (hereinafter abbreviated as KARC). [Effects of the Invention]

[0026] Advantageous Effects of the Invention The composition of the present invention, which contains aldehyde dehydrogenase encoded by the gene of SEQ ID NO: 1, including SEQ ID NO: 2, contained in KARC, which is a dried lysate of any Saccharomyces cerevisiae selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, exhibits the effect of improving behavioral and motor function, and also exhibits the effect of preventing and inhibiting Parkinson's disease. [Brief explanation of the drawings]

[0027] Detailed Description of the Drawings [Figure 1] [Figure 1] is a chemical formula showing the process of generating and decomposing endogenous aldehydes from ethanol and monoamines in vivo.

[0028] 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).

[0029] Endogenous monoamines (R-C2H4-NH2) are converted to aldehydes (R-CH2-CHO) by the monoamine oxidase (MAO) enzyme, which are subsequently detoxified to acetate (R-CH2-CO2H) by aldehyde dehydrogenase and alcohol dehydrogenase reactions, in the same manner as alcohol metabolism.

[0030] [Figure 2] Figure 2 shows the chemical formula for the synthesis and metabolism of dopamine. Dopamine (DA), a typical monoamine neurotransmitter, is converted by monoamine oxidase (MAO) into toxic aldehyde structures such as DOPAL and MOPAL. These are then decomposed by aldehyde dehydrogenase (ALDH) and ultimately metabolized to the relatively less toxic homovanillic acid (HVA).

[0031] Dopamine is also converted by monoamine oxidase to dopanal via norepinephrine (NE), such as dopegal, a known toxic substance, which is finally decomposed into an acid compound by aldehyde dehydrogenase.

[0032] Additionally, dopamine metabolism may be impaired for various reasons, including a decrease in ALDH during dopamine metabolic degradation, leading to increased accumulation of dopanol (dopamine-induced alcohols) such as DOPET in vivo, because dopanol cannot be converted to less toxic acid compounds.

[0033] 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.

[0034] Despite the existence of various dopamine enzymatic metabolic pathways, when enzymatic dopamine metabolism fails, dopamine is metabolized via non-enzymatic reactions, where it is spontaneously converted into a quinone derivative by reactive oxygen species (ROS), which is then converted into neuromelanin. In this case, rapid changes in melanin distribution disrupt homeostasis, leading to various diseases.

[0035] [Figure 3] [Figure 3] is a graph showing the ability of KARC to decompose acetaldehyde in vivo.

[0036] [Figure 4] [Figure 4] is a graph showing the ability of KARC to degrade malondialdehyde in vivo.

[0037] In animal experiments in which blood levels of acetaldehyde [Figure 3] and malondialdehyde [Figure 4], endogenous toxic aldehydes, were increased by alcohol consumption, KARC's effects of reducing aldehydes and oxidative stress were confirmed.

[0038] [Figure 5][Figure 5] is a graph showing the ability of KARC to decompose acetaldehyde in the human body.

[0039] [Figure 6-7] [Figure 6] and [Figure 7] are graphs showing the ability of KARC to decompose malondialdehyde in the human body.

[0040] In studies to confirm the reduction of endogenous blood acetaldehyde [Figure 5] and blood malondialdehyde [Figure 6] in humans, the effects of KARC administration appear to be reducing acetaldehyde and malondialdehyde, which are biomarkers for hangovers, fatigue, and cardiovascular disease. [Figure 7] shows the effect of KARC administration in reducing oxidative stress by lowering malondialdehyde, a biomarker for oxidative stress and reactive oxygen species, in a state where oxidative stress is increased due to medication, etc.

[0041] [Figure 8] [Figure 8] Protocol for the MPTP model: preparation of MPTP mice and dosage.

[0042] [Figure 9] [Figure 9] Rotenone model protocol: general preparation and dosage.

[0043] [Figure 10] [Figure 10] shows the protocol for an acute neuronal inflammation model induced by LPS (lipopolysaccharide).

[0044] [Figure 11] FIG. 11 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).

[0045] [Figure 12] [Figure 12] shows the results of the dopamine turnover index in an animal model of Parkinson's disease (PD).

[0046] When Parkinson's disease was induced in animals using rotenone, dopamine secretion decreased. L-Dopa had no effect on the recovery of the decreased dopamine metabolism. On the other hand, when KARC of the present invention was administered, the decreased dopamine metabolism was recovered and DOPAL was also reduced (Figures 11 and 12).

[0047] [Figure 13] [FIG. 13] shows the results of immunostaining of changes in TH-positive neurons in the substantia nigra pars compacta (SNpc) and TH-positive fibers in the striatum of a PD animal model induced by oral administration of KARC.

[0048] Rotenone administration caused massive neuronal death in dopaminergic neurons, which secrete dopamine. KARC treatment clearly prevented neuronal cell death and restored the therapeutic effect on neurons.

[0049] [Figure 14] [Figure 14] shows the results of TH-positive neurons in the SNpc of PD mice treated with KARC.

[0050] [Figure 15] [Figure 15] shows the results of changes in TH-positive fibers in PD mice after KARC administration.

[0051] Induction of Parkinson's disease by MPTP resulted in a decrease in the density of dopaminergic neurons in the SNpc, which produce and secrete dopamine, due to neuronal cell death, and a decrease in the expression levels of enzymes that promote dopamine production. Finally, dopamine secretion was reduced, resulting in a whitening phenomenon in the substantia nigra (SNpc).

[0052] Induction of Parkinson's disease by MPTP resulted in a decrease in the density of dopaminergic neurons in the SNpc, which produce and secrete dopamine, due to neuronal cell death, and a decrease in the expression levels of enzymes that promote dopamine production.Finally, dopamine secretion was reduced, resulting in a whitening phenomenon in the substantia nigra (SNpc).

[0053] However, in the KARC-treated group, the density of dopaminergic neurons increased [Figures 13 and 15], the expression level of enzymes that promote dopamine production increased [Figure 14], and the color of the substantia nigra was restored due to increased dopamine secretion [Figure 13].

[0054] [Figure 16] [FIG. 16] shows the results of immunostaining for changes in TH and α-synuclein in neurons of a PD model following administration of KARC.

[0055] [Figure 17] Figure 17 shows the results of alpha-synuclein immunostaining in neurons of a KARC-administered PD model. Abnormal accumulation of alpha-synuclein aggregates was confirmed in the substantia nigra of rotenone-induced Parkinson's disease model animals.

[0056] Alpha-synuclein aggregates, a neurotoxic substance, were reduced in both the L-dopa (dopamine precursor) and KARC groups (Figure 16).

[0057] Examination of the density of dopamine neurons confirmed that the KARC-administered group recovered in a similar manner to the L-Dopa-administered group [Figure 17].

[0058] [Figure 18] [Fig. 18] shows the results of a rotarod test of a PD model using the KARC of the present invention.

[0059] In Parkinson's model animals, muscle endurance improved in a dose-dependent manner, with the high-dose group (20 units / kg) showing a higher recovery rate compared with the reference drug rasagiline.

[0060] [Figure 19] FIG. 19 shows the results of a pole test in a PD animal model using the KARC of the present invention.

[0061] In an animal model of Parkinson's disease, motor neuron damage was reduced in all drug treatment groups (rasagiline and KARC). In particular, the high dose of KARC (20 units / kg) showed a similar protective effect against motor neuron damage caused by Parkinson's disease as the reference drug, rasagiline.

[0062] [Figure 20] FIG. 20 shows the results of a postural instability test in a PD animal model using the KARC of the present invention.

[0063] [Figure 21]

[0043] FIG. 21 shows the results of a toe drag test in a PD animal model using the KARC of the present invention.

[0064] [Figure 22] [Figure 22] shows the results of a pasta handling test in a PD animal model using the KARC of the present invention.

[0065] Experiments on the behavior of model animals, such as postural instability [Figure 20], stride length [Figure 21], and pasta grip strength [Figure 22], confirmed that Parkinson's disease symptoms improved in all groups (L-Dopa and KARC). The KARC group, administered for preventative purposes, showed better effects in improving Parkinson's disease symptoms than the reference drug L-Dopa.

[0066] [Figure 23] [Figure 23] shows the results of immunostaining for the inflammatory response of microglia in a PD animal model using the KARC of the present invention.

[0067] [Figure 24] FIG. 24 shows the results of measuring changes in inflammatory responses of crogliata in a PD animal model using the KARC of the present invention.

[0068] In an animal model of PD with neuronal inflammation induced by LPS (lipopolysaccharide), the activation level of microglia, which is known to be the cause of brain neuron death, was observed via Iba-1 immunostaining. When KARC was administered, the activation level of microglia in the KARC-treated group was reduced [Figure 23], and similarly, the number of activated microglia was also reduced [Figure 24].

[0069] [Figure 25] [Figure 25] shows the results of immunostaining for inflammatory responses of astrocytes in a PD animal model using the KARC of the present invention.

[0070] [Figure 26]

[0073] FIG. 26 shows the results of measuring changes in the inflammatory response of astrocytes in a PD animal model using the KARC of the present invention.

[0071] In PD animal models, astrocytic inflammation induces neuronal death and simultaneously leads to impaired brain homeostasis through the creation and maintenance of neuronal synapses. Astrocyte activation was measured via GFAP immunostaining, a measure of astrocyte inflammatory responses. KARC administration reduced the abnormal inflammatory activation of astrocytes [Figure 25], as well as the number of activated astrocytes [Figure 26]. Notably, the inflammation-reducing effect increased dose-dependently with KARC administration.

[0072] [Figure 27] [Fig. 27] shows the change in enzyme activity when the KwonP-1 strain was orally administered.

[0073] [Figure 28][Figure 28] shows the change in enzyme activity when the KwonP-2 strain was orally administered.

[0074] [Figure 29] [Figure 29] shows the change in enzyme activity when the KwonP-3 strain was orally administered.

[0075] [Figure 30] [Figure 30] shows the change in enzyme activity when the Pico YP strain was orally administered.

[0076] [Figure 31] [Figure 31] shows the change in enzyme activity when the PicoYP-01 strain was orally administered.

[0077] [Figure 32] [Figure 32] shows the change in enzyme activity when the PicoYP-02 strain was orally administered.

[0078] [Figures 27, 28, 29, 30, 31, and 32] show that KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02 were orally administered for 90 minutes under conditions similar to the human gastric digestion process (1 < pH < 5). The change in ALDH enzyme activity was measured. [[ID=​​​​​​​​​​​​​​​

[0082] [Figure 35] [Figure 35] shows the growth curve and enzyme activity of the KwonP-3 strain cultured in a 5 L fermenter.

[0083] [Figure 36] [Figure 36] shows the growth curve and enzyme activity of the PicoYP strain cultured in a 5 L fermenter.

[0084] [Figure 37] [Figure 37] shows the growth curve and enzyme activity of the PicoYP-01 strain cultured in a 5 L fermenter.

[0085] [Figure 38] [Figure 38] shows the growth curve and enzyme activity of the PicoYP-02 strain cultured in a 5 L fermenter.

[0086] In Figures 33, 34, 35, 36, 37, and 38, the novel mutant strains KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-01 were cultured in a 5-liter fermenter using YPD medium under the same conditions at 30°C and 200 rpm for 48 hours.

[0087] When the growth curves (OD660nm) and ALDH enzyme activity of each strain were compared with those of the type strain, 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.

[0088] [Figure 39] [Figure 39] shows the results of oral administration of KARC in Camk-PARIS Parkinson's disease transgenic mice.

[0089] [Figure 40]FIG. 40 shows changes in the expression of Parkinson's disease marker proteins such as PARIS, TH, GFAP, and Neun in brain tissue of Camk-PARIS Parkinson's disease transgenic mice when KARC was orally administered.

[0090] When KARC (20 units / kg / day) was orally administered to Camk-PARIS Parkinson's disease transgenic mice for 4 weeks, the results of the pole test showed that the T-LA value was 23 seconds, which was a 36.08% reduction compared to the KARC-free group [Figure 39].

[0091] Additionally, protein expression levels were confirmed using PARIS (Parkin-interacting substrate), TH (tyrosine hydroxylase), GFAP (glial fibrillary acidic protein), and NeuN (neuronal nucleus) antibodies, respectively, to measure the number of surviving neurons after KARC administration. As NeuN activity increased and GFAP activity decreased, neuronal damage was confirmed to be reversed. Finally, in Camk-PARIS Parkinson's disease transgenic mice, astrocyte inflammation was reduced and neuronal damage was reversed. [Figure 40]

[0092] [Figure 41] [Figure 41] is an HPLC spectrum of a mixture of distilled water and DOPAL.

[0093] [Figure 42] [Figure 42] is an HPLC spectrum of a mixture of KARC and DOPAL of the present invention after being kept at 30°C for 1 hour.

[0094] [Figure 43] [Figure 43] is an HPLC spectrum of a mixture of KARC and DOPAL of the present invention after being kept at 30°C for 3 hours.

[0095] [Figure 44] [Figure 44] is an HPLC spectrum of a mixture of KARC and DOPAL of the present invention after being kept at 37°C for 1 hour.

[0096] [Figure 45] [Figure 45] shows the HPLC spectrum of a mixture of KARC and DOPAL after being kept at 37°C for 3 hours.

[0097] In Figures 41, 42, 43, 44, and 45, 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.

[0098] DOPAC increased at 6 minutes, confirming that KARC oxidizes DOPAL and converts it to DOPAC.

[0099] [Figure 46] [Figure 46] is an HPLC spectrum of a mixture of distilled water and succinic semialdehyde (SSA).

[0100] [Figure 47] [Figure 47] shows an HPLC spectrum of a mixture of KARC and SSA of the present invention after being kept at 37°C for 1 hour.

[0101] [Figure 48] [Figure 48] shows the HPLC spectrum of a mixture of KARC and SSA after being kept at 37°C for 3 hours.

[0102] In Figures 46, 47, and 48, 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.

[0103] [Figure 49] [Figure 49] is an HPLC spectrum of a mixture of distilled water and acetaldehyde.

[0104] [Figure 50][Figure 50] is an HPLC spectrum of a mixture of KARC of the present invention and acetaldehyde after being kept at 30°C for 1 hour.

[0105] [Figure 51] [Figure 51] is an HPLC spectrum of a mixture of KARC of the present invention and acetaldehyde after being kept at 30°C for 3 hours.

[0106] [Figure 52] [Figure 52] is an HPLC spectrum of a mixture of KARC of the present invention and acetaldehyde after being kept at 37°C for 1 hour.

[0107] [Figure 53] [Figure 53] shows the HPLC spectrum of a mixture of KARC and acetaldehyde after being kept at 37°C for 3 hours.

[0108] When acetaldehyde, a representative endogenous aldehyde and known carcinogen, was treated with KARC for 1 hour, it was oxidized 100% not only at 30°C but also at 37°C [Figures 49, 50, 51, 52, 53].

[0109] [Figure 54] [Figure 54] is an HPLC spectrum of a mixture of distilled water and glyoxal.

[0110] [Figure 55] [Figure 55] is an HPLC spectrum of a mixture of KARC of the present invention and glyoxal after being kept at 30°C for 1 hour.

[0111] [Figure 56] [Figure 56] is an HPLC spectrum of a mixture of KARC of the present invention and glyoxal after being kept at 30°C for 3 hours.

[0112] [Figure 57][Figure 57] is an HPLC spectrum of a mixture of KARC of the present invention and glyoxal after being kept at 37°C for 1 hour.

[0113] [Figure 58] [Figure 58] shows the HPLC spectrum of a mixture of KARC and glyoxal after being kept at 37°C for 3 hours.

[0114] 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 54, 55, 56, 57, 58].

[0115] [Figure 59] [Figure 59] is an HPLC spectrum of a mixture of distilled water and trans-cinnamaldehyde.

[0116] [Figure 60] [Figure 60] 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.

[0117] [Figure 61] [Figure 61] is an HPLC spectrum of a mixture of KARC of the present invention and trans-cinnamaldehyde after being kept at 30°C for 3 hours.

[0118] [Figure 62] [Figure 62] 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.

[0119] [Figure 63] [Figure 63] shows the HPLC spectrum of a mixture of KARC and trans-cinnamaldehyde after being kept at 37°C for 3 hours.

[0120] 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 59, 60, 61, 62, 63]

[0121] [Figure 64] [Figure 64] is an HPLC spectrum of a mixture of distilled water and benzaldehyde.

[0122] [Figure 65] [Figure 65] is an HPLC spectrum of a mixture of KARC of the present invention and benzaldehyde after maintaining it at 30°C for 1 hour.

[0123] [Figure 66] [Figure 66] is an HPLC spectrum of a mixture of KARC of the present invention and benzaldehyde after keeping it at 30°C for 3 hours.

[0124] [Figure 67] [Figure 67] is an HPLC spectrum of a mixture of KARC of the present invention and benzaldehyde after being kept at 37°C for 1 hour.

[0125] [Figure 68] [Figure 68] shows the HPLC spectrum of a mixture of KARC and benzaldehyde after keeping it at 37°C for 3 hours.

[0126] 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 64, 65, 66, 67, 68] DETAILED DESCRIPTION OF THE INVENTION

[0127] 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.

[0128] 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]

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] [Table 1]

[0140] [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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] [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.

[0145] 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.

[0146] 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.

[0147] 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).

[0148] 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.

[0149] [Table 2] TIFF2025539868000006.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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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 This was more than double the rate of increase.

[0156] 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.

[0157] 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.

[0158] [Table 3]

[0159] [Table 4]

[0160] [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).

[0161] The characteristics and novelty of the carbon source selection of the strains were analyzed by API 50 CHL kit (API systems, BIOMERIEUX, SA, France).

[0162] 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.

[0163] 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.

[0164] 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 +++.

[0165] 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.

[0166] 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.

[0167] 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].

[0168] [Table 5]

[0169] [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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] [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.

[0181] 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).

[0182] 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).

[0183] 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).

[0184] 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].

[0185] 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].

[0186] 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).

[0187] [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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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).

[0195] 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%.

[0196] 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%.

[0197] KARC4 was produced from PicoYP. The enzymatic activities of ADH and ALDH in 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%.

[0198] 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%.

[0199] 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%.

[0200] 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%.

[0201] 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).

[0202] [Table 6]

[0203] [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.

[0204] 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.

[0205] 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%).

[0206] 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).

[0207] 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.

[0208] 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.

[0209] 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].

[0210] [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.

[0211] After administration was completed, 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 3, 4].

[0212] 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).

[0213] 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 3).

[0214] 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.

[0215] 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-treated group (F) were 0.232 and 0.137 μM, respectively, representing decreases of 80.4% and 86.3% compared to the control group (Figure 4).

[0216] [Example 10] Effect of oral administration of KARC on behavior and motor function improvement We established a mouse model of Parkinson's disease using MPTP and rotenone, and observed the effects of oral administration of KARC on behavioral and motor function improvement. The pharmacological effects of KARC on Parkinson's disease were verified by behavioral and motor performance (rotarod test, pole test, postural instability test, pasta handling test) and by alterations in brain substances (DOPAL, DOPAC, HVA, and α-synuclein).

[0217] 10-1: Preparation of PD mouse model via MPTP injection Male C57 / BL6 mice (8 weeks, 19-23 g) were divided into six groups (n = 5 / group) for the experiment: a control group receiving PBS injections (CTL, vehicle), a control group receiving PBS injections along with oral administration of KARC (20 units / kg / day), and experimental groups receiving MPTP injections along with oral administration of either KARC (0, 10, or 20 units / kg / day) or rasagiline (0.1 mg / kg / day). All animals had free access to food and water.

[0218] Mice were orally administered KARC (0, 10, or 20 units / kg / day) or rasagiline (0.1 mg / kg / day) daily for 19 days. Starting on the 10th day of KARC administration, they were given intraperitoneal injections of MPTP (30 mg / kg / day) for a period of 5 days (Figure 8).

[0219] 10-2: Preparation of PD mouse model via rotenone injection 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.

[0220] To confirm the preventive and therapeutic effects of KARC on Parkinson's disease, two administration methods were employed. KARC (20 units / kg) was orally administered simultaneously with rotenone to observe the preventive effects on Parkinson's disease. KARC (20 units / kg) or L-dopa was orally administered two weeks after rotenone to observe the therapeutic effects on Parkinson's disease. Brain tissue was isolated and stored in liquid nitrogen at -80°C to quantify dopamine. [Figure 9]

[0221] 10-3: Preparation of a neuroinflammatory mouse model via lipopolysaccharide (LPS) injection Male C57 / BL6 mice (8 weeks, 19-23 g) were divided into six groups (n = 5 / group) for the experiment: a control group receiving PBS injections (CTL, vehicle), a control group receiving PBS injections along with oral administration of KARC (20 units / kg / day), and experimental groups receiving LPS injections along with oral administration of either KARC (0, 10, or 20 units / kg / day) or quercetin (10 mg / kg / day). All animals had free access to food and water.

[0222] Mice were orally administered ARC (0, 10, or 20 units / kg / day) or quercetin (10 mg / kg / day) daily for 3 days, followed by injections of LPS (10 mg / kg / day) or PBS. Mice were sacrificed 3 hours after injection of LPS or PBS (Figure 10).

[0223] 10-4: Changes in behavior and motor function MPTP-treated PD model mice were subjected to the rotarod and pole tests to assess balance and leg movement ability, while rotenone-treated PD model rats were subjected to balance, foot-dragging, and pasta handling tests to assess leg movement ability.

[0224] 10-4-1:Rotarod test To observe the motor and balance abilities of the mice, a rotarod test was performed. On days 18 and 19 after KARC administration, the mice underwent preparatory training on the rotating rod of the rotarod unit set (ROTA-ROD, SK-RO).

[0225] During the pre-training period, a rotation speed of 2 rpm was maintained on the rotating rod (100 mm diameter) for 5 minutes. Once stabilized, the speed was gradually increased to 1 rpm per 6 seconds, and the time remaining on the rod and the time to fall off the device were recorded. Pre-training was conducted three times a day, and the main experiment was conducted in the same manner as pre-training on the 20th day after KARC administration.

[0226] MPTP-injected mice showed a 65.9% reduction in latency to fall on the rotorod, from 218.4 seconds to 74.5 seconds, compared with normal mice [Figure 18]. Mice treated with KARC showed a concentration-dependent increase of 52.0% and 61.0% in the time spent on the rotorod, to 155.1 seconds and 190.8 seconds, respectively, compared with MPTP-injected mice. The reference drug rasagiline had an MPTP of 175.8 seconds, a 57.6% increase over the group that received MPTP.

[0227] 10-4-2: Pole test A pole test was performed to observe the motor skills and balance abilities of the mice's forelimbs and hindlimbs. The mice were attached to the top of a sturdy pole (8 mm diameter, 55 cm high) that provided sufficient grip strength for descending, and the time it took for all four paws to contact the ground was recorded as the descending latency (T-LA).

[0228] MPTP-injected mice showed a 46.5% increase in time to bed (T-LA), from 6.9 seconds to 10.1 seconds, compared with normal mice [Figure 19]. KARC (10, 20 units / kg / day) treatment reduced T-LA by 29.8% and 36.7%, to 7.1 and 6.4 seconds, respectively, compared with MPTP treatment. The reference drug, rasagiline (0.1 mg / kg / day), shortened T-LA by 35.7% compared with MPTP, at 6.5 seconds.

[0229] 10-4-3: Postural instability test To assess rat balance sensitivity, a postural instability test was performed. The rat's body and one front paw were gently held. The rear end was elevated, and the rat was gently moved forward from a height where the front paw barely touched the surface. The rat's center of gravity shifted forward, and the rat moved its front paw to regain balance. This process was repeated twice, and the distance traveled by the paw to regain balance was measured on the left and right sides, respectively, and averaged.

[0230] 10-4-4: Pasta handling test A pasta handling test was conducted to determine the rats' forepaw coordination and motor skills. In this test, a piece of pasta was placed on the floor near the front of the cage. This was measured by recording the time it took the rat to eat the pasta piece, the forepaw used, and the position of the forepaw, as seen on a pasta handling score sheet.

[0231] As Parkinson's disease progresses, "balance impairment" and "rigidity" are observed, and "balance impairment" was confirmed by "rigidity" in tapping, toe dragging, and pasta handling tests. In the evaluation of postural instability and observation of toe dragging, a significant reduction in stride length was observed in a rat model of Parkinson's disease (PD) using rotenone. L-dopa, a treatment for Parkinson's disease, improved balance impairment.

[0232] The KARC of the present invention exhibited similar effects to L-dopa in improving balance deficits. It demonstrated significantly greater efficacy in preventing balance deficits compared with L-dopa. "Toe dragging" was observed in 71.4% of the rotenone-administered group, but in less than 14.3% of the L-dopa and KARC-administered group (Figures 20 and 21). In the pasta handling test, rotenone significantly induced paw stiffness, resulting in a 40.0% increase in pasta consumption time compared with the control group (Figure 22). The L-dopa group showed substantial improvement in paw stiffness, with a 35.7% reduction in pasta consumption time compared with the rotenone group (vehicle). Administration of the KARC of the present invention reduced pasta consumption time by 42.9%, significantly preventing Parkinson's disease.

[0233] The treatment group administered the KARC of the present invention showed no significant improvement in rigidity, but had a 14.3% increase. This data is significant because it demonstrates that the KARC of the present invention can improve behavioral symptoms associated with Parkinson's disease, such as balance deficits and rigidity, and is more effective than the therapeutic agent L-dopa when used for preventative purposes.

[0234] 10-5: Changes in brain neurotransmitters 10-5-1: 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 brain striatum and muscle samples was measured by HPLC / MMS. Samples were dissolved in trichloroacetic acid (3.0 M / 100 μl), and then isoproterenol (1 nmol / ml, 100 μl) was added. The samples were then centrifuged using a Toyopack SP carton (Toso, Tokyo, Japan).

[0235] 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 solution was injected into HPLC to measure dopamine.

[0236] 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 via HPLC / MMS. The DOPAC content was calculated as ng / g tissue.

[0237] To investigate the changes in dopamine metabolism in the brains of PD model animals, rotenone, DA, DOPAL, DOPAC, and HVA were measured using HPLC [Figure 11].

[0238] 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.

[0239] 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.

[0240] 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 to the rotenone-treated group, while DOPAL was relatively decreased.

[0241] 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.

[0242] 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 12].

[0243] 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.

[0244] 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.

[0245] This indicates that the metabolic intermediate DOPAL accumulates in vivo. KARC administration inhibited the accumulation of the neurotoxin DOPAL, restored normal dopamine metabolism, and accelerated 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.

[0246] 10-5-2: Preparation of brain tissue Mice were anesthetized by injection of chloral hydrate (40 mg / kg, i.p.), perfused transcardially with saline containing 0.5% sodium nitrate and heparin (10 U / ml), and then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.2).

[0247] Whole brain tissue was dissected from the skull and post-fixed overnight in 4% paraformaldehyde in 0.1 M PB at 4° C. It was stored at 4° C. in a 30% sucrose solution in 0.05 M PBS until it sank.

[0248] Brains were cryosectioned into 30 μm-thick coronal sections on a Cryostat (Microsystems AG, Leica, Wetzlar, Germany) and stored at 4°C in cryoprotectant (25% ethylene glycol, 25% glycerol, 0.2 M PB, and water) until use.

[0249] Striatum (ST) brain sections and substantia nigra (SN) sections were designated as one series of 6 sections, resulting in a total of 36 brain sections, and brain changes were observed via immunostaining.

[0250] * 10-5-3: Immunohistochemical staining Brain sections (30 μm thick) containing the SN and ST were incubated with rabbit anti-tyrosine hydroxylase (TH, 1:2000; Pel-Freez Biologicals, Rogers, AR, USA) for dopaminergic neurons.

[0251] Brain sections (30 μm thick) containing the SN or hippocampus were incubated with rabbit anti-glial fibrillary acidic protein (anti-GFAP, 1:5000; Neuromics, Edina, MN, USA) or rabbit anti-ionized calcium-binding adapter molecule 1 (anti-Iba-1, 1:1000; Wako, Osaka, Japan), respectively, followed by staining with biotinylated anti-rabbit IgG and an avidin-biotin peroxidase complex (ABC) standard kit (Vector Laboratories, Burlingame, CA, USA).

[0252] Signals were detected by incubating sections with 0.5 mg / ml 3,3′-diaminobenzidine (Sigma, St. Louis, MO, USA) in 0.1 M PBS containing 0.003% H O .

[0253] To quantify GFAP- or Iba-1-positive cells, stained brain sections were imaged under a bright-field microscope (Olympus Optical, Tokyo, Japan). Results were quantified by counting the number of TH-immunopositive neurons in the SN in brain tissue sections (5 sections / series) at ×100 magnification. TH-immunopositivity in the ST was measured by the optical density of TH-positive fibers at ×40 magnification using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

[0254] 10-5-4: Three-dimensional cell counting An unbiased stereoscopic estimation of the total number of TH-immunopositive neurons in the SN was performed using the optical fractionator method implemented on Olympus CAST (Computer-Aided Stereoscopic Toolbox System) version 2.1.4. Actual counting was performed using a ×100 objective. The estimate of the total number of neurons was calculated according to the optical fractionator equation:

[0255] 10-5-5: Protective effect on dopaminergic neurons To examine the effect of KARC on dopaminergic neurons, mouse brains were immunostained using anti-TH antibody [Figure 13]. Dopaminergic neurons were quantified by stereological counting of TH-positive neurons in the SNpc and absorbance of TH-positive fibers in the striatum.

[0256] In PD model mice treated with MPTP, there was a decrease in TH-positive neuronal death in the SNpc (38.40±12.31%, p<0.001, n=5) [Figure 14] and a decrease in TH-positive fibers in the striatum (36.82±12.25%, p<0.001) [Figure 15] compared to the control group.

[0257] Mice treated with KARC (10 or 20 units / kg) showed a significant increase in TH-positive neurons in the SN (1 g / kg: 59.4 ± 30.3%, p < 0.001; 2 g / kg: 66.37 ± 19.00%, p < 0.001) and TH-positive fibers in the striatum (1 g / kg: 74.02 ± 13.49%, p < 0.001; 2 g / kg: 85.14 ± 10.43%, p < 0.001) compared to vehicle controls.

[0258] Rasagiline also increased TH-positive neurons in the SNpc (74.87±16.19%, p<0.01) and TH-positive fibers in the striatum (105.3±10.74%, p<0.01), but showed a weaker effect compared with KARC.

[0259] 10-5-6: Inhibitory effect on α-synuclein aggregation in dopaminergic neurons We investigated the effect of KARC on intracellular α-synuclein accumulation in the SNpc of rotenone-treated PD rat models. Substantia nigra sections were co-immunostained with antibodies against TH (red) and α-synuclein (green). Sections were analyzed using a laser scanning confocal microscope (Figure 16).

[0260] Abnormal aggregation of α-synuclein in the substantia nigra of the brain leads to increased endoplasmic reticulum stress and neuroinflammation, resulting in neuronal death. In normal rats, no accumulation of α-synuclein was observed in TH-positive neurons. In the rotenone-treated group, a decrease in TH-positive neurons and accumulation of α-synuclein were observed compared to the control group.

[0261] KARC and L-dopa treatment groups significantly reduced dopaminergic neuron loss and α-synuclein accumulation compared with rotenone treatment. Preventive treatment with KARC reduced α-synuclein accumulation compared with L-dopa treatment [Figure 17].

[0262] 10-5-7: Inhibitory effects on neuroinflammatory microglial and astrocyte activation We observed the effects of KARC on the activation of neurons, microglia, and astrocytes in the SN of an LPS-induced neuroinflammatory PD mouse model. Substantia nigra sections were analyzed by immunostaining with Iba-1 (microglia) [Figure 23] and GFAP (astrocytes) [Figure 25] antibodies.

[0263] In the SN tissue of mice injected with LPS (10 mg / kg), morphological changes were observed in activated microglia or astrocytes, with enlarged cytoplasm and sharpened, elongated neurites, while in the SN tissue of mice treated with KARC (10 units / kg, 20 units / kg) or quercetin (10 mg / kg), activated microglia and astrocytes showed a resting morphology with smaller cell bodies and shorter neurites, indicating that their activation was inhibited [Figures 23, 25].

[0264] In LPS-injected mice, the numbers of activated GFAP- or Iba-1-positive cells increased more than fourfold compared to the total numbers of microglia (344.3 ± 51.13%, p < 0.001) and astrocytes (325.0 ± 55.28%, p < 0.05). In the SN treated with KARC, the numbers of Iba-1-positive microglia (1 g / kg: 138.6 ± 16.23%, p < 0.001; 2 g / kg: 107.1 ± 8.571%, p < 0.001) and GFAP-positive astrocytes (2 g / kg: 127.5 ± 7.5%, p < 0.01) were largely reduced.

[0265] Quercetin similarly reduced GFAP-positive astrocytes (115.0±15.61%, p<0.001) and Iba-1-positive microglia (107.1±19.64%, p<0.001). The number of activated microglia and astrocytes (per section) confirmed that KARC inhibited the activation of both cells (Figures 24 and 26).

[0266] Neuroinflammation is known to be a factor leading to dopamine neuron degeneration and neuronal cell death. Common features of neurodegenerative diseases caused by neuroinflammation include microglial activation, astrogliosis, and lymphocyte infiltration. Under normal conditions, microglia protect the nervous system by removing protein fragments, killing pathogens, and regulating innate and adaptive immune responses.

[0267] After nerve injury, stroke, trauma, and various neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis, microglia become activated and release proinflammatory cytokines and neurotoxic substances, including TNF-α (tumor necrosis factor alpha), IL-1 alpha (interleukin-1 alpha), IL-6 (interleukin-6), and NO (nitric oxide).

[0268] It has been suggested that enhanced activation of NF-kappa B (nuclear factor-kappa B) may contribute to the maintenance of the proinflammatory microglial activation state and progressive neurodegeneration in PD. Therapeutic intervention in the inflammatory response is proposed to be useful in preventing disease progression or halting the pathological process of PD.

[0269] Iba-1 (ionized calcium-binding adaptor molecule 1), a type of allograft inflammatory factor-1 (AIF-1), is a protein specifically expressed in microglia in the central nervous system. Iba-1 is overexpressed in microglia following neuronal injury, CNS ischemia, and various other brain disorders.

[0270] The reduction in Iba-1 expression indicates that KARC has neuroprotective effects and ameliorates CNS ischemia, further pointing to the improvement of various brain disorders.

[0271] GFAP (glial fibrillary acidic protein) is a fibrillary acidic protein present in brain neurons and is used as a marker for astrocytes, stellate cells distributed throughout the central nervous system (CNS). GFAP is involved in various functions, including the formation of neuronal myelin, regulating motility, and is important for maintaining the shape of astrocytes.

[0272] In mammals, GFAP expression is specifically increased in damaged astrocytes in the brain following various physical and chemical causes. Ultimately, the increase in damaged astrocytes leads to the induction of inflammation and the death of brain neurons, accelerating the progression of neurodegenerative brain diseases.

[0273] The reduction in GFAP indicates the restoration of damaged astrocytes by KARC, pointing to a preventive and therapeutic effect on neurodegenerative brain diseases.

[0274] Treatment with KARC significantly inhibited LPS-induced neuroinflammatory responses, as assessed by the degree of microglial and astrocyte activation. These data suggest that KARC has potential for the treatment of neuroinflammation in vivo.

[0275] These results show that KARC improved behavioral and motor function in MPTP-treated, rotenone-treated, and LPS-treated PD mouse models. It also had significant effects on regulating PD biomarkers in the brain. The data suggest that when administered prophylactically, KARC is more effective in preventing and ameliorating PD than L-dopa, the currently used treatment.

[0276] [Example 11] 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.

[0277] 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.

[0278] Blood acetaldehyde concentrations were measured over time after alcohol consumption [Figure 5]. The area under the curve (AUC) of blood acetaldehyde (Ach) was 13.02 ± 1.18 mgh / dL after alcohol consumption alone. When administered at a dose of 10 units / kg of KARC, the area under the curve (AUC) of blood acetaldehyde (Ach) was significantly reduced by 26.13% compared to alcohol consumption alone, measuring 9.39 ± 1.07 mgh / dL (P = 0.005).

[0279] 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.

[0280] The reduction in blood acetaldehyde (Ach) levels resulting from KARC administration has a positive impact on reducing oxidative stress and promoting health.

[0281] The blood malondialdehyde (MDA) concentration was measured during chemotherapy (Figure 6). 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).

[0282] 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 7.

[0283] Various factors, such as drug intake, stress, and strenuous physical exercise, lead to an increase in intracellular reactive oxygen species, which trigger 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 (Ach), and dopamine-derived aldehydes, accumulate intracellularly and exacerbate oxidative stress.

[0284] 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, further enhancing oxidative stress.

[0285] 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). Aldehyde-related chain reactions result in the accumulation of stable end glycoxidation 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.

[0286] 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 reinforces and amplifies each other, leading to increased endoplasmic reticulum stress (ER stress).

[0287] 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 concentrations in the bloodstream, demonstrating its ability to reduce reactive oxygen species and oxidative stress.

[0288] 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 ROS and oxidative stress. This suggests that by regulating intracellular ROS and oxidative stress, KARC inhibits neuronal cell apoptosis, thereby suppressing and preventing Parkinson's disease. This leads to improvements in behavior and motor function.

[0289] [Example 12] Acute oral administration test 12-1. Preparation of experimental animals *The experimental animals were female and male ICR mice (7 weeks old). The mice were allowed to acclimate for 7 days. The general symptoms of the adopted mice were observed during the acclimatization period, and only healthy animals were used for the short-term toxicity test. Food and water were consumed 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.

[0290] 12-2. 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.

[0291] 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.

[0292] 13-3. Observation and autopsy All animals in the test groups were observed for symptoms at least once daily 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 necropsy, changes in each organ were observed macroscopically.

[0293] 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 abnormalities, such as changes in weight gain or food intake, were observed in the mice. Necropsies performed after the end of the observation period revealed no abnormal findings.

[0294] [Example 13] Effect of KARC on CamK-PARIS Parkinson's disease transgenic mice To observe the effects of oral administration of KARC on motor function and neuronal recovery, we generated Camk-PARIS PD transgenic mice. The efficacy of KARC was confirmed through motor performance (pole test) and neuronal changes in brain tissue (TH, GFAP, and Neun).

[0295] 13-1: Production of CamK-PARIS Parkinson's disease transgenic mice Camk-PARIS PD Tg mice were generated by crossing CamKIIa-tTA mice with TetP-PARIS mice (CamKIIa-tTA;TetP-PARIS). To suppress PARIS expression, Camk-PARIS PD Tg mice were fed a doxycycline-containing chow diet for 1.5–1.8 months and then switched to a normal chow diet to induce PARIS expression.

[0296] All groups of mice were orally administered either water or KARC (20 units / kg / day) daily for 4 weeks, starting 1 week before the onset of PARIS expression. All animals had free access to food and water. KARC was orally administered for 4 weeks before behavioral evaluation of Camk-PARIS PD Tg mice. Mice were sacrificed, and brain regions were isolated for histopathological evaluation. Mouse brains were immediately frozen and stored in liquid nitrogen at -80°C.

[0297] 13-2: Changes in motor ability in CamK-PARIS Parkinson's disease transgenic mice The pole test was performed to assess forelimb and hindlimb motor skills and balance in Camk-PARIS PD Tg mice. Mice were fixed to the top of a pole (8 mm diameter, 55 cm high) with a rough surface that provided sufficient grip for the mice to descend. The time required for all four paws to land on the floor was recorded as the time of spontaneous motor activity (T-LA).

[0298] The effectiveness of KARC in behavioral testing related to motor impairment caused by Parkinson's disease was evaluated in Camk-PARIS PD Tg mice using the pole test. There was no difference in the T-LA time of the pole test between groups of WT normal mice orally administered water and KARC (20 units / kg / day). The T-LA time of Camk-PARIS PD Tg mice was significantly increased, with an average of 23 seconds, compared with the WT mouse group. The T-LA time of mice orally treated with KARC (20 units / kg / day) was significantly shortened by 36.08%, with an average of 8.3 seconds [Figure 39].

[0299] 13-3: Preparation of brain tissue Proteins were isolated from frozen brain tissue at -80°C. Proteins were quantified by BCA assay, and 30µg of protein was subjected to Western blot analysis using 12% SDS-PAGE. Protein expression levels were monitored using PARIS (Parkin-interacting substrate), TH (tyrosine hydroxylase), GFAP (glial fibrillary acidic protein), and NeuN (neuronal nucleus) antibodies. Expression of each protein was compared using the control protein alpha [Figure 40].

[0300] A consistent neurochemical abnormality in PD is the degeneration of dopaminergic neurons in the substantia nigra, leading to reduced striatal dopamine (DA) levels. Because TH catalyzes the formation of L-DOPA (L-dihydroxyphenylalanine), the rate-limiting step in DA biosynthesis, this is considered a striatal TH deficiency syndrome. TH activity and protein expression can be used to determine the progression and severity of Parkinson's disease.

[0301] GFAP is a cytoskeletal intermediate filament protein primarily expressed in astrocytes. In Parkinson's disease, GFAP is hyperphosphorylated and overexpressed, and this astrocyte degeneration is thought to be part of the pathogenesis of Parkinson's disease. Increased GFAP levels can be used as an astrocyte marker to diagnose Parkinson's disease. This was also selected and applied as a diagnostic tool in this experiment. The reduction of GFAP indicates that astrocyte damage is restored by KARC, which means that KARC has the effect of preventing and ameliorating neurodegenerative diseases.

[0302] NeuN (neuronal nucleus) is a protein expressed in most mammalian neurons. NeuN has been used in Parkinson's disease models as a marker of actual dopamine neuron loss or simply reduced TH expression. NeuN is also used to measure protein expression in living neurons. Increased NeuN expression indicates that administration of KARC resulted in the recovery of brain neuronal damage and the improvement of various brain disorders.

[0303] The expression of GFAP, a cell marker associated with inflammatory responses, was increased in the brain tissue of Camk-PARIS PD Tg mice. The expression of neuronal markers NeuN and TH in dopaminergic neurons was decreased. Oral administration of KARC decreased the expression of GFAP and increased the expression of NeuN and TH in the brain tissue of Camk-PARIS PD Tg mice. These findings suggest that KARC has anti-inflammatory and neuronal therapeutic effects in Camk-PARIS PD Tg mice.

[0304] [Example 14] Observation of in vitro reduction of various aldehydes by KARC The present invention confirmed the effectiveness of KARC in reducing exogenous and endogenous aldehydes.

[0305] When KARC (300 mg / ml) was reacted with various aldehydes (1 mM) for 3 hours at 37°C, 3,4-dihydroxyphenylacetaldehyde (DOPAL) was reduced by 24.4%, succinic semialdehyde (SSA) by 74.9%, glyoxal by 23.8%, cinnamaldehyde by 99.6%, and benzaldehyde by 97.1%. In the case of acetaldehyde, a 100.0% reduction was observed even after 1 hour of reaction at 30°C. [Figures 41-68]

[0306] 14-1: 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 were dispensed into a microtube. + Aqueous solutions, 10 μl of DW or 100 mM aldehyde in acetonitrile solution, and 10 μl of DW were dispensed into microtubes.

[0307] 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.

[0308] The reactions were shaken using a thermoshaker at 30°C or 37°C for 1 or 3 hours.

[0309] 14-2: Pre-processing before HPLC analysis For experiments using representative aliphatic aldehydes: SSA, acetaldehyde, and glyoxal, 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 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 6N 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.

[0310] For experiments with DOPAL, cinnamaldehyde, and benzaldehyde, which represent aromatic aldehydes, 10 μl of the solution reacted with KARC was aliquoted without heating with DNPH or DHBA and injected into the HPLC for analysis.

[0311] 14-3: HPLC analysis A 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.

[0312] The gradient started with 80% water (1% v / v trifluoroacetic acid) and developed in reverse phase at 20% after 15 minutes. Absorbance was analyzed at wavelengths of 254 nm, 310 nm, or 360 nm using a UV detector. Results were confirmed by the progress of the reaction, in which aldehydes were consumed through the reduction of DNPH-aldehyde conjugate or DHBA-aldehyde conjugate in the experimental group compared to the negative control group.

[0313] [Example 15] Examples of producing food and pharmaceutical compositions for improving behavior and motor function by restoring dopamine metabolism in vivo. Food and pharmaceutical compositions containing KARC as an active ingredient for improving behavior and motor function were prepared. Food or pharmaceutical compositions containing KARC powder with various composition ratios can be prepared. As an example, the powder composition of the present invention has the function of improving behavior and motor function through the intake of 13 g of the composition twice daily. The weight ratios of the ingredients and phases of the food or pharmaceutical composition containing the powder composition are shown in Table 7.

[0314] [Table 7] [Industrial Applicability]

[0315] Industrial Applicability The food and pharmaceutical compositions contained KARC dry powder, excipients, and natural sweeteners such as fructooligosaccharides, enzyme-treated stevia (stevia), citric acid anhydrous, isomaltodextrin (isomalt), and xylitol, citrus juice powder, and citrus flavor powder. Processing and testing of the raw materials and final products of the food or pharmaceutical compositions were performed in accordance with the General Test Methods and the Health Functional Food Act as described in the Korean Food Code.

[0316] A KARC-containing food or pharmaceutical composition can prevent or improve the deterioration of behavioral and motor function.

[0317] The above examples provide a detailed description of mutant yeast compositions containing aldehyde dehydrogenase for suppressing and preventing Parkinson's disease, including 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 effectiveness of KARC has been described in detail in the above examples, these are merely examples of the present invention.

[0318] 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.

[0319] 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 food composition for improving behavior and motor function, comprising an aldehyde dehydrogenase encoded by a gene having more than 98% homology to the gene of SEQ ID NO:

1.

2. 2. The food composition for improving behavior and motor function according to claim 1, wherein the aldehyde dehydrogenase is an aldehyde dehydrogenase encoded by the gene of SEQ ID NO: 1, which includes SEQ ID NO:

2.

3. The food composition for improving behavior and motor function according to claim 1 or 2, wherein the aldehyde is an endogenous aldehyde.

4. The food composition for improving behavior and motor function according to claim 3, wherein the aldehyde is an endogenous aldehyde produced by oxidation of alcohol or an endogenous amine compound.

5. 5. The food composition for improving behavior and motor function according to claim 4, wherein the endogenous amine compound is selected from the group consisting of dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid (GABA).

6. 4. The food composition for improving behavior and motor function 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, 4-oxo-nonena, malondialdehyde (MDA), propionaldehyde, hexanal, palmitic aldehyde, succinic aldehyde, and acrylaldehyde.

7. 3. The food composition for improving behavioral and motor function according to claim 1 or 2, wherein the aldehyde dehydrogenase is contained in any one or a mixture thereof selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP.

8. A food composition for improving behavior and motor function, characterized in that it contains 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.

9. A pharmaceutical composition for suppressing and preventing Parkinson's disease, comprising an aldehyde dehydrogenase encoded by a gene having more than 98% homology to the gene of SEQ ID NO:

1.

10. 10. The pharmaceutical composition for suppressing and preventing Parkinson's disease according to claim 9, wherein the aldehyde dehydrogenase is an aldehyde dehydrogenase encoded by the gene of SEQ ID NO: 1, which includes SEQ ID NO:

2.

11. The pharmaceutical composition for suppressing and preventing Parkinson's disease according to claim 9 or 10, wherein the aldehyde is an endogenous aldehyde.

12. 12. The pharmaceutical composition for suppressing and preventing Parkinson's disease according to claim 11, wherein the endogenous aldehyde is an endogenous aldehyde produced by oxidation of an alcohol or an endogenous amine compound.

13. 13. The pharmaceutical composition for suppressing and preventing Parkinson's disease according to claim 12, wherein the endogenous amine compound is selected from the group consisting of dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid (GABA).

14. 13. The pharmaceutical composition for suppressing and preventing Parkinson's disease according to claim 12, wherein the endogenous aldehyde is selected from the group consisting of formaldehyde, acetaldehyde, 4-hydroxy-2-nonenal, non-2-enal, 4-hydroxy-hexanal, 4-oxo-nonena, malondialdehyde (MDA), propionaldehyde, hexanal, palmitic aldehyde, succinic aldehyde, and acrylaldehyde.

15. A pharmaceutical composition for suppressing and preventing Parkinson's disease according to claim 9 or 10, wherein the aldehyde dehydrogenase is contained in a lysate of any one or a mixture thereof selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP.

16. A pharmaceutical composition for suppressing and preventing Parkinson's disease, characterized by 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.

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