Food and pharmaceutical compositions containing novel aldehyde dehydrogenase for improving memory and cognitive function

Abstract

The present invention relates to food and pharmaceutical compositions containing a novel aldehyde dehydrogenase for improving memory and cognitive function. The present invention relates to food and pharmaceutical compositions for preventing Alzheimer's disease and Huntington's disease by reducing the accumulation of pathological proteins in brain tissue. The food or pharmaceutical composition of the present invention contains a lysate of any one or a mixture of Saccharomyces cerevisiae strains selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP.

Description

[Technical Field]

[0001] The present invention relates to a food or pharmaceutical composition containing a novel aldehyde dehydrogenase (ALDH) for improving memory and cognitive function. The composition of the present invention reduces oxidative stress and inflammation in brain tissue, thereby reducing the abnormal aggregation of pathogenic or pathological proteins in the brain. That is, the present invention is directed to a food or pharmaceutical composition that inhibits or prevents various neurodegenerative diseases caused by the accumulation of pathological proteins in brain tissue.

[0002] More specifically, the compositions of the present invention contain a novel aldehyde dehydrogenase derived from a novel mutant yeast that improves memory and cognitive function.The present invention relates to food and pharmaceutical compositions for preventing Alzheimer's disease and Huntington's disease by reducing the accumulation of pathological proteins in brain tissue.

[0003] The composition of the present invention contains aldehyde dehydrogenase (ALDH), coenzymes (NAD, NADP), glutathione, etc., thereby inhibiting Alzheimer's disease. More specifically, the food or pharmaceutical composition of the present invention contains a lysate of any one or a mixture of Saccharomyces cerevisiae, KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP. [Background technology]

[0004] Dementia refers to a clinical syndrome in which cognitive functions such as memory, language, and judgment deteriorate, resulting in an inability to function normally. Representative examples of dementia include Alzheimer's disease, vascular dementia, dementia with Lewy bodies, and frontotemporal dementia.

[0005] According to histopathological research, it is known that dementia begins with the damage to neurons in the hippocampus, which is a specific part of the brain responsible for memory and cognition, and then spreads throughout the brain.Dementia is a delayed disease symptom that is caused by various causes, such as environmental factors such as drinking alcohol, smoking and taking medicine, and the effects of various diseases.It is known that any disease or lifestyle that can cause damage to brain function can be the cause of dementia.

[0006] Excessive alcohol consumption produces excessive amounts of acetaldehyde in the human body, leading to oxidative stress in brain tissue, which has been reported to induce neuroinflammation, produce abnormal amyloid protein aggregates, and cause dementia.

[0007] In addition to the amyloid protein aggregates mentioned above, synaptic dysfunction caused by hyperphosphorylation of tau protein, which plays an important role in maintaining the morphology of brain cell tissue, is known to cause dementia.In addition, increased brain neuroinflammation, increased oxidative stress, mitochondrial and synaptic dysfunction, and inhibition of neurotransmitter metabolism are also known to damage brain cells and cause dementia.

[0008] Senile plaques, a typical brain pathology, are associated with the aggregation of beta-amyloid protein, and neurofilament bundles are associated with hyperphosphorylation of tau protein.

[0009] In addition, abnormal accumulation of α-synuclein (α-Syn) protein aggregates in neurons, nerve fibers, or glial cells is known to cause neurodegenerative diseases.

[0010] As a result, modified proteins such as beta-amyloid protein, hyperphosphorylated tau protein, or abnormal aggregates of alpha-synuclein accumulate abnormally in neurons or glial cells, inducing inflammation or increasing oxidative stress in the nerves, leading to dementia. These modified proteins act as toxins to nerve cells, resulting in degenerative neurological diseases.

[0011] Amyloid proteins are formed by the polymerization of several monomeric peptides to form fibrillar morphologies. Amyloids and amyloid plaques, which are abnormal aggregates of these fibril-forming proteins, are generally toxic to nerve cells.

[0012] Amyloid plaques are toxic to cells by reducing the function of cellular calcium ion channels and intracellular mitochondria and increasing the concentration of reactive oxygen species (ROS) within the cell.

[0013] Amyloid plaques reduce the function of cellular calcium ion channels and intracellular mitochondria, increasing the concentration of reactive oxygen species (ROS) within cells, which leads to brain cell death.

[0014] Tau protein is mainly present in the axonal region of neurons and plays a role in stabilizing the structure of neurons by attaching to the cytoskeleton (microtubules).

[0015] However, when tau proteins are separated from their cytoskeleton, environmental factors cause them to aggregate, leading to the death of neurons. As a result, when amyloid beta and tau proteins accumulate in brain tissue, they damage synapses, or the connections between neurons that store memories in the brain. Such damage is known to cause cognitive disorders such as memory loss.

[0016] Lewy body dementia is characterized by the development of Lewy bodies, cells containing high concentrations of aggregated alpha-synuclein, and Lewy neuritis.

[0017] Alpha-synuclein is the causative agent in the early stages of Alzheimer's disease. As amyloid plaques and tau protein aggregate, Alzheimer's disease becomes more severe.

[0018] No drug has yet been developed that can completely treat dementia, and research and development of various drugs for treating dementia is currently underway. Current dementia treatments are drugs that aim to alleviate symptoms caused by dementia or prevent the symptoms of dementia from worsening.

[0019] Acetylcholinesterase inhibitor (ACE inhibitor) drugs such as donepezil, rivastigmine, galantamine, and NMDA receptor antagonist (N-methyl-D-aspartate receptor antagonist) drugs such as memantine are used to treat dementia.

[0020] These drugs for treating dementia improve the symptoms of dementia patients by inhibiting the action of an enzyme that breaks down acetylcholine in brain tissue, thereby increasing the amount of acetylcholine in brain tissue.

[0021] However, the therapeutic effectiveness of these drugs for progressive dementia is reduced, and they cause side effects such as nausea, diarrhea, loss of appetite, muscle spasms, and sleep disorders.

[0022] Recently, many clinical trials to verify the effectiveness of dementia treatments aimed at blocking protein aggregation have failed. Therefore, investigations to determine the exact cause of dementia and new treatment strategies for dementia are being discussed.

[0023] The present inventors have prepared a composition containing a novel aldehyde dehydrogenase enzyme that reduces oxidative stress in brain neurons, alleviates inflammation in neural tissue, and reduces amyloid plaque accumulation in brain tissue. The composition of the present invention can be used as a food composition or pharmaceutical composition for suppressing Alzheimer's disease by reducing apoptosis of brain cells. [Prior art documents] [Patent documents]

[0024] [Patent Document 1] U.S. Patent Application Publication No. 2021-0254023A1, dated August 21, 2021 [Non-patent literature]

[0025] TIFF2025540043000001.tif152108 Summary of the Invention [Problem to be solved by the invention]

[0026] Disclosure of the Invention Technical issues Therefore, a basic object of the present invention is to provide a food composition that improves memory and cognitive function, and a pharmaceutical composition that suppresses the accumulation of abnormal amyloid proteins in brain nerve tissues, which contain a novel aldehyde dehydrogenase that promotes the acid conversion of endogenous aldehydes generated by the in vivo decomposition of alcohol or the oxidation of endogenous amine compounds such as dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid (GABA).

[0027] Another object of the present invention is to provide a food composition for improving memory and cognitive function, and a pharmaceutical composition for treating Alzheimer's dementia, which contains 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). [Means for solving the problem]

[0028] Solutions to problems The primary object of the present invention as described above can be achieved by providing a composition containing a novel aldehyde dehydrogenase that promotes the decomposition of endogenous alcohol or the acid conversion of endogenous aldehydes produced by the oxidation of endogenous amine compounds such as dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid (GABA).

[0029] Another object of the present invention can also be achieved by providing a food composition for improving memory and cognitive function, and a pharmaceutical composition for treating Alzheimer's disease, each containing any one selected from the group consisting of Saccharomyces cerevisiae (KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP, or a mixture thereof. [Effects of the Invention]

[0030] Advantageous Effects of the Invention In the brains of Alzheimer's mice orally administered a dry powder or solution of any one or a mixture thereof selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP of the present invention, oxidative stress was reduced, astrocytes and microglia activated by inflammatory responses were decreased, and amyloid aggregation and plaques were reduced.In addition to these effects, the pharmaceutical effects of restoring memory and cognitive ability were also verified. [Brief explanation of the drawings]

[0031] Detailed Description of the Drawings [Figure 1] FIG. 1 is a graph showing the ability of KARC to decompose acetaldehyde in vivo.

[0032] [Figure 2] [Figure 2] is a graph showing the ability of KARC to degrade malondialdehyde in vivo.

[0033] In animal experiments in which blood levels of acetaldehyde [Figure 1] and malondialdehyde [Figure 2], endogenous toxic aldehydes, were increased by alcohol consumption, KARC's effects of reducing aldehydes and oxidative stress were confirmed.

[0034] [Figure 3] [Figure 3] is a graph showing the ability of KARC to decompose acetaldehyde in the human body.

[0035] [Figure 4] [Figure 4] is a graph showing the ability of KARC to decompose malondialdehyde in the human body.

[0036] [Figure 5] [Figure 5] is a graph showing the stabilization of malondialdehyde in the human body by KARC.

[0037] In studies to confirm the reduction of endogenous blood acetaldehyde [Figure 3] and blood malondialdehyde [Figure 4] in humans, the effects of KARC administration appear to be reducing acetaldehyde and malondialdehyde, which are biomarkers for hangovers, fatigue, and cardiovascular disease. [Figure 5] 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.

[0038] [Figure 6] [Figure 6] shows the schedule and drug content for producing an Alzheimer's model by using 5xFAD mice.

[0039] [Figure 7] [FIG. 7] shows the results of the Y-maze test in the Alzheimer's model using KARC.

[0040] In a maze experiment to evaluate short-term memory after induction of Alzheimer's disease using 5xFAD mice, the number of times they visited a new maze decreased by 47.4%, indicating a decline in cognitive ability.

[0041] However, in the group of mice administered KARC, cognitive ability was observed to be restored by increasing to 65.02%, indicating that KARC can improve memory deficits caused by Alzheimer's disease, is a treatment for preventing cognitive decline caused by degenerative brain diseases, and can improve impaired cognitive function.

[0042] [Figure 8] [Figure 8] shows the effect of KARC in reducing amyloid plaques in the whole brain of an Alzheimer's model.

[0043] [Figure 9][Figure 9] shows the results of KARC reducing the intensity of amyloid plaques in the hippocampus region of an Alzheimer's model.

[0044] [Figure 10] [Figure 10] shows the results of KARC reducing the number of amyloid plaques in the hippocampus of an Alzheimer's model.

[0045] After Alzheimer's disease induction using 5xFAD mice, the amount of amyloid plaques deposited in brain tissue increased abnormally, but was reduced by KARC [Figure 8], and the size of the actual amyloid plaques decreased [Figure 9] and the number also decreased [Figure 10]. Therefore, the effectiveness of KARC for preventing and treating neurodegenerative diseases such as dementia by reducing modified proteins and amyloid plaques is demonstrated.

[0046] [Figure 11] [Figure 11] shows the results of immunostaining for increased inflammatory response due to microglial activation in the hippocampus of Alzheimer's model mice.

[0047] [Figure 12] [Figure 12] shows the results of measuring the number of activated microglia in the CA1 region of the hippocampus of Alzheimer's model mice administered KARC.

[0048] [Figure 13] [FIG. 13] shows the results of measuring the number of activated microglia in the CA3 region of the hippocampus of Alzheimer's model mice administered KARC.

[0049] In Alzheimer's disease mice, the degree of microglial activation, an indicator of microglial inflammation known to cause neuronal death in the brain, was measured via Iba-1 immunostaining. In the KARC-treated group, the degree of activated microglia was reduced [Figure 11], and the number of activated microglia in the CA1 and CA3 regions of the hippocampus, where memory is stored, was also reduced [Figures 12 and 13]. Thus, KARC administration in Alzheimer's disease model animals is effective in preventing and treating brain neuroinflammatory responses.

[0050] [Figure 14] [FIG. 14] shows the results of immunostaining for changes in inflammatory astrocytes in the hippocampus of Alzheimer's model mice following administration of KARC.

[0051] [Figure 15] FIG. 15 shows the results of measuring the number of activated astrocytes in the CA1 region of the hippocampus of Alzheimer's model mice administered KARC.

[0052] [Figure 16] FIG. 16 shows the results of measuring the number of activated astrocytes in the CA3 region of the hippocampus of Alzheimer's model mice administered KARC.

[0053] In Alzheimer's mice, astrocytic inflammation causes the death of brain neurons and simultaneously leads to a weakening of brain homeostasis through the creation and maintenance of cranial neural synapses.

[0054] The level of astrocyte activation was confirmed through immunohistochemistry for GFAP, a measure of the inflammatory response of astrocytes. The level of astrocyte activation was reduced in the KARC-treated group [Figure 14], and the activation level of the hippocampus, which stores memory, was also reduced. The number of astrocytes in the CA1 and CA3 regions was also reduced [Figures 15 and 16], demonstrating the reduced inflammation effect of the KARC-treated group.

[0055] [Figure 17] [Fig. 17] shows the change in enzyme activity when the KwonP-1 strain was orally administered.

[0056] [Figure 18] [Figure 18] shows the change in enzyme activity when the KwonP-2 strain was orally administered.

[0057] [Figure 19] [Figure 19] shows the change in enzyme activity when the KwonP-3 strain was orally administered.

[0058] [Figure 20] [Figure 20] shows the change in enzyme activity when the Pico YP strain was orally administered.

[0059] [Figure 21] [Figure 21] shows the change in enzyme activity when the PicoYP-01 strain was orally administered.

[0060] [Figure 22] [Figure 22] shows the change in enzyme activity when the PicoYP-02 strain was orally administered.

[0061] [Figures 17, 18, 19, 20, 21, and 22] 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. The ALDH enzyme activity was maintained at a minimum of 37.29 units / g and a maximum of 52.24% at pH = 5 (similar to the conditions observed during food intake). When KARC was orally administered, it was confirmed that the enzyme activity was maintained.

[0062] [Figure 23] [Figure 23] shows the growth curve and enzyme activity of the KwonP-1 strain cultured in a 5 L fermentation tank.

[0063] [Figure 24] [Figure 24] shows the growth curve and enzyme activity of the KwonP-2 strain cultured in a 5 L fermentation tank.

[0064] [Figure 25] [Figure 25] shows the growth curve and enzyme activity of the KwonP-3 strain cultured in a 5 L fermenter.

[0065] [Figure 26] [Figure 26] shows the growth curve and enzyme activity of the PicoYP strain cultured in a 5 L fermenter.

[0066] [Figure 27] [Figure 27] shows the growth curve and enzyme activity of the PicoYP-01 strain cultured in a 5 L fermenter.

[0067] [Figure 28] [Figure 28] shows the growth curve and enzyme activity of the PicoYP-02 strain cultured in a 5 L fermenter.

[0068] * In Figures 23, 24, 25, 26, 27, and 28, the novel mutant strains KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-01 were cultured in 5L fermenters using YPD medium under the same conditions. The cultures were grown at 30°C and 200 rpm for 48 hours. The growth curves (OD660nm) and ALDH enzyme activity of each strain were compared with those of the type strain, revealing that the ALDH enzyme activity was at least 10.5-fold and up to 18.75-fold higher. PicoYP-01 had the highest ALDH activity at 52.68 units / g, while KwonP-3 had the lowest at 29.5 units / g. DETAILED DESCRIPTION OF THE INVENTION

[0069] 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.

[0070] 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]

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] [Table 1]

[0082] [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.

[0083] 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.

[0084] 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 a reverse completion process.

[0085] 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.

[0086] [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.

[0087] 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.

[0088] 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.

[0089] 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).

[0090] 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.

[0091] [Table 2] TIFF2025540043000004.tif35108

[0092] 3-2: Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] In six mutants (K-1, 4, 6, 7, 8, and 9), NADP 合計 The rate of increase in NAD 合計 The NADP of the three novel mutants (PicoYP, PicoYP-01, and PicoYP-02) increased less than twofold. 合計 The increase rates of NAD content were 25.7, 22.9, and 27.6 times, respectively. 合計 The increase rates of NADP content were 10.8, 9.9, and 11.3 times, respectively. 合計 The increase rate is NAD 合計 The rate of increase was more than double.

[0100] 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.

[0101] 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 with the existing deposited strains.

[0102] [Table 3]

[0103] [Table 4]

[0104] [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).

[0105] The characteristics and novelty of the carbon source selection of the strains were analyzed by API 50 CHL kit (API systems, BIOMERIEUX, SA, France).

[0106] 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.

[0107] 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.

[0108] 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 +++.

[0109] 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.

[0110] 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.

[0111] 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].

[0112] [Table 5]

[0113] [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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] [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.

[0125] 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).

[0126] 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).

[0127] 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).

[0128] 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].

[0129] 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].

[0130] 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).

[0131] [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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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).

[0139] 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%.

[0140] 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%.

[0141] 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%.

[0142] 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%.

[0143] 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%.

[0144] 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%.

[0145] 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).

[0146] [Table 6]

[0147] [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.

[0148] Among the ALDs (yeast aldehyde dehydrogenases) in the novel mutant strain, ALD2 (SEQ ID NO: 3) was found to be condensed with ALD3 (SEQ ID NO: 4) on chromosome 13. A non-coding region of 689 nucleotides was located between the genes encoding ALD2 and ALD3. ALD2 and ALD3 existed contiguously in the same genome. ALD2 and ALD3 encoded aldehyde dehydrogenases, respectively. The gene encoding ALD2 consisted of 1,521 nucleotides and 506 amino acids and was nearly identical to ALD3, but had an 8.2% sequence difference. ALD2 and ALD3 were identified as distinct aldehyde dehydrogenases, differing from each other by 125 base pairs (8.2%).

[0149] 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).

[0150] 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.

[0151] 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.

[0152] 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].

[0153] [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.

[0154] After completion of administration, blood samples were collected from the tail vein at 0, 1, 3, 5, and 8 hours after KARC injection, and after centrifugation, the plasma was stored at −80°C [Figures 1, 2].

[0155] 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).

[0156] 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 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 1).

[0157] 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.

[0158] 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 2).

[0159] These results indicated that oral administration of KARC was effective in reducing various endogenous aldehydes, such as acetaldehyde and malondialdehyde, in the blood.

[0160] [Example 10] Effect of reducing oxidative stress. Drinking alcohol increases reactive oxygen species or oxidative stress due to excess acetaldehyde (Ach) produced by alcohol dehydrogenase (ADH). Aldehyde dehydrogenase (ALDH) converts acetaldehyde to acetic acid and excretes it from the body. Excess aldehydes, caused by aldehyde dehydrogenase gene mutations or excessive alcohol consumption, can lead to lipid peroxidation.

[0161] 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.

[0162] Blood acetaldehyde concentrations were measured over time after alcohol consumption [Figure 3]. The area under the curve (AUC) of blood acetaldehyde (Ach) was 13.02 ± 1.18 mg·h / dL after alcohol consumption alone. When a 10 unit / kg KARC dose was administered, the area under the curve (AUC) of blood acetaldehyde (Ach) was measured at 9.39 ± 1.07 mg·h / dL, a significant decrease of 26.13% compared to alcohol consumption alone (P = 0.005).

[0163] 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.

[0164] The reduction in blood acetaldehyde (Ach) levels resulting from KARC administration has a positive impact on reducing oxidative stress and promoting health.

[0165] The blood malondialdehyde (MDA) concentration was measured during chemotherapy (Figure 4). 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).

[0166] 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 5.

[0167] 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.

[0168] These aldehydes then react with surrounding proteins and undergo secondary metabolic processes to form stable end products, such as malondialdehyde-acetaldehyde adducts (MAA) and malondialdehyde-lysine adducts (M-lys adducts), known as advanced lipid peroxidation end products. The accumulation of these products exerts toxic effects on various cells and further enhances oxidative stress.

[0169] 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.

[0170] Increased reactive oxygen species and oxidative stress are associated with elevated levels of reactive aldehydes, such as HNE and MDA, and modified proteins, such as advanced glycation end products (AGEs) and advanced lipid peroxidation end products (ALEs). This series of events is known to reinforce and amplify each other, leading to increased endoplasmic reticulum stress (ER stress).

[0171] [Example 11] Acute oral administration test 11-1. Preparation of experimental animals The experimental animals were female and male ICR mice (7 weeks old). The received ICR mice were acclimated for 7 days. The general symptoms of the adopted mice were observed during the acclimation period, and only healthy animals were used for the short-term administration toxicity test. Food and water were 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.

[0172] 11-2. Administration of test substance The test substances were prepared by dissolving the mutant yeast lysate KARC of the present invention in physiological saline so that the doses administered to the experimental animals were 0, 750, 3,000 and 5,000 mg / kg, respectively, based on the content of the mutant yeast lysate KARC of the present invention.

[0173] 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.

[0174] 11-3. 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.

[0175] 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.

[0176] [Example 12] Effect of oral administration of KARC for alleviating memory deficits and abnormal amyloid proteins To confirm the effects of KARC on improving memory deficits and cognitive function and to verify the reduction of abnormal amyloid beta protein deposition, a 5xFAD mouse model was generated. Administration of KARC improved the performance of 5xFAD mice in a Y-maze behavioral test to assess memory, and reduced inflammatory cells and amyloid plaques in brain tissue.

[0177] Preparation of the 12-1:5xFAD mouse model and administration of KARC 5xFAD mice express three human APP mutations [Swedish (K670N / M671L), Florida (I716V), and London (V717I)] and two PSEN1 mutations [M146L and L286V]. A total of five mutations in 5xFAD mice are closely associated with Alzheimer's disease.

[0178] Expression of these two genes is regulated by neural-specific elements in the mouse Thy1 promoter, leading to overexpression in the brain. The 6799 line of mice has the highest expression of amyloid beta precursor protein (APP), which is associated with the rapid accumulation of the 42-amino acid beta amyloid.

[0179] 5xFAD mice accumulate amyloid plaques beginning at 1.5 months of age, with gliosis evident in mice at approximately 2 months of age. Amyloid pathology is more severe in females than in males. Using primarily female mice, neuronal loss occurs in multiple brain regions, with amyloidosis occurring most prominently at approximately 6 months of age. Mice develop Alzheimer's disease, with a variety of cognitive and motor deficits.

[0180] The tails of the mice born through double mating were clipped, and the expression of APP and PSEN1 was confirmed via genomic DNA PCR. 5xFAD mice expressing both genes were then selected for the experiment.

[0181] Seventeen-month-old Alzheimer's model mice and non-transgenic (Ntg) mice selected from 5xFAD female mice were divided into four groups (n = 5 per group). In the control group, Ntg mice received PBS alone daily for one month. In the vehicle group, 5xFAD mice received PBS alone.

[0182] Ntg or 5xFAD mice were orally administered PBS together with KARC (20 units / kg / day), respectively, under the same conditions [Figure 6].

[0183] 12-2: Behavioral and memory assessment results following oral administration of KARC A Y-maze test was performed to assess short-term memory in 5xFAD mice, a model of Alzheimer's disease. Rodents typically prefer to explore new maze arms rather than return to previously visited ones. Many parts of the brain are involved in this task, including the hippocampus, septum, basal forebrain, and prefrontal cortex.

[0184] Testing was performed in a Y-shaped maze with three plastic arms at 120° angles to each other. Mice were allowed to freely explore the three arms from the central hub of the maze. A ratio, expressed as a percentage, was calculated based on the number of times the mice visited a new location compared to the total number of visits.

[0185] Cognitive deficits were observed as Alzheimer's disease progressed. In 5xFAD mice, an Alzheimer's model, the rate of visits to novel locations in the maze using the Y-maze test decreased to 47.4%. In mice administered KARC, this increased to 65.02%. This suggests that KARC has potential as a treatment for improving memory deficits caused by Alzheimer's disease and restoring cognitive function due to Alzheimer's dementia [Figure 7].

[0186] 12-3-1: Preparation of brain tissue Mice were anesthetized by injection of chloral hydrate (40 mg / kg, i.p.) and transcardially perfused with saline containing 0.5% sodium nitrate and heparin (10 U / ml). Brain tissue was fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.2).

[0187] 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.

[0188] Brains were cryosectioned into 30 μm-thick coronal sections on a Cryostat (Microsystems AG, Leica, Wetzlar, Germany). Brains were stored at 4°C in cryoprotectant (25% ethylene glycol, 25% glycerol, 0.2 M PB, and water) until use. Changes in hippocampal sections in the brain were observed via immunostaining.

[0189] 12-3-2: Immunohistochemical staining Thioflavin S (Sigma-Aldrich, St. Louis, MO, USA) was used to stain abnormally aggregated amyloid plaques in brain sections (30 μm) containing the hippocampus of 5xFAD mice, an Alzheimer's disease model. Thioflavin S is used in histological staining and biophysical investigations to visualize protein aggregates such as amyloid plaques. A 1% thioflavin S solution was prepared by dissolving thioflavin S in distilled water and then filtering through a 0.45 μm filter. Brain tissue, including the hippocampus, was stained with the 1% thioflavin S solution for 5 minutes, washed three times with PBS, cover-slipped, and analyzed using fluorescent microscopy (Figure 8).

[0190] Astrocytes and neuroinflammatory cells from brain sections (30 μm thick) containing the hippocampus were incubated with rabbit anti-glial fibrillary acidic protein (anti-GFAP, 1:5000; Neuromics, Edina, MN, USA) or rabbit anti-ionized calcium-binding adaptor molecule 1 (anti-Iba-1, 1:1000; Wako, Osaka, Japan), respectively.

[0191] The incubated astrocytes were stained with biotinylated anti-rabbit IgG or avidin-biotin peroxidase complex (ABC) standard kit (Vector Laboratories, Burlingame, CA, USA), respectively.

[0192] Signals were detected after incubating the stained sections with 0.5 mg / ml 3,3'-diaminobenzidine (Sigma, St. Louis, MO, USA) in 0.1 M PBS containing 0.003% H2O2. Images of stained brain sections were analyzed under a bright-field microscope (Olympus Corporation, Tokyo, Japan) to quantify GFAP- or Iba-1-positive cells, respectively.

[0193] 12-3-3: Methods for amyloid plaque intensity and counting. The fluorescence intensity and total number of amyloid plaques were measured in the hippocampus of 5xFAD mice, an Alzheimer's model. The fluorescence intensity and total number of stained amyloid plaques in the hippocampus were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA) on images captured at 40x magnification.

[0194] 12-4: Oral administration of KARC induces changes in brain substances in 5xFAD mice, an Alzheimer's model 12-4-1: Effect of KARC on amyloid plaque reduction in the whole brain of an Alzheimer's disease model To measure abnormally aggregated amyloid proteins in 5xFAD mice, an Alzheimer's model, amyloid plaques in brain tissue, including the hippocampus, were stained with white thioflavin S. Amyloid plaques are misfolded protein aggregates consisting of a mixture of degenerating neurons and beta-amyloid aggregates. These plaques gradually accumulate in the brain because they cannot be destroyed or removed. The accumulation of these amyloid plaques in specific organs or tissues, leading to functional defects and deterioration, is known as amyloidosis. When amyloid plaques deposit in brain neurons, they initiate various neurodegenerative diseases. Abnormally structured proteins, such as amyloid plaques, play a central role in Alzheimer's disease.

[0195] In 17-month-old female non-transgenic (Ntg) mice, similar to normal mice, no amyloid plaques were observed in the brain. In 17-month-old female 5xFAD mice (vehicle group), amyloid plaque accumulation was observed throughout the brain, including the hippocampus. In 5xFAD mice orally administered KARC (20 units / kg / day), amyloid plaques were reduced overall compared to the vehicle group (Figure 8).

[0196] The Image J program was used to quantify the intensity and number of amyloid plaques accumulated in the hippocampus of the brain.

[0197] In 17-month-old female Ntg mice, no amyloid plaques were observed in the control group. The amyloid plaque intensities were 11.47 in the vehicle group and 12.37 in the KARC-treated group, which were not significant compared to the control group. In 17-month-old female 5xFAD mice, the amyloid plaque intensities were 52.36 in the vehicle group and 34.76 in the KARC-treated group, a significant decrease of 66.38% compared to the vehicle group (p<0.001) [Figure 9].

[0198] Quantification of amyloid plaque counts revealed no pathological differences, with 319 in 17-month-old female non-transgenic mice (control group) and 501 in the KARC-treated group. Using 17-month-old female 5xFAD mice, the number of amyloid plaques was 2272 in the KARC-treated group compared with 3202 in the vehicle group, a significant reduction of 70.09% (p<0.001) [Figure 10].

[0199] 12-4-2: Oral administration of KARC reduces neuronal inflammation in microglia and astrocytes in 5xFAD mice, a model of Alzheimer's disease The efficacy of KARC in alleviating neuroinflammation in the 5xFAD mouse model was evaluated by staining microglia and astrocytes. The hippocampus of brain tissue was immunostained using Iba-1 (microglia) antibody [Figure 11] and GFAP (astrocyte) antibody [Figure 14] and then analyzed.

[0200] In 17-month-old non-transgenic (Ntg) mice, both microglia and astrocytes were found to be in a quiescent state in the CA1 and CA3 regions of the hippocampus. In the CA1 and CA3 regions of the hippocampus of 17-month-old 5xFAD mice, microglia and astrocytes were morphologically enlarged upon activation. Furthermore, neurites, with dendritic processes, became brighter and longer. This was due to the induction of neuroinflammation by the accumulation of abnormal amyloid plaques.

[0201] Neuroinflammatory activation of hippocampal microglia and astrocytes was suppressed in 5xFAD mice orally administered KARC (20 units / kg / day) [Figures 11, 14].

[0202] In 17-month-old 5xFAD mice, the number of microglia with positive activated Iba-1 was 125 ± 45 in the CA1 region and 118 ± 14 in the CA3 region. In the hippocampus of 17-month-old 5xFAD mice orally administered KARC, the number of Iba-1-positive activated microglia was reduced to 59 ± 11 in the CA1 region and 80 ± 12 in the CA3 region (Figures 12 and 13).

[0203] In the hippocampus of 17-month-old 5xFAD mice, the number of GFAP-positive activated astrocytes was 137 ± 1 in the CA1 region and 114 ± 17 in the CA3 region. In the hippocampus of 17-month-old 5xFAD mice orally administered KARC, the number of GFAP-positive activated astrocytes was reduced to 90 ± 9 in the CA1 region and 87 ± 5 in the CA3 region (Figures 15 and 16). Quantification of activated cells confirmed that oral administration of KARC suppressed neuroinflammatory activation of hippocampal microglia and astrocytes.

[0204] It is well documented that neuroinflammation plays a key role in inducing various pathological features observed in Alzheimer's disease. Persistent activation of microglia and other immune cells in the brain has been shown to exacerbate both amyloid and tau pathology, potentially acting as a contributor to disease manifestations.

[0205] Cytokines such as IL-1 and IL-6, which mediate the inflammatory response, have been shown to increase the amount of amyloid-beta or induce hyperphosphorylation of tau protein, as well as trigger chronic inflammatory processes in microglia and astrocytes, indicating that the inflammatory response may directly contribute to the pathological manifestations of Alzheimer's disease.

[0206] Iba-1 (ionized calcium-binding adaptor molecule 1) is a subtype of allograft inflammatory factor-1 (AIF-1). Iba-1 is a protein specifically expressed in immune cells and microglia of the central nervous system. Iba-1 is overexpressed in microglia in response to neuronal injury, such as in central nervous system ischemia and various other brain disorders.

[0207] Reduction of Iba-1 expression by KARC administration demonstrates its potential to reduce cranial nerve damage and ameliorate central nervous system ischemia, suggesting the possibility of ameliorating various brain disorders.

[0208] GFAP (glial fibrillary acidic protein) is a fibrillary acidic protein present in brain neurons. GFAP is a marker for astrocytes distributed throughout the central nervous system (CNS). GFAP plays an important role in regulating astrocyte motility and morphological maintenance by contributing to myelination in neurons.

[0209] In mammals, GFAP expression in astrocytes is also known to be specifically increased in brains damaged by a variety of causes, both physical and chemical. The increase in abnormal astrocytes in the damaged brain leads to neurodegenerative diseases through increased inflammation and induced death of brain neurons, accelerating disease progression.

[0210] The decrease in abnormal GFAP indicates that astrocyte damage was reversed by KARC administration, and KARC administration may be effective in preventing and ameliorating neurodegenerative diseases.

[0211] Treatment with KARC significantly suppressed the neuroinflammatory response in 5xFAD mice, as measured by the degree of activation in microglia and astrocytes. These data indicate that KARC has a therapeutic effect on neuroinflammation in vivo.

[0212] In conclusion, KARC significantly improved memory and cognitive function, alleviated brain inflammation, and downregulated the abnormal accumulation of amyloid plaques in 5xFAD mice. This study suggests that KARC may be an effective treatment for dementia.

[0213] [Example 13] An example of the manufacture of food and pharmaceutical compositions for memory impairment and cognitive function by reducing amyloid beta plaques and endogenous acetaldehyde. Food and pharmaceutical compositions containing KARC as an active ingredient for improving memory impairment and cognitive function were prepared. Food or pharmaceutical compositions containing KARC powder with various composition ratios can be prepared. For example, the powder composition of the present invention has the function of improving memory impairment and cognitive function through oral ingestion 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.

[0214] [Table 7] [Industrial Applicability]

[0215] 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.

[0216] A KARC-containing food or pharmaceutical composition can prevent or improve the deterioration of cognitive function.

[0217] The above examples have described in detail the mutant yeast composition KARC containing aldehyde dehydrogenase: its pharmacological effects on Alzheimer's disease treatment or Lewy body dementia, its administration method, its therapeutically effective dose for disease models, its acute oral toxicity, and representative examples of food or pharmaceutical compositions. The effectiveness of KARC has been described in detail through the above examples, but these are merely examples of the present invention.

[0218] 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.

[0219] * 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 memory and cognitive function, comprising an aldehyde dehydrogenase encoded by a gene having more than 98% homology to the gene of SEQ ID NO:

1.

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

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

5. 5. The food composition for improving memory and cognitive 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 memory and cognitive 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-hydroxy-hexanal), 4-oxo-nonena, malondialdehyde (MDA), propionaldehyde, hexanal (hexanal), palmitic aldehyde, succinic aldehyde and acrylaldehyde.

7. 3. The food composition for improving memory and cognitive function according to claim 1 or 2, 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.

8. A food composition for improving memory and cognitive 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 Alzheimer's or Lewis Body dementia, 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 Alzheimer's disease or Lewis body dementia according to claim 9, wherein the aldehyde dehydrogenase is an aldehyde dehydrogenase encoded by the gene of SEQ ID NO: 1, including SEQ ID NO:

2.

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

12. 12. The pharmaceutical composition for suppressing and preventing Alzheimer's disease or Lewis body dementia 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 Alzheimer's or Lewis body dementia 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 Alzheimer's or Lewis body dementia 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 Alzheimer's or Lewis body dementia as described in 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 Alzheimer's disease or Lewis body dementia, 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.

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