Food and pharmaceutical composition for improving memory and cognitive function comprising novel aldehyde dehydrogenase

Abstract

Compositions of the present invention comprise novel aldehyde dehydrogenases derived from novel mutant yeasts, which improve memory and cognitive functions. The present invention relates to food and pharmaceutical compositions for preventing Alzheimer's disease and Huntington's disease by reducing accumulation of diseased proteins in brain tissue. The food or pharmaceutical composition of the present invention comprises a lysate of any one selected from the group consisting of Saccharomyces cerevisiae KCTC 13925BP, KCTC 14122BP, KCTC 14123BP, KCTC 14983BP, KCTC 14984BP, and KCTC 14985BP, or a mixture thereof. The food or pharmaceutical composition of the present invention comprises a lysate of any one selected from the group consisting of KCTC 13925BP, KCTC 14122BP, KCTC 14123BP, KCTC 14984BP, and KCTC 14985BP.

Description

Technical Field

[0001] The present invention relates to food and pharmaceutical compositions containing novel aldehyde dehydrogenases (ALDHs) for improving memory and cognitive function. The compositions of the present invention reduce abnormal aggregation of pathogenic or diseased proteins in the brain by reducing oxidative stress and inflammation in brain tissue. Specifically, the present invention relates to food or pharmaceutical compositions that inhibit or prevent various neurodegenerative diseases caused by the accumulation of diseased proteins in brain tissue.

[0002] More specifically, the compositions of the present invention comprise novel aldehyde dehydrogenases derived from novel mutant yeast that improve 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 diseased proteins in brain tissue.

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

[0004] Dementia refers to a clinical syndrome in which cognitive functions such as memory, language, and judgment are weakened, making it impossible to lead a normal life. Representative examples of dementia include Alzheimer's disease, vascular dementia, Lewy body dementia, and frontotemporal dementia.

[0005] Based on histopathological studies, dementia begins with damage to neurons in the hippocampus (a specific part of the brain responsible for memory and cognition) and spreads throughout the brain. Dementia is a late-onset disease symptom caused by a variety of factors, including environmental factors such as alcohol, smoking, and medication, as well as the effects of various diseases. Any disease or lifestyle known to impair brain function may be a cause of dementia.

[0006] Excessive drinking produces excessive acetaldehyde in the human body, leading to oxidative stress in brain tissue. Oxidative stress in the brain has been reported to cause neuroinflammation, abnormal aggregates of amyloid protein, and lead to dementia.

[0007] In addition to the aforementioned amyloid aggregates, synaptic dysfunction caused by hyperphosphorylation of tau protein (which plays an important role in maintaining the shape of brain cell tissue) is known to contribute to dementia. Furthermore, increased brain neuroinflammation, increased oxidative stress, mitochondrial and synaptic dysfunction, and suppressed neurotransmitter metabolism are known to damage brain cells and lead to dementia.

[0008] Senile plaques, a representative brain pathology, are associated with the aggregation of β-amyloid protein, and neurofibrillary bundling is associated with the hyperphosphorylation of tau protein.

[0009] Furthermore, 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, abnormal aggregates of modified proteins, such as beta-amyloid, hyperphosphorylated tau, or alpha-synuclein, accumulate in neurons or glial cells, inducing neuroinflammation or increasing oxidative stress, leading to dementia. These modified proteins act as toxins to nerve cells, causing degenerative neurological diseases.

[0011] Amyloid proteins are formed by the aggregation of multiple monomeric peptides into fibrils. Amyloid proteins and amyloid plaques are abnormal aggregates of these fibrils and are often toxic to nerve cells.

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

[0013] Amyloid plaques reduce the function of cellular calcium channels and intracellular mitochondria and increase the concentration of intracellular reactive oxygen species (ROS), leading to brain cell death.

[0014] Tau protein is mainly present in the axon region of nerve cells and plays a role in stabilizing the structure of nerve cells by attaching to the intracellular skeleton (microtubules).

[0015] However, when tau protein detaches from the cytoskeleton within the cell, environmental factors cause it to aggregate, leading to the death of nerve cells. Therefore, when amyloid-β and tau accumulate in brain tissue, they damage synapses (the brain's memory storage sites), or the connections where communication occurs between nerve cells. This type of damage is known to lead to cognitive impairments such as memory loss.

[0016] Lewy body dementia is characterized by the presence of Lewy bodies and Lewy neuritis in cells containing high concentrations of aggregated α-synuclein.

[0017] In the early stages of Alzheimer's disease, alpha-synuclein is a pathogenic substance, which becomes more severe as amyloid plaques and tau proteins accumulate.

[0018] A drug that completely cures dementia has not yet been developed, and various drugs for treating dementia are currently being researched and developed. Current dementia treatments are drugs that aim to alleviate symptoms caused by dementia or prevent dementia symptoms from worsening.

[0019] Acetylcholinesterase inhibitor (ACE inhibitor) drugs such as donepezil, rivastigmine, and galantamine, as well as 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 symptoms in patients with dementia 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, these agents have reduced therapeutic effects on advanced dementia, and they can cause side effects such as nausea, diarrhea, loss of appetite, muscle cramps, and sleep disturbances.

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

[0023] The present inventors have prepared a composition containing a novel aldehyde dehydrogenase that can reduce oxidative stress in brain nerve cells, alleviate inflammation in neural tissue, and reduce the accumulation of amyloid plaques in brain tissue. The composition of the present invention can be used as a food composition and a pharmaceutical composition to suppress Alzheimer's disease by reducing brain cell apoptosis.

[0024] [Prior art literature]

[0025] [Patent Document]

[0026] U.S. Patent Application Publication No. US2021-0254023-A1, dated August 21, 2021

[0027] [Non-patent literature]

[0028] Chen et al, "Targeting aldehyde dehydrogenase 2: new therapeutic opportunities". PhysiolRev. 2014; 94:1-34.

[0029] AU Joshi et al."Aldehyde dehydrogenase2activity and aldehydic loadcontribute to neuroinflammation and Alzheimer′s disease related pathology"Acta Neuropathol Commun.2019 Dec;7:190.

[0030] ZY Zhu et al."Mitochondrial aldehyde dehydrogenase(ALDH2)rescuescardiac contractile dysfunction in an APP / PS1 murine model of Alzheimer′sdisease via in-hibition of ACSL4-dependent ferroptosis"Acta PharmacolSin.2022Jan;43(1):39-49.

[0031] Chen et al,"Association between Aldehyde dehydrogenase-2polymorphismsand risk of Alzheimer′s disease and Parkinson′s Disease;A Meta-Analysis Basedon5,315Individuals"Front Neurol.2019Mar;28290.

[0032] W HUANG et al."Role of oxidative stress in Alzheimer′s disease"BiomedRep.2016May;4(5):519-522.

[0033] D Twohig et al."α-synuclein in the pathophysiology of Alzheimer′sdisease"Mol Neurodegener.2019Jun11;14(1):23.

[0034] E Grunblatt et al. "Aldehyde dehydrogenase (ALDH) in Alzheimer's and Parkinson's disease" J Neural Transm. 2016 Feb; 123(2):83-90.

[0035] EE Tuppo et al. "The role of inflammation in Alzheimer's disease" Int JBiochem Cell Biol. 2005 Feb;37(2):289-305.

[0036] Z Chen et al. "Oxidative stress in Alzheimer's disease" NeurosciBull.2014Apr;30(2):271-81.

[0037] I Blasko et al. "How chronic inflammation can affect the brain and support the de-velopment of Alzheimer's disease in old age: the role of microglia and astrocytes" AgingCell. 2004 Aug; 3(4): 169-76.

[0038] Butterfield DA, et al. "Evidence of oxidative damage in Alzheimer's disease brain: central role for amyloid beta-peptide" Trends Mol Med. 2001 Dec; 7(12): 548-54. Summary of the Invention Technical issues

[0039] Therefore, the basic object of the present invention is to provide a food composition for improving memory and cognitive function and a pharmaceutical composition for inhibiting abnormal amyloid accumulation in brain neural tissue, which comprises a novel aldehyde dehydrogenase that 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)) in the body.

[0040] 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 comprises any one or a mixture thereof selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP (hereinafter referred to as KARC).

[0041] Solution to the problem

[0042] The main object of the present invention as described above can be achieved by providing a composition comprising a novel aldehyde dehydrogenase that promotes the acid conversion of endogenous aldehydes produced by the decomposition of endogenous alcohols or the oxidation of endogenous amine compounds such as dopamine, norepinephrine, serotonin and gamma-aminobutyric acid (GABA).

[0043] 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 dementia, wherein the composition comprises any one selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof. Beneficial effects of the present invention

[0044] Oral administration of a dry powder or lysate of any one of the yeast Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP, or a mixture thereof, to the brains of Alzheimer's mice revealed reduced oxidative stress, decreased astrocyte and microglia activation due to inflammatory responses, and decreased amyloid aggregation and plaques. In addition to these effects, the drug also demonstrated efficacy in restoring memory and cognitive abilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] [ Figure 1 ] is a graph showing the ability of KARC to decompose acetaldehyde in the body.

[0046] [ Figure 2 ] is a graph showing the ability of KARC to decompose malondialdehyde in vivo.

[0047] In alcohol consumption, acetaldehyde is an endogenous toxic aldehyde in the blood. Figure 1 ] and blood malondialdehyde[ Figure 2 ]In animal experiments, the aldehyde-reducing and oxidative stress-reducing effects of KARC were confirmed.

[0048] [ Figure 3 ] is a graph showing the ability of KARC to break down acetaldehyde in the human body.

[0049] [ Figure 4 ] is a graph showing the ability of KARC to decompose malondialdehyde in the human body.

[0050] [ Figure 5 ] is a graph showing that KARC stabilizes malondialdehyde in the human body.

[0051] In the confirmation of reducing endogenous acetaldehyde in human blood[ Figure 3 ] and reduce blood malondialdehyde [ Figure 4 ] trial, the effects of KARC administration appeared to reduce acetaldehyde and malondialdehyde, biomarkers of hangover, fatigue, and cardiovascular disease. Figure 5 ] showed that in a state where oxidative stress is increased due to taking drugs, KARC administration can reduce oxidative stress and malondialdehyde, a biomarker of reactive oxygen species, thereby reducing oxidative stress.

[0052] [ Figure 6 ] shows a protocol and drug contents for generating an Alzheimer's disease model by using 5xFAD mice.

[0053] [ Figure 7 ] show the results of KARC's Y-maze test on an Alzheimer's disease model.

[0054] In a maze test evaluating short-term memory after induction of Alzheimer's disease using 5xFAD mice, the number of visits to a new maze decreased to 47.4%, indicating a decline in cognitive ability.

[0055] However, in the group of mice administered KARC, cognitive ability was observed to recover by increasing to 65.02%. This suggests that KARC can improve memory impairment caused by Alzheimer's disease and may be used as a treatment for preventing cognitive decline caused by degenerative brain diseases and improving cognitive function decline.

[0056] [ Figure 8 ] showed the effect of KARC in reducing amyloid plaques throughout the brain of an Alzheimer's disease model.

[0057] [ Figure 9 ] showed the results that KARC reduced the intensity of amyloid plaques in the hippocampus of an Alzheimer's disease model.

[0058] [ Figure 10 ] showed results that KARC reduced the number of amyloid plaques in the hippocampus of an Alzheimer's disease model.

[0059] After Alzheimer's disease induction in 5xFAD mice, amyloid plaques deposited in brain tissue increased abnormally but were reduced by KARC [ Figure 8 ], actual amyloid plaque size decreased [ Figure 9 ], and the number also decreased[ Figure 10 ]. Thus, the effectiveness of KARC in preventing and treating neurodegenerative diseases such as dementia by reducing modified proteins and amyloid plaques is revealed.

[0060] [ Figure 11 ] show immunostaining results of increased inflammatory response caused by microglial activation in the hippocampus of Alzheimer's disease model mice.

[0061] [ Figure 12 ] show the results of measuring the number of activated microglia in the CA1 region of the hippocampus of Alzheimer's disease model mice administered with KARC.

[0062] [ Figure 13 ] show the results of measuring the number of activated microglia in the CA3 region of the hippocampus of Alzheimer's disease model mice administered with KARC.

[0063] In Alzheimer's disease mice, the degree of microglial activation was visualized by immunostaining for Iba-1, which indicates the degree of microglial inflammation known to be a cause of brain neuron death. In the KARC-administered group, the degree of activated microglial cells was reduced. Figure 11 ], and the number of activated microglia in the CA1 and CA3 regions of the hippocampus, where memories are stored, also decreased [ Figure 12 and Figure 13 ]. Thus, the preventive and therapeutic effects of KARC administration on cranial nerve inflammatory responses in Alzheimer's disease model animals were revealed.

[0064] [ Figure 14 ] show the results of immunostaining measuring changes in inflammatory astrocytes in the hippocampus of Alzheimer's disease model mice administered with KARC.

[0065] [ Figure 15 ] show the results of measuring the number of activated astrocytes in the CA1 region of the hippocampus of Alzheimer's disease model mice administered with KARC.

[0066] [ Figure 16 ] show the results of measuring the number of activated astrocytes in the CA3 region of the hippocampus of Alzheimer's disease model mice administered with KARC.

[0067] In Alzheimer's disease mice, inflammation of astrocytes leads to brain neuron death and simultaneously weakens brain homeostasis through the generation and maintenance of cranial nerve synapses.

[0068] The degree of astrocyte activation was confirmed by immunostaining of GFAP as a measure of the astrocytic inflammatory response, and it was found that the degree of astrocyte activation in the KARC administration group was reduced Figure 14 , and the activation level of the hippocampus, which stores memories, was reduced. In the KARC administration group, the number of astrocytes in the CA1 and CA3 regions decreased Figure 15 、 Figure 16 , and an anti-inflammatory effect was observed.

[0069] Figure 17 shows the change in enzyme activity upon oral administration of the KwonP-1 strain.

[0070] Figure 18 shows the change in enzyme activity upon oral administration of the KwonP-2 strain.

[0071] Figure 19 shows the change in enzyme activity upon oral administration of the KwonP-3 strain.

[0072] Figure 20 shows the change in enzyme activity upon oral administration of the Pico YP strain.

[0073] Figure 21 shows the change in enzyme activity upon oral administration of the PicoYP-01 strain.

[0074] Figure 22 shows the change in enzyme activity upon oral administration of the PicoYP-02 strain.

[0075] Figure 17 、 Figure 18 、 Figure 19 、 Figure 20 、 Figure 21 、 Figure 22 shows that KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02 were orally administered for 90 minutes under conditions similar to the digestive process of the human stomach (1 < pH < 5). The change in ALDH enzyme activity was measured. At pH = 5 (similar to the conditions observed during food intake), the ALDH enzyme activity remained at a minimum of 37.29 units / g and a maximum of 52.24%. It was confirmed that the enzyme activity was maintained when KARC was orally administered.

[0076] Figure 23 ​​​​​​​​] shows the growth curve and enzyme activity of KwonP-1 strain cultured in a 5 L fermentor.

[0077] [ Figure 24 ] shows the growth curve and enzyme activity of KwonP-2 strain cultured in a 5 L fermentor.

[0078] [ Figure 25 ] shows the growth curve and enzyme activity of KwonP-3 strain cultured in a 5 L fermentor.

[0079] [ Figure 26 ] shows the growth curve and enzyme activity of the PicoYP strain cultured in a 5 L fermentor.

[0080] [ Figure 27 ] shows the growth curve and enzyme activity of PicoYP-01 strain cultured in a 5 L fermentor.

[0081] [ Figure 28 ] shows the growth curve and enzyme activity of PicoYP-02 strain cultured in a 5 L fermentor.

[0082] *exist[ Figure 23 、 Figure 24 、 Figure 25 、 Figure 26 、 Figure 27 、 Figure 28 ], the new mutant strains KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-01 were cultured in 5 L fermentors using YPD medium under the same conditions. This was carried out at 30°C and 200 rpm for 48 hours. When the growth curve (OD660nm) and ALDH enzyme activity of each strain were compared with the model strain, the ALDH enzyme activity was at least 10.5 times and at most 18.75 times higher. PicoYP-01 had the highest ALDH activity at 52.68 units / g, and KwonP-3 had the lowest value at 29.5 units / g. DETAILED DESCRIPTION

[0083] Hereinafter, the method for producing the KARC dry powder of the present invention, i.e., the lysate of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, KCTC14985BP, will be described in more detail.

[0084] However, these examples are only for the purpose of illustrating compositions capable of achieving the objects of the present invention, and therefore, the scope of the present invention is not limited only to the compositions described in the following examples.

[0085] Example

[0086] [Example 1] Screening of wild yeast parent strains for mutation.

[0087] In the present invention, each Makgeolli (Korean traditional liquor) suspension was prepared by mixing various types of Makgeolli with a 0.9% NaCl solution. The Makgeolli suspension was stirred at 200 rpm for 1 hour. The supernatant containing the wild yeast strain was diluted with YPD (yeast extract peptone dextrose broth) medium. The diluted solution was prepared to be 10% of the original solution. -6 times.

[0088] The diluted solution was spread onto YPD agar medium. The agar medium was incubated statically at 30°C under aerobic conditions for one week. Preliminary screening of Saccharomyces cerevisiae was performed based on colony morphology, growth characteristics in YPD agar, and microscopic observation.

[0089] The ALDH activity and glutathione content of the screened Saccharomyces cerevisiae were measured. Parent strains were selected based on ALDH activity and glutathione production.

[0090] 1-1: Measurement of aldehyde dehydrogenase

[0091] Acetaldehyde reacts with dinitrophenylhydrazine (DNPH) to form an acetaldehyde-hydrazone (Ach-DNPH) compound. The Ach-DNPH compound is detected by HPLC equipped with a C18 column at 360 nm. The amount of Ach-DNPH compound detected quantifies the amount of aldehyde reduced by the decomposition reaction by aldehyde dehydrogenase (ALDH).

[0092] The enzyme reaction was performed at 30°C by adding 10 μL of yeast lysate to 990 μL of a reaction mixture [50 mM potassium phosphate buffer (pH 8.0), 1.5 mM acetaldehyde, and 3 mM NADP+]. After the enzyme reaction was complete, 50 μL of 10 mM DNPH was added to induce the formation of Ach-DNPH. Ach-DNPH formation was performed at 22°C for 1 hour.

[0093] The formation of Ach-DNPH was terminated by adding 3M sodium acetate (pH 9). The formed Ach-DNPH compound was isolated by adding two volumes of acetonitrile. The isolated Ach-DNPH compound (in ACN) was analyzed by injection HPLC.

[0094] The concentration of Ach-DNPH compounds was analyzed by HPLC at a wavelength of 360 nm using a mobile phase (acetonitrile, water) developed on a C18 column at a rate of 1 mL / min. The area values ​​of the chromatograms obtained from the HPLC results were converted using a standard curve for aldehyde-DNPH (Sigma-Aldrich) to quantify the concentration of Ach-DNPH compounds. A decrease of 1 mM in Ach-DNPH concentration per minute was calculated as 1 unit of ALDH. ALDH activity was normalized to units per mg of protein.

[0095] 1-2: Glutathione measurement

[0096] Yeast cells were harvested by centrifuging 1 mL of Saccharomyces cerevisiae culture medium. A suspension was prepared by adding 1 mL of water to the harvested yeast cells. Glutathione was extracted by stirring the suspension at 85°C and 1,000 rpm for 2 hours. The suspension was centrifuged to remove the yeast cells, and the supernatant was filtered through a 0.22 μm filter to obtain a glutathione-containing sample.

[0097] The glutathione concentration in the sample was analyzed by HPLC (Shimazu LC-20AD) equipped with a C18 column. Glutathione concentration was analyzed at a wavelength of 210 nm under conditions of developing a mobile phase (2.02 g / L sodium 1-heptanesulfonate monohydrate, 6.8 g / L potassium dihydrogen phosphate, pH 3.0, methanol mixture) at a rate of 1 mL / min. The area value of the chromatogram obtained as the HPLC result was analyzed using a standard curve analysis of glutathione.

[0098] The ALDH activity and glutathione content of 200 different yeasts obtained from Korean Makgeolli were analyzed. The 10 yeasts listed in [Table 1] showed higher ALDH activity or glutathione production capacity than other yeasts.

[0099] Yeast #97 had an ALDH activity of 0.10 units / mg protein, the second highest overall. Yeast #97 also had a glutathione content of 0.42%, the highest of all strains. Yeast #97 was selected as the parent strain and subjected to the mutation induction procedure.

[0100] Table 1

[0101] [Example 2] Identification of parent strains used in the mutagenesis process

[0102] Identification was performed to confirm the exact species of the wild-type parent strain (yeast #97, wild-type yeast). To ensure sufficient yeast cells for DNA extraction, only a single yeast colony was plated on YPD agar medium. DNA was extracted using a genomic DNA preparation kit (HiGene™, BIOFACT Co., Ltd., Daejeon, South Korea) according to the manufacturer's instructions.

[0103] To amplify the rRNA gene in the yeast ITS region, polymerase chain reaction (PCR) was performed on yeast chromosomal DNA using ITS5 (forward) and ITS4 (reverse) primers. PCR results were analyzed by DNA sequencing.

[0104] The DNA sequence of the parent strain was isolated using the Bioedit program. The reverse strand of the PCR result was converted to a paired base sequence using the reverse completion process.

[0105] The forward strand sequence matched its reverse strand counterpart using the Cluster X program. Using the BLAST database provided by the National Center for Biotechnology Information (NCBI), the parent strain was identified as matching the sequence identified in the experimental procedure. The rRNA in the parent strain's ITS was found to be 100% identical to that of Saccharomyces cerevisiae.

[0106] [Example 3] Selection of mutants with improved aldehyde dehydrogenase production

[0107] The mutagenesis procedure for the wild-type S. cerevisiae parent strain was performed according to the methods described in US Patent Application No. 17 / 176,365.

[0108] To induce mutations in the yeast parent strain, wild yeast strains that produce both ALDH and glutathione were treated with ethyl methanesulfonate (EMS) or nitrosoguanidine (NGD). The induced mutant yeast strains were exposed to varying concentrations of methylglyoxal. Mutants with excellent adaptability to methylglyoxal were screened. The selected yeast strains were exposed to varying concentrations of lysine. Mutants with excellent adaptability to lysine were screened. 30 mutants with excellent adaptability to both methylglyoxal and lysine were obtained. Each of the 30 yeast strains was evaluated for five properties: growth curve, ALDH activity, ADH activity, coenzyme content, and glutathione content.

[0109] 3-1: Growth characteristics

[0110] Saccharomyces cerevisiae is a Crabtree-positive microorganism that produces ethanol while growing under aerobic conditions. Cultivating high-yield yeast requires Saccharomyces cerevisiae with high ethanol tolerance.

[0111] YPD medium with different ethanol concentrations (no ethanol, 5%, 7% and 10%) was prepared. Saccharomyces cerevisiae (yeast) medium adjusted to OD=1 at 660nm was prepared. Each mixture of the prepared YPD medium and yeast medium was diluted in a ratio of 99:1. Finally, YPD medium containing yeast with four different alcohol concentrations was prepared. Each YPD medium mixed with yeast was shaken at 30°C and 200rpm. The growth curve of the mutant strain was measured every 3 hours for 48 hours. The growth curve of each mutant strain was evaluated by three characteristics: the time (or period) of the lag phase, the specific growth rate (OD660nm / hr) in the exponential phase and the maximum density (OD660nm).

[0112] The higher the ethanol concentration in YPD medium, the longer the lag phase. The maximum density and specific growth rate decreased. Comparing the maximum density of mutant strains at low (5%) and high (10%) ethanol concentrations revealed that, for nine mutant strains, the high concentration maintained 50% growth compared to the low concentration. The nine mutant strains are distinguished from other strains by their short lag phase and high specific growth rate.

[0113] [Table 2]

[0114] 3-2: Activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH)

[0115] The activity of alcohol dehydrogenase (ADH) was measured by adding 10 μL of yeast lysate to 990 μL of a reaction mixture consisting of 50 mM potassium phosphate buffer (pH 8.0), 2 mM NAD+, and 1% ethanol. The activity of aldehyde dehydrogenase (ALDH) was measured by adding 10 μL of yeast lysate to 990 μL of a reaction mixture consisting of 50 mM potassium phosphate buffer (pH 8.0), 3 mM NAD+, and 1.5 mM acetaldehyde. The enzyme reaction of ADH and ALDH was carried out at 30°C for 5 minutes, and the concentration of NAD(P)H generated by the enzyme reaction was measured by absorbance at 340 nm.

[0116] The enzyme activities of the nine mutant strains (K-1 to K-9) selected in the present invention were measured. The ADH activity of the mutant strains was as low as 382.69 units / g and as high as 975.29 units / g. Compared with the model strain (reference yeast, Saccharomyces cerevisiae KCTC7296), the ADH activity of the mutant strains increased by at least 5.1 times and as much as 13.1 times. The ALDH activity of the mutant strains was as low as 15.23 units / g and as high as 72.16 units / g. Compared with the enzyme activity of the model strain, the ALDH activity of the mutant strains increased by at least 5.3 times and as much as 24.9 times.

[0117] Six mutant strains (K-1, K-4, K-6, K-7, K-8, and K-9) showed similar rates of increase in ADH and ALDH enzyme activities compared to the model strain. The ALDH enzyme activities in three mutant strains (K-2, K-3, and K-5) were 18.3-, 23.2-, and 24.9-fold higher than those in the model strain, respectively. The ADH enzyme activities in three mutant strains (K-2, K-3, and K-5) were 9.7-, 11.6-, and 13.1-fold higher than those in the model strain, respectively. The rate of increase in ALDH enzyme activity in the three mutant strains (K-2, K-3, and K-5) was twice that of ADH.

[0118] The present inventors named three new mutants (K-2, K-3, and K-5) adapted to increase aldehyde dehydrogenase (ALDH) activity PicoYP, PicoYP-01, and PicoYP-02, respectively. These three new mutants were deposited with the Bioresource Center of the Korea Institute of Bioscience and Biotechnology under the accession numbers KCTC14983BP, KCTC14984BP, and KCTC14985BP, respectively.

[0119] 3-3: Content of coenzymes (NAD and NADP)

[0120] NAD total and NADP total were measured in lysates extracted from the mutant strain using the NADH / NAD+ assay kit and the NADPH / NADP+ assay kit, respectively. NAD(P) in the sample was converted to NAD(P)H using an NAD(P) cycling buffer and an NAD(P) cycling enzyme mix. A chromogenic reaction was induced with an NAD(P) chromogen, and the absorbance at 450 nm was measured. The chromogenic reaction was measured by absorbance at 450 nm. The sample absorbance was substituted into the equation corresponding to the standard curve to calculate the NAD(P) total in the yeast lysate.

[0121] The coenzyme content of the nine mutant strains (K-1 to K-9) selected in the present invention was measured. The NADtotal of the mutant strains was as low as 126 nmol / g and as high as 195 nmol / g. Compared with the model strain, the NADtotal of the mutant strain increased by at least 7.3 times and as much as 10.8 times. The NADPtotal content of the mutant strain was as low as 2.4 nmol / g and as high as 5.8 nmol / g. Compared with the model strain, the NADPtotal content of the mutant strain increased by at least 11.4 times and as much as 27.6 times.

[0122] In the six mutants (K-1, K-4, K-6, K-7, K-8, and K-9), the rate of increase in NADPtotal was less than twice the rate of increase in NADtotal. The rates of increase in total NADP content in the three new mutants (PicoYP, PicoYP-01, and PicoYP-02) were 25.7-fold, 22.9-fold, and 27.6-fold, respectively. The rates of increase in total NAD content in the three new mutants were 10.8-fold, 9.9-fold, and 11.3-fold, respectively. The rate of increase in NADPtotal in the three new mutants was greater than twice the rate of increase in NADtotal.

[0123] 3-4: Glutathione (GSH) content

[0124] Glutathione content was measured in nine mutant strains using the same method as in Examples 1-2. Glutathione content ranged from a low of 0.85% to a high of 1.05%. Compared to the model strain, glutathione content increased by at least 2.7 times and as much as 3.3 times in the mutants. Three of the new mutants (PicoYP, PicoYP-01, and PicoYP-02) showed higher increases in ALDH activity and coenzyme content than in the other strains.

[0125] The three new mutant yeasts (PicoYP, PicoYP-01, and PicoYP-02) have similar glutathione production capabilities to existing deposited strains (Kwon P-1, Kwon P-2, and Kwon P-3). Compared to existing deposited strains, the three new mutant yeasts have significantly increased ADH and ALDH enzyme activities and coenzyme content.

[0126] [Table 3]

[0127] [Table 4]

[0128] [Example 4] Comparison of carbon source preferences

[0129] The carbon source preferences of three mutants (KwonP-1, KwonP-2, and KwonP-3) with elevated ALDH and glutathione levels were investigated, for which a domestic patent application was filed on February 18, 2020. The carbon source preferences of a reference yeast strain (KCTC7296) were measured. To determine the maximum capacity for ALDH production, the carbon source preferences of three new mutants (PicoYP, PicoYP-01, and PicoYP-02) were investigated.

[0130] The carbon source preference of the strains was analyzed for specificity and novelty using the API 50CHL kit (API Systems, BIOMERIEUX, SA, France).

[0131] Prepare 15 mL conical tubes containing 8 mL of YPD medium. Inoculate each of the seven mutant strains into the prepared conical tubes.

[0132] After incubating the inoculated conical tubes at 30°C and 200 rpm for 24 hours, each of the seven mutant strains was isolated from the exponential growth phase. To eliminate the effects of residual carbon source in the YPD medium, the yeast was washed three times using a centrifuge. A yeast suspension at a 2 McFarland concentration was prepared using API 50CHL medium. The prepared yeast suspension was placed in a test strip tube. The test strips, upon which the suspension was dispensed, were incubated at 30°C for 24 hours.

[0133] API 50CHL medium, used for API testing, is purple. As acids are produced through energy metabolism, API 50CHL medium turns blue, then green, and finally yellow. The carbon source used by the mutant is recorded based on the color change, such as purple x, blue +, green ++, and yellow +++.

[0134] All seven mutant strains tested used 19 carbon sources for energy production and growth: L-arabinose, ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, N-acetylglucosamine, arbutin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, raffinose, and gentiobiose.

[0135] Only three mutants used rhamnose: KwonP-1, PicoYP-01, and PicoYP-02. Four mutants used sorbitol: KwonP-1, KwonP-3, PicoYP-01, and PicoYP-02. Four mutants used α-methyl-D-mannoside: the model strain, KwonP-1, KwonP-2, and PicoYP-02. Six mutants used amygdalin: KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02. Four mutants used D-turanose: the model strain, KwonP-1, KwonP-3, and PicoYP-02. Three mutants used D-tagatose: the model strain, KwonP-3, and PicoYP-3. Only the model strain used gluconate.

[0136] Mannitol and sorbitol, corresponding to alcoholic carbon sources, had a significant effect on yeast growth. The three new mutants differed from the other four yeast strains in the types of sugars they utilized for growth. The three new mutants (PicoYP, PicoYP-01, and PicoYP-02) showed slight differences in their preferred alcoholic carbon sources [Table 5].

[0137] [Table 5]

[0138] [Example 5] Changes in ALDH activity of mutant strains in gastric juice When KARC is administered orally, in order to maintain enzyme activity in the intestine, the enzyme activity must pass safely without being destroyed by gastric acid, which secretes strong proteolytic enzymes such as pepsin.

[0139] NaOH solution was added to artificial gastric fluid at pH 1.17 to create two simulated solutions at pH 3 and pH 5, similar to the human gastric environment during food digestion. 1g of KARC was added to 7mL of artificial gastric fluid and 7mL of each simulated solution and mixed at 36.5°C for 5, 30, 60, and 90 minutes, respectively. NaOH solution was added to each reaction mixture to adjust the acidity to pH 7. 10mL samples were collected from each of the adjusted pH 7 solutions for analysis. ALDH activity was assayed for each sample.

[0140] At pH 1.17, ALDH activity in samples decreased by over 92.88% compared to the control within 5 minutes of reaction. At pH 1.17, ALDH activity in samples decreased by an average of 98.89% over 90 minutes. At pH 3, ALDH activity decreased by an average of 96.66% after 90 minutes, and at pH 5, it decreased by 56.83%. Finally, ALDH activity at pH 3 and 5 remained relatively higher than at pH 1.17 during the 90-minute reaction.

[0141] Specifically, at pH 1.17, the ALDH activity of KwonP-1 (KCTC13925BP) decreased by 90.94% to 5.57 units / g after 5 minutes of reaction compared to the control group. The ALDH activity of KwonP-1 decreased by 98.57% to 0.88 units / g after 90 minutes of reaction. Figure 17 The enzyme activity at pH = 3 and pH = 5 remained relatively higher than that at pH = 1.17. After 90 minutes of reaction, the ALDH activity of KwonP-1 decreased by 96.66% to 5.57 units / g at pH = 3, and decreased by 98.57% to 0.88 units / g at pH = 5.

[0142] At pH = 1.17, the ALDH activity of KwonP-2 (KCTC14122BP) decreased by 91.18% to 5.43 units / g after 5 minutes of reaction. The ALDH activity of KwonP-2 decreased by 98.81% to 0.73 units / g after 90 minutes of reaction. Figure 18 At pH 3 and pH 5, higher enzyme activity was maintained than at pH 1.17. After 90 minutes of reaction, ALDH activity decreased by 97.62% to 1.47 units / g at pH 3, and decreased by 56.11% to 26.99 units / g at pH 5.

[0143] At pH = 1.17, the ALDH activity of KwonP-3 (KCTC14123BP) decreased by 89.99% to 6.16 units / g after 5 minutes of reaction. The ALDH activity of KwonP-3 decreased by 97.85% to 1.32 units / g after 90 minutes of reaction. Figure 19 At pH 3 and pH 5, higher enzyme activity was maintained than at pH 1.17. After 90 minutes of reaction, ALDH activity decreased by 92.61% to 4.55 units / g at pH 3, and decreased by 62.31% to 22.18 units / g at pH 5.

[0144] At pH = 1.17, the ALDH activity of PicoYP (KCTC14983BP) decreased by 92.84% to 4.40 units / g after 5 minutes of reaction. The ALDH activity of PicoYP decreased by 98.33% to 1.03 units / g after 90 minutes of reaction. Figure 20 High enzyme activity was maintained at both pH 3 and pH 5. After 90 minutes of reaction, ALDH activity decreased by 96.66% at pH 3 to 2.05 units / g, and decreased by 53.97% at pH 5 to 28.31 units / g.

[0145] At pH = 1.17, the ALDH activity of PicoYP-01 (KCTC14984BP) decreased by 95.71% to 2.64 units / g after 5 minutes of reaction. The ALDH activity of PicoYP-01 decreased by 99.76% to 0.15 units / g after 90 minutes of reaction. Figure 21 At pH 3 and pH 5, higher enzyme activity was maintained than in gastric juice. After 90 minutes of reaction, ALDH activity decreased by 98.21% at pH 3 to 1.10 units / g, and decreased by 58.74% at pH 5 to 25.38 units / g.

[0146] At pH = 1.17, the ALDH activity of PicoYP-02 (KCTC14985BP) decreased by 96.66% to 2.05 units / g after 5 minutes of reaction. The ALDH activity of PicoYP-02 decreased by 99.76% to 0.15 units / g after 90 minutes of reaction. Figure 22 At pH 3 and pH 5, higher enzyme activity was maintained than in gastric juice. After 90 minutes of reaction, at pH = 3, ALDH activity decreased by 98.21% to 1.10 units / g, and at pH = 5, ALDH activity decreased by 62.08% to 23.32 units / g.

[0147] The pH of secreted gastric juice is 1.17. When food is consumed, the pH of the original gastric juice and food mix in the stomach, raising it from 3 to 5, making it unlikely to reach a pH of 1.17. Despite this, ALDH activity in the mutant strain is maintained even under this extreme pH condition.

[0148] Finally, the ALDH enzyme activity of the new mutants (PicoYP, PicoYP-01, PicoYP-02) remained at 2 to 5 units / g, despite a 92% to 97% decrease under the strongly acidic conditions of pH 1.17. Maintaining 2-5 units of enzyme activity is sufficient to function in the intestine. Compared to pH 1.17, it was even higher at pH 3 and pH 5. This leads to the conclusion that the new mutants (PicoYP, PicoYP-01, PicoYP-02) can be administered orally.

[0149] [Example 6] Growth characteristics of 5L fermenter culture

[0150] Be inoculated in YPD substratum (2% peptone, 1% yeast extract, 2% glucose) respectively, and carry out primary seed culture 18 hours at 30 ℃ and 200rpm.The seed of 20mL culture is inoculated in 1980mL YPD substratum, and in 5L, cultivate again.The cultivation in 5L culture tank was carried out 48 hours at 30 ℃ and 200rpm.Use the 10mL sample analysis OD660nm growth curve and the enzyme activity under collecting from secondary culture.

[0151] The maximum density (OD660nm) of KwonP-1 (KCTC13925BP) was 134.4. The maximum density of KwonP-1 was 4.35% higher than that of the model strain (KCTC7296). The growth curve characteristics and specific growth rate (OD660nm / hr) of KwonP-1 were similar to those of the model strain. The ALDH activity of KwonP-1 was 33.6 units / g. The ALDH activity of KwonP-1 was 11.96 times higher than that of the model strain. Figure 23 ].

[0152] The maximum density (OD660nm) of KwonP-2 (KCTC14122BP) was 133.8. The maximum density of KwonP-2 was 3.88% higher than that of the model strain. The growth of KwonP-2 ended earlier than that of the model strain. The specific growth rate (OD660nm / hr) of KwonP-2 was 14.8% higher than that of the model strain. The ALDH activity of KwonP-2 was 31.5 units / g. The ALDH activity of KwonP-2 was 11.21 times higher than that of the model strain. Figure 24 ].

[0153] The maximum density (OD660nm) of KwonP-3 (KCTC14123BP) was 134.1. The maximum density of KwonP-3 was 4.12% higher than that of the model strain. The growth of KwonP-3 ended earlier than that of the model strain. The specific growth rate (OD660nm / hr) of KwonP-3 was 6.08% higher than that of the model strain. The ALDH activity of KwonP-3 was 29.5 units / g. The ALDH activity of KwonP-3 was 10.5 times higher than that of the model strain. Figure 25 ].

[0154] The maximum density (OD660nm) of PicoYP (KCTC14983BP) was 123.8. The maximum density of PicoYP was 3.88% higher than that of the model strain. The growth curve characteristics of PicoYP were similar to those of the model strain. The specific growth rate (OD660nm / hr) of PicoYP was 6.22% higher than that of the model strain. The ALDH activity of PicoYP was 44.2 units / g. The ALDH activity of PicoYP was 15.73 times higher than that of the model strain. Figure 26 ].

[0155] The maximum density (OD660nm) of PicoYP-01 (KCTC14984BP) was 126.9. The maximum density of PicoYP-01 was 1.47% higher than that of the model strain. The growth curve characteristics of PicoYP-01 were similar to those of the model strain. The specific growth rate (OD660nm / hr) of PicoYP-01 was 2.14% higher than that of the model strain. The ALDH activity of PicoYP-01 was 47.1 units / g. The ALDH activity of PicoYP-01 was 16.76 times higher than that of the model strain. Figure 27 ].

[0156] The maximum density (OD660nm) of PicoYP-02 (KCTC14985BP) was 148.1. The maximum density of PicoYP-02 was 14.99% higher than that of the model strain. Compared with the model strain, the growth curve of PicoYP-02 was at the top. The specific growth rate (OD660nm / hr) of PicoYP-02 was 9.64% lower than that of the model strain. The ALDH activity of PicoYP-02 was 52.68 units / g. The ALDH activity of PicoYP-02 was 18.75 times higher than that of the model strain. Figure 28 ].

[0157] [Example 7] Preparation of mutant strain lysate (KARC)

[0158] To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysate, proteases are removed and inhibited. To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysate, cell debris is removed. The dried products or lysates of the mutant strains are mixed to prepare a KARC composition.

[0159] The mutant strain and the culture medium containing it contain a variety of substances, such as yeast metabolites and proteolytic enzymes secreted by the yeast. To extract and preserve the ALDH, coenzymes, and glutathione present in the yeast, it is necessary to fully remove substances outside the yeast. To this end, a washing procedure was performed. Washing of the mutant strain was performed by dispensing 40 mL of culture medium into a 50 mL conical tube, centrifuging at 13,000 rpm for 15 minutes, and removing the supernatant.

[0160] As a result of centrifugation, the yeast cells aggregated into a pellet containing residual culture medium. Add 30 mL of purified water and vortex to loosen the pellet. Repeat this procedure three times to fully remove the residual culture medium.

[0161] Yeast is known to have an ethanol tolerance of up to 13%, and yeast will die when exposed to high ethanol concentrations. Dissolve the washed pellet thoroughly in 10 mL of 20% ethanol solution to induce yeast death. Stir the pellet in ethanol at 100 rpm for 30 minutes to induce yeast death. At the end of the reaction time, add 30 mL of purified water to bring the ethanol concentration to 5%. Repeat this washing procedure three times to fully remove the ethanol.

[0162] To prevent the degradation of ALDH and ADH by proteases present in yeast cells, 10 mL of 1X PBS was prepared by dissolving two protease inhibitor tablets (Pierce Protease Inhibitor Tablets, EDTA-free, Thermo Scientific). The solution was added to the washed yeast pellet and distributed thoroughly.

[0163] To prepare the lysate of the mutant strain prepared in the present invention, 4 g of glass beads were added and stirred to disrupt the yeast cell walls. To prevent heat generated during yeast disruption from denaturing the enzyme, vortexing for 30 seconds and incubating on ice for 30 seconds were repeated 6 times.

[0164] After the yeast cell wall was broken, 10 mL of 100 mM potassium phosphate buffer was added and mixed by vortexing for 3-5 seconds. Centrifuge 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 ( The KARC composition was prepared by filtration through a syringe filter (Sartorius, Goettingen, Germany).

[0165] To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysates, remove and inhibit intracellular proteases, and remove cellular debris such as cell walls, KARC compositions were prepared using lysates of six mutant strains (KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, PicoYP-02) or mixtures thereof in a free ratio [Table 6].

[0166] KARC 1 is made from Kwon P-1. KARC 1's ADH and ALDH enzyme activities are 461.4 units / g and 28.6 units / g, respectively. The NAD and NADP total coenzyme contents in KARC 1 are 176.2 nanomoles / g and 5.1 nanomoles / g, respectively. KARC 1 also contains 0.98 wt% GSH.

[0167] KARC 2 is made from Kwon P-2. KARC 2's ADH and ALDH enzyme activities are 482.1 units / g and 29.8 units / g, respectively. KARC 2's NAD total and NADP total coenzyme contents are 175.4 nanomoles / g and 5.2 nanomoles / g, respectively. KARC 2 also contains 0.96 wt% GSH.

[0168] KARC 3 is made from Kwon P-3. KARC 2 has ADH and ALDH enzyme activities of 477.5 units / g and 28.1 units / g, respectively. KARC 3 has NAD total and NADP total coenzyme contents of 177.2 nanomoles / g and 5.1 nanomoles / g, respectively. KARC 3 also has a GSH content of 1.00 wt%.

[0169] KARC 4 is made from PicoYP. KARC 2 has ADH and ALDH enzyme activities of 586.8 units / g and 33.8 units / g, respectively. KARC 4 has NAD total and NADP total coenzyme contents of 184.3 nanomoles / g and 5.7 nanomoles / g, respectively. KARC 4 also has a GSH content of 0.84 wt%.

[0170] KARC 5 is produced from PicoYP-01. KARC 5 exhibits ADH and ALDH enzyme activities of 621.6 units / g and 38.2 units / g, respectively. The NAD and NADP total coenzyme contents in KARC 5 are 186.9 nanomoles / g and 5.6 nanomoles / g, respectively. KARC 5 also contains 0.84 wt% GSH.

[0171] KARC 6 is made from PicoYP-02. KARC 5 has ADH and ALDH enzyme activities of 664.1 units / g and 41.6 units / g, respectively. KARC 6 has NAD total and NADP total coenzyme contents of 195.0 nanomoles / g and 5.8 nanomoles / g, respectively. KARC 6 also has a GSH content of 0.88 wt%.

[0172] KARC was prepared by freely mixing dry powders and lysates prepared from six deposited strains. The average enzyme activities of ADH and ALDH in the KARC composition were 547.6 units / g and 33.1 units / g, respectively. The average contents of the coenzyme NAD total and coenzyme NADP total in the KARC composition were 180.4 nanomoles / g and 5.4 nanomoles / g, respectively. The average glutathione content in the KARC composition was 0.84 wt%.

[0173] The aldehyde decomposition ability of KARC was maintained during the lysate production process. KARC exhibited the ability to remove endogenous aldehydes such as HNE, MDA, and 3,4-dihydroxyphenylacetaldehyde (DOPAL).

[0174] [Table 6]

[0175] [Example 8] Sequence analysis of ALDH contained in mutant strains.

[0176] The differences in ALD (yeast aldehyde dehydrogenase) between the mutants and the parent strain were investigated. Whole-genome sequencing was performed on the parent strain and mutants KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02. Mutant cells were isolated by culturing pure strains on solid culture medium. The resulting mutant genome sequences were then analyzed.

[0177] In the ALD (yeast aldehyde dehydrogenase) of the new mutant strain, ALD2 (SEQ ID NO: 3) and ALD3 (SEQ ID NO: 4) were found to be densely distributed on chromosome 13. A 689-nucleotide noncoding region was located between the ALD2 and ALD3 encoding genes.

[0178] ALD2 and ALD3 coexist in the same genome. ALD2 and ALD3 encode separate aldehyde dehydrogenases. The ALD2 gene is nearly identical to ALD3, consisting of 1,521 nucleotides and 506 amino acids, but differs by 8.2% in sequence. ALD2 and ALD3 were identified as separate aldehyde dehydrogenases, differing by 125 bases (8.2%).

[0179] In six mutant strains (KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02), the ALD2 sequence lacks a stop codon, allowing protein synthesis to proceed. Consequently, a new, larger ALDH enzyme was created by linking a portion of ALD2 to ALD3 [SEQ ID NO: 1].

[0180] ALD2 [SEQ ID NO. 3] of the model strain (KCTC7296) consists of a 30-nucleotide sequence (5'-GTTCACATAAATCTCTCTTTGGACAACTAA-3') encoding 9 amino acids (N-VHINLSLDN-C) at the terminal, excluding the stop codon.

[0181] ALD2 of the six mutants consists of a specific 42-nucleotide sequence (5'-AGATATAGATTATACACATTTAGAAAATTAGCCAAAAGAAAA-3') encoding 14 amino acids (N-RYRLYTFRKLAKRK-C) between the 5' end of ALD2 and ALD3 [SEQ ID NO. 2].

[0182] The ALD2 gene encoding ALD2 is deleted from nucleotide 1492 to nucleotide 647 in the noncoding region, eliminating the termination codon. Ultimately, the six deposited mutants contain a novel mutant gene consisting of 3,054 bases encoding a novel ALD2 gene [SEQ ID NO: 1].

[0183] [Example 9] Effect of Oral Administration of KARC on Reducing Acetaldehyde (Ach) and Malondialdehyde (MDA) in Vivo

[0184] For the acetaldehyde and MDA animal experiments, 5-week-old male Sprague Dawley (SD) rats were used. The KARC composition was orally administered to the rats at 10 units / kg or 20 units / kg, and alcohol (3 g / kg) was orally administered to the rats 30 minutes after KARC injection.

[0185] After administration, blood samples were collected from the tail vein at 0, 1, 3, 5, and 8 hours after KARC injection, and the plasma was stored at −80°C after centrifugation. Figure 1 、 Figure 2 ].

[0186] 9-1: Acetaldehyde-reducing effect of oral administration of KARC

[0187] The total acetaldehyde reduction effect of oral administration of KARC was evaluated using an acetaldehyde assay kit (LSBio, Seattle, WA, USA). 20 μL of each sample was distributed to two wells of a 96-well plate. 80 μL of working reagent (75 μL assay buffer, 8 μL NAD / MTT, 1 μL enzyme A, 1 μL enzyme B) was distributed to one well. In the remaining wells, 80 μL of blank working reagent (75 μL assay buffer, 8 μL NAD / MTT, 1 μL enzyme B) was distributed. The distributed plate was gently mixed and reacted at room temperature for 30 minutes. After the reaction was complete, the absorbance was measured at 565 nm (520-600 nm).

[0188] In the KARC composition-administered group, acetaldehyde concentration reached its maximum 1 hour after ethanol administration and then showed a downward trend. In the KARC-administered group, acetaldehyde concentrations were significantly lower 1, 3, and 5 hours after ethanol administration compared to the control group (vehicle). In the high-dose KARC-administered group (F), blood acetaldehyde concentrations were 0.356, 0.224, and 0.091 mM, respectively, representing decreases of 39.2%, 58.4%, and 72.1% compared to the control group. Figure 1 ].

[0189] 9-2: MDA-reducing effect of oral administration of KARC

[0190] According to the manufacturer's protocol (ZeptoMetric, Buffalo, NY, USA), the total malondialdehyde content in the blood was analyzed using the OxiTecTM TBARS assay kit. 100 μL of sample, 100 μL of 8.1% SDS solution and 4 mL of color indicator (TBA, 10% NaOH solution, 20% acetic acid) were added to a conical tube and then reacted in a 95°C thermostatic water bath for 60 minutes. After the reaction was complete, the sample was centrifuged at 4°C and 1,600 rpm for 10 minutes and stabilized at room temperature for 30 minutes. 150 μL of supernatant was transferred to a 96-well plate and absorbance was measured at 530-540 nm.

[0191] In the control group (vehicle), the MDA concentration in the blood reached its maximum 3 hours after ethanol administration, while in the KARC administration group, it reached its maximum 1 hour after ethanol administration. The MDA concentration in the blood decreased 3 and 5 hours after ethanol administration, showing a significant difference from the control group. The blood MDA concentrations in the high-dose KARC administration group (F) were 0.232 and 0.137 μM, respectively, which were decreased by 80.4% and 86.3% compared to the control group. Figure 2 ].

[0192] These results indicate that oral administration of KARC is effective in reducing multiple endogenous aldehydes such as acetaldehyde and malondialdehyde in the blood.

[0193] [Example 10] Effect of reducing oxidative stress.

[0194] When drinking alcohol, excessive acetaldehyde (ACh) is produced by alcohol dehydrogenase (ADH), leading to increased reactive oxygen species and oxidative stress. Aldehyde dehydrogenase (ALDH) converts ACh into acetic acid and excretes it from the body. Excessive aldehyde production due to mutations in the ALDH gene or from excessive alcohol consumption can lead to lipid peroxidation.

[0195] The resulting acetaldehyde and malondialdehyde exacerbate oxidative stress and interfere with mitochondrial energy metabolism. Endoplasmic reticulum stress is caused by the accumulation of denatured proteins in cells, leading to cell death.

[0196] Blood acetaldehyde concentration was measured over time after drinking. Figure 3 The area under the curve (AUC) for blood acetaldehyde (Ach) was 13.02 ± 1.18 mg·h / dL for alcohol consumption alone. When administered at a dose of 10 units / kg of KARC, the area under the curve (AUC) for blood acetaldehyde (Ach) decreased significantly by 26.13% compared to alcohol consumption alone, measuring 9.39 ± 1.07 mg·h / dL (P = 0.005).

[0197] When administered at a dose of 20 units / kg, the AUC of blood acetaldehyde (ACh) significantly decreased by 55.71% compared to alcohol consumption alone, reaching 5.22 ± 0.99 mg·h / dL (P < 0.001). Compared to the group administered 10 units / kg of KARC, the 20 units / kg group showed a significant decrease in blood acetaldehyde (ACh) (P = 0.034). KARC demonstrated a dose-dependent reduction in total blood acetaldehyde (ACh) over time.

[0198] The reduction in blood acetaldehyde (Ach) concentration due to KARC administration has a positive impact on reducing oxidative stress and promoting health.

[0199] Measurement of blood malondialdehyde (MDA) concentration during chemotherapy Figure 4 The blood MDA concentration in the control group was 0.607±0.161 μM. The group receiving KARC treatment showed a significant 63.3% decrease in blood MDA concentration compared to the control group, measuring 0.223±0.033 μM (P<0.001).

[0200] In the control group, blood MDA concentration ranged from 0.427 μM to 0.885 μM, showing a wide variation. In the KARC-administered group, the range was significantly narrowed, with values ​​ranging from 0.158 μM to 0.269 μM. This confirms not only the effect of lowering blood MDA concentration but also stabilizing it, as shown in [ Figure 5 ] confirmed.

[0201] Various factors, including drug ingestion, stress, and intense physical exercise, lead to an increase in intracellular reactive oxygen species (ROS). This triggers lipid peroxidation and the oxidation of endogenous amines (such as dopamine, norepinephrine, serotonin, and histamine). Reactive aldehyde compounds, including 4-hydroxynonenal (HNE), malondialdehyde (MDA), acetaldehyde (ACh), and dopamine-induced aldehydes, accumulate within cells, exacerbating oxidative stress.

[0202] These aldehydes then react with surrounding proteins and undergo secondary metabolic processes to form stable end products such as malondialdehyde-acetaldehyde adducts (MAA) and malondialdehyde-lysine adducts (M-lys adducts), known as advanced lipid peroxidation end products. The accumulation of these products has toxic effects on various cells, further exacerbating oxidative stress.

[0203] This cumulative oxidative stress disrupts mitochondrial energy metabolism within the cell and leads to the accumulation of aldehyde intermediates in aldose sugar metabolism, including methylglyoxal (MG) and glyceraldehyde-3-phosphate (GA3P). Chain reactions involving aldehydes lead to the accumulation of stable final sugar oxidation products, known as advanced glycation end products (AGEs), which weaken intracellular antioxidant defense systems such as glutathione (GSH). These processes increase endoplasmic reticulum (ER) stress, leading to increased apoptosis in neuronal cells.

[0204] 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).

[0205] [Example 11] Acute oral administration test

[0206] 11-1. Preparation of experimental animals

[0207] The experimental animals were female and male ICR mice (7 weeks old). The received ICR mice were adapted for 7 days. The general symptoms of the adopted mice during the adaptation period were observed, and only healthy animals were used for short-term toxicity testing. Feed and water were available freely. Based on an average body weight of about 20g the day before oral administration, the group was divided into 10 groups of 5 mice each.

[0208] 11-2. Administration of test substance

[0209] The test substances were prepared by dissolving in physiological saline so that the doses for experimental animals were 0, 750, 3,000, and 5,000 mg / kg, respectively, based on the content of the mutant yeast lysate KARC of the present invention.

[0210] The dosage standards were in accordance with the Korea National Toxicology Program (KNTP) Toxicity Test Manual of the Ministry of Food and Drug Safety. The maximum dose of 5,000 mg / kg, as specified in the KNTP Manual, was used as the maximum concentration for this experiment. Samples prepared for each group were administered orally once to each test animal. The normal group (G1) received normal saline.

[0211] 11-3. Observation and autopsy

[0212] For all test groups, mice were observed for symptoms at least once daily from the date of acquisition until the date of necropsy. Symptoms were observed for 7 days after oral administration. Necropsy was performed after observing the symptoms of the rats. During the rat necropsy, changes in various organs were observed visually.

[0213] Single-dose toxicity testing of the ALDH-containing KARC composition of the present invention was conducted in mice. Results showed that no mouse mortality was observed within 7 days at concentrations of up to 5,000 mg / kg of mutant yeast KARC. No abnormalities, such as weight gain or changes in feed intake, were observed in the mice. Post-observation autopsies revealed no abnormal findings.

[0214] [Example 12] Effect of Oral Administration of KARC on Alleviating Memory Impairment and Abnormal Amyloid Protein

[0215] The 5xFAD mouse model was created to confirm the effects of KARC on improving memory impairment and cognitive function, as well as to verify the reduction of abnormal amyloid-β protein deposition. Administration of KARC improved the performance of 5xFAD mice in a Y-maze behavioral test assessing memory and reduced inflammatory cells and amyloid plaques in brain tissue.

[0216] 12-1: Preparation of 5xFAD Mouse Model and Administration of KARC

[0217] 5xFAD mice express three human APP variants (Swedish (K670N / M671L), Florida (I716V), and London (V717I)) and two PSEN1 variants (M146L and L286V). A total of five mutations in 5xFAD mice are strongly associated with Alzheimer's disease.

[0218] The expression of both genes is regulated by neural-specific elements of the mouse Thy1 promoter, inducing overexpression in the brain. The 6799 strain of mice has the highest expression of amyloid-β precursor protein (APP), which is associated with the rapid accumulation of the 42-amino acid form of amyloid-β.

[0219] 5xFAD mice begin to accumulate amyloid plaques at 1.5 months of age, with gliosis observed in mice around 2 months of age. Amyloid pathology is more severe in females than in males. Female mice are primarily used, and neuronal loss occurs in multiple brain regions, with the most pronounced neuronal loss in the amyloid-affected areas starting around 6 months of age. Mice develop Alzheimer's disease, with multiple cognitive and motor impairments.

[0220] The tails of mice born from double mating were clipped and the expression of APP and PSEN1 was confirmed by GENOMIC DNA PCR. Then, 5xFAD mice expressing both genes were selected for the experiment.

[0221] 17-month-old Alzheimer's disease model mice and non-transgenic (Ntg) mice selected from 5xFAD female mice were divided into 4 groups (n=5 / group). In the control group, Ntg mice were administered only PBS every day for one month. In the vehicle group, 5xFAD mice were administered.

[0222] Under the same conditions, Ntg or 5xFAD mice were orally administered PBS with KARC (20 units / kg / day) [ Figure 6 ].

[0223] 12-2: Results of behavioral tests and memory assessments by oral administration of KARC

[0224] A Y-maze test was used to assess short-term memory in 5xFAD mice, a model of Alzheimer's disease. Rodents typically prefer to explore new arms of the maze rather than return to previously visited arms. Many parts of the brain, including the hippocampus, septum, basal forebrain, and prefrontal cortex, are involved in this task.

[0225] The test took place in a Y-shaped maze with three plastic arms at 120° angles to each other. Mice were allowed to freely explore the three mazes from the central hub of the maze. The rate, expressed as a percentage, was calculated based on the number of times the mouse visited a new location compared to the total number of visits.

[0226] As Alzheimer's disease progresses, cognitive impairment is observed. In the Y-maze test in 5xFAD mice, a model of Alzheimer's disease, the rate of visiting new locations in the maze decreased to 47.4%. In mice administered KARC, this rate increased to 65.02%. This suggests that KARC has the potential to improve memory impairment caused by Alzheimer's disease and restore cognitive function caused by Alzheimer's dementia. Figure 7 ].

[0227] 12-3-1: Preparation of brain tissue

[0228] Mice were anesthetized by injection of chloral hydrate (40 mg / kg, ip) and perfused intracardially with saline solution containing 0.5% sodium nitrate and heparin (10 U / mL). Brain tissue was fixed with 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB, pH 7.2).

[0229] The whole brain tissue was dissected from the skull and post-fixed with 4% paraformaldehyde in 0.1 M PBS at 4° C. overnight and stored in 30% sucrose solution in 0.05 M PBS at 4° C. until sinking.

[0230] The brains were cryosectioned on a Cryostat (Microsystems AG, Leica, Wetzlar, Germany) with coronal sections of 30 μm thickness. The brains were stored in a cryoprotectant (25% ethylene glycol, 25% glycerol, 0.2 M PB, and water) at 4°C until use. Changes in the brain in hippocampal sections were observed by immunostaining.

[0231] 12-3-2: Immunohistochemical staining

[0232] Thioflavin S (Sigma-Aldrich, St. Louis, MO, USA) was used to stain abnormal aggregated amyloid plaques in brain sections (30 μm) including the hippocampus of 5xFAD mice (an Alzheimer's disease model). Thioflavin S is used in histological staining and biophysical studies 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 1% Thioflavin S solution for 5 minutes, washed three times with PBS, covered with a coverslip, and then analyzed using a fluorescent microscope. Figure 8 ].

[0233] Astrocytes (a type of neuroinflammatory cell) in brain sections (30 μm thickness) including 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 1 (anti-Iba-1, 1:1000; Wako, Osaka, Japan).

[0234] The incubated astrocytes were stained with biotinylated anti-rabbit IgG or avidin-biotin peroxidase complex (ABC) standard kit (Vector Laboratories, Burlingame, CA, USA).

[0235] Signals were detected after incubation of stained sections with 0.5 mg / mL 3,3'-diaminobenzidine (Sigma, St. Louis, MO, USA) in 0.1 M PBS containing 0.003% H2O2. To quantify GFAP or Iba-1-positive cells, respectively, images of stained brain sections were analyzed under a bright-field microscope (Olympus Optical, Tokyo, Japan).

[0236] 12-3-3: Intensity and counting methods of amyloid plaques.

[0237] Fluorescence intensity and total number of amyloid plaques were measured in the hippocampus of 5xFAD mice, an Alzheimer's disease model. Fluorescence intensity and total number of stained amyloid plaques in the hippocampus were analyzed on images taken at 40x magnification using Image J software (National Institutes of Health, Bethesda, MD, USA).

[0238] 12-4: Changes in brain mass induced by oral administration of KARC in 5xFAD mice, a model of Alzheimer's disease

[0239] 12-4-1: Effects of KARC on the Reduction of Amyloid Plaques in the Whole Brain of an Alzheimer's Disease Model

[0240] To measure abnormally aggregated amyloid proteins in 5xFAD mice, a model of Alzheimer's disease, amyloid plaques in brain tissues, including the hippocampus, were stained with white thioflavin S. Amyloid plaques are collections of misfolded proteins composed of a mixture of degenerating neurons and beta-amyloid aggregates. These plaques gradually accumulate in the brain because they cannot be broken down or removed. The accumulation of these amyloid plaques in specific organs or tissues, leading to functional impairment and deterioration, is called amyloidosis. When amyloid plaques are deposited in brain neurons, they trigger various neurodegenerative diseases. Structurally abnormal proteins (such as amyloid plaques) play a central role in Alzheimer's disease.

[0241] In 17-month-old female non-transgenic (Ntg) mice, no amyloid plaques were observed in the brain, similar to normal mice. In 17-month-old female 5xFAD mice (vehicle group), amyloid plaques were observed to accumulate throughout the brain, including the hippocampus. In 5xFAD mice orally administered KARC (20 units / kg / day), there was an overall reduction in amyloid plaques compared to the vehicle group. Figure 8 ].

[0242] The intensity and number of amyloid plaques accumulated in the hippocampus were quantified using Image J program.

[0243] No amyloid plaques were observed in 17-month-old female Ntg mice in the control group. The amyloid plaque intensity was 11.47 in the vehicle group and 12.37 in the KARC administration group, which were not significantly different from the control group. The amyloid plaque intensity of 17-month-old female 5xFAD mice was 52.36 in the vehicle group and 34.76 in the KARC administration group, which was significantly reduced by 66.38% compared with the vehicle group (p < 0.001). Figure 9 ].

[0244] There was no pathological difference in the quantitative number of amyloid plaques, with 319 amyloid plaques in the 17-month-old female non-transgenic mice (control group) and 501 in the KARC administration group. In the case of amyloid plaques in 17-month-old female 5xFAD mice, the number of plaques in the KARC administration group was significantly reduced by 2272 compared to 3202 in the vehicle group, representing a 70.09% decrease (p<0.001). Figure 10 ].

[0245] 12-4-2: Oral administration of KARC reduces neuronal inflammation in microglia and astrocytes in 5xFAD mice, an Alzheimer's disease model

[0246] The efficacy of KARC in reducing neuroinflammation in the 5xFAD mouse model was evaluated by staining microglia and astrocytes. Figure 11 ] and GFAP (astrocyte) antibodies [ Figure 14 ]The hippocampus in brain tissue was immunostained and then analyzed.

[0247] In 17-month-old non-transgenic (Ntg) mice, microglia and astrocytes in the CA1 and CA3 regions of the hippocampus were found to be in a quiescent state. In the CA1 and CA3 regions of the hippocampus of 17-month-old 5xFAD mice, microglia and astrocytes were found to be enlarged due to activation. Furthermore, neurites, including dendritic processes, became brighter and longer. This is due to the induction of brain neuroinflammation by the accumulation of abnormal amyloid plaques.

[0248] Oral administration of KARC (20 units / kg / day) suppressed neuroinflammatory activation of hippocampal microglia and astrocytes in 5xFAD mice [ Figure 11 、 Figure 14 ].

[0249] 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 with KARC, the number of microglia with positive activated Iba-1 was reduced to 59 ± 11 in the CA1 region and 80 ± 12 in the CA3 region. Figure 12 、 Figure 13 ].

[0250] In the hippocampus of 17-month-old 5xFAD mice, the number of GFAP-positive activated astrocytes in the CA1 region was 137 ± 1, and in the CA3 region was 114 ± 17. In the hippocampus of 17-month-old 5xFAD mice orally administered with KARC, the number of GFAP-positive activated astrocytes in the CA1 region was reduced to 90 ± 9, and in the CA3 region was reduced to 87 ± 5 [ Figure 15 、 Figure 16 ]. By quantification of activated cells, it was confirmed that oral administration of KARC inhibited neuroinflammatory activation of hippocampal microglia and astrocytes.

[0251] There is strong evidence that neuroinflammation plays a crucial role in inducing multiple pathological features observed in Alzheimer's disease. Sustained activation of microglia and other immune cells in the brain has been shown to exacerbate amyloid and tau pathologies and may be involved in the development of the disease.

[0252] Inflammatory cytokines, such as IL-1 and IL-6, have been shown to increase the amount of amyloid-β or induce hyperphosphorylation of tau, as well as trigger chronic inflammatory processes in microglia and astrocytes. This suggests that inflammation may directly contribute to the pathological manifestations of Alzheimer's disease.

[0253] Iba-1 (ionized calcium-binding adaptor 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 neurological damage, such as central nervous system ischemia and various other brain diseases.

[0254] The reduction of Iba-1 expression by KARC administration suggests that it has the potential to reduce brain nerve damage and improve central nervous system ischemia, and indicates the possibility of improving various brain diseases.

[0255] GFAP (glial fibrillary acidic protein) is a fibrillary acidic protein present in brain nerve cells. GFAP is a marker for astrocytes, which are distributed in the central nervous system (CNS). GFAP plays a crucial role in regulating astrocyte motility and maintaining their morphology by promoting myelination in nerve cells.

[0256] In mammals, GFAP expression in astrocytes is known to increase specifically in brains damaged by various factors, including physical and chemical damage. The increase in abnormal astrocytes in damaged brains can lead to neurodegenerative diseases by increasing inflammation and inducing brain neuronal cell death, accelerating disease progression.

[0257] The decrease in abnormal GFAP indicated that astrocyte damage was repaired by KARC administration, which may be effective in preventing and improving neurodegenerative diseases.

[0258] Analysis of microglial and astrocyte activation revealed that KARC treatment significantly suppressed neuroinflammatory responses in 5xFAD mice. These data suggest that KARC has a therapeutic effect on neuroinflammation in vivo.

[0259] In summary, 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 could be an effective treatment for dementia.

[0260] [Example 13] An example of producing a food and pharmaceutical composition for memory impairment and cognitive function by reducing amyloid β plaques and endogenous acetaldehyde.

[0261] 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 in various composition ratios can be prepared. As an example, the powder composition according to the present invention improves memory impairment and cognitive function by taking 13 g of the composition twice daily. The weight ratios of the components and phases of the food or pharmaceutical compositions containing the powder composition are shown in Table 7.

[0262] [Table 7] Industrial Applicability

[0263] KARC dry powder, excipients, and natural sweeteners such as oligofructose, enzyme-treated stevia, anhydrous citric acid, isomaltodextrin, and xylitol, as well as citrus juice powder and citrus flavoring powder, are added to food and pharmaceutical compositions. The processing and testing of the raw materials and final products of the food or pharmaceutical compositions are conducted in accordance with general testing methods and the Health Functional Food Act as outlined in the Korean Food Code.

[0264] Foods or pharmaceutical compositions containing KARC can prevent or improve the deterioration of cognitive function.

[0265] The above examples have detailed the pharmacological effects of KARC, a mutant yeast composition containing aldehyde dehydrogenase, for treating Alzheimer's disease or Lewy body dementia, along with administration methods, therapeutically effective doses for disease models, acute oral toxicity, and representative examples of food or pharmaceutical compositions. While the above examples clearly demonstrate the efficacy of KARC, they are merely exemplary of the present invention.

[0266] 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.

[0267] *Even foods or therapeutic agents containing modified forms of aldehyde dehydrogenase that embody the technical gist of the present invention as stated in the patent claims fall within the scope of legal protection of the present invention.

[0268]

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 including 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. 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. 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-oxo-nonenal, malondialdehyde (MDA), propionaldehyde, hexanal, palmitaldehyde, succinaldehyde and acrolein.

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

8. A food composition for improving memory and cognitive function, characterized in that The food composition comprises aldehyde dehydrogenase contained in a lysate of any one selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

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

1.

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

12. The pharmaceutical composition for inhibiting and preventing Alzheimer's dementia or Lewy body dementia according to claim 11, wherein The endogenous aldehyde is an endogenous aldehyde produced by oxidation of alcohol or endogenous amine compounds.

13. The pharmaceutical composition for inhibiting and preventing Alzheimer's dementia or Lewy 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. The pharmaceutical composition for inhibiting and preventing Alzheimer's dementia or Lewy 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-nonenal, malondialdehyde (MDA), propionaldehyde, hexanal, palmitaldehyde, succinaldehyde and acrolein.

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

16. A pharmaceutical composition for inhibiting and preventing Alzheimer's dementia or Lewy body dementia, characterized in that The pharmaceutical composition comprises aldehyde dehydrogenase contained in a lysate of any one selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof.

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