KR20230007252A - A composition which relieves endo plasmic reticulum stress - Google Patents

Abstract

The present invention relates to a composition for inhibiting or preventing various disease phenomena caused by the occurrence of endoplasmic reticulum stress by relieving endoplasmic reticulum stress, and relates to a composition derived from mutant yeast that inhibits or prevents fatty liver or steatohepatitis. More specifically, the present invention relates to a food or pharmaceutical composition derived from mutant yeast, which suppresses or prevents fatty liver or steatohepatitis by alleviating endoplasmic reticulum stress.

Description

Composition which relieves endoplasmic reticulum stress {A composition which relieves endo plasmic reticulum stress}

The present invention relates to a composition for inhibiting or preventing various disease phenomena caused by the occurrence of endoplasmic reticulum stress (ER Stress) by alleviating endoplasmic reticulum stress, which inhibits or prevents fatty liver or steatohepatitis, derived from mutant yeast It is about the composition to be.

More specifically, the present invention alleviates ER stress to inhibit liver inflammation, alcoholic fatty liver disease (AFLD), or none-alcoholic fatty liver disease (NAFLD) It relates to a food or pharmaceutical composition derived from mutant yeast that prevents.

More specifically, the present invention contains aldehyde dehydrogenase (ALDH), coenzymes (NAD, NADP), glutathione, etc. to suppress fatty liver and hepatitis caused by endoplasmic reticulum stress, saccharide It relates to a fatty liver inhibitory food composition or preventive and therapeutic composition containing a lysate of any one or mixtures thereof selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP, and KCTC14985BP in Roma Ices Celebijie. .

Fatty liver is caused by drinking and obesity, and fatty liver occurs in patients with metabolic diseases such as hyperlipidemia or diabetes with high blood lipid concentrations, and increases in toxins such as toxic aldehydes caused by oxidative stress and causes endoplasmic reticulum stress. Medications such as drugs or corticosteroids or female hormones may be the cause.

When alcohol is ingested, fat synthesis is enhanced due to an increase in ER stress in the liver, so fat is accumulated in the liver tissue, resulting in fatty liver, preventing normal energy metabolism.

Non-alcoholic fatty liver symptoms caused by factors other than drinking may also occur. Although drugs with various pharmacological effects on fatty liver are being developed, there is no drug whose effect has been clearly identified in the treatment of fatty liver to date.

Steatohepatitis refers to a case in which lipids are accumulated in liver tissue due to insulin resistance and obesity, which is accompanied by inflammatory signs of hepatocyte necrosis. Steatohepatitis may develop into chronic hepatitis and liver cirrhosis in some cases. Non-alcoholic steatohepatitis accompanies metabolic syndrome that causes abnormal fat metabolism.

The endoplasmic reticulum (ER) is a membrane structure present in the cytoplasm of eukaryotic cells and is connected to the nuclear membrane. The inner space of the endoplasmic reticulum occupies 10% of the cell volume and more than 50% of the total cell membrane, and many ribosomes that synthesize proteins are attached to the surface, resulting in rough endoplasmic reticulum (rER) and liposomes There are two types of smooth endoplasmic reticulum (sER), which are not attached and have a smooth surface, and are classified according to their shape.

The smooth endoplasmic reticulum (sER) is where membrane lipids and steroid hormones are synthesized and where detoxification takes place, where lipids, phospholipids, and steroids are synthesized. In the smooth endoplasmic reticulum, glucose-6-phosphate is dephosphorylated to be separated and discharged into glucose, and it is also responsible for oxidizing and detoxifying organic substances including alcohol.

The smooth endoplasmic reticulum is an endoplasmic reticulum to which ribosomes are not bound, and is present in most cells, but is mainly present in cells synthesizing fat. The detoxification process mainly occurs in hepatocytes. In order to rapidly expel potentially toxic substances such as reactive aldehydes in medicines or foods out of the body, an oxidation reaction is performed to dehydrogenate endogenous aldehydes using dehydrogenases in vivo. Smooth endoplasmic reticulum is well developed mainly in liver, muscle cells, and steroid-making cells.

In the rough endoplasmic reticulum (rER) membrane, ribosomes are attached to the surface of the endoplasmic reticulum and are in charge of mRNA translation during protein synthesis, and folding to create secondary structures in the lumen surrounded by membranes. ) process and processes that cause protein maturation and transformation. In the rough endoplasmic reticulum, chaperone proteins and enzymes that enable protein maturation and transformation are distributed.

Among proteins synthesized by ribosomes, proteins having an ER signal peptide at the N-terminus migrate into the endoplasmic reticulum. At this time, lipid-soluble membrane proteins stay in the endoplasmic reticulum membrane, and water-soluble proteins enter the endoplasmic reticulum lumen.

Among proteins synthesized by ribosomes, unfolded proteins (UP) or abnormally folded proteins (MP), which have not been transformed into their own secondary structure, are not removed and accumulate in the endoplasmic reticulum In this case, endoplasmic reticulum homeostasis is disrupted, resulting in ER stress.

A response phenomenon in which unfolded proteins and abnormal proteins, which are the cause of endoplasmic reticulum stress, are restored or degraded and removed, is called an unfolded protein response (UPR).

Cells respond to endoplasmic reticulum stress by activating the unfolded protein response so that endoplasmic reticulum stress is alleviated or homeostasis is maintained, and Grp78, Ire-1α, ATF6, PERK and the like are known as proteins related to the activation of the unfolded protein response.

When homeostasis is not maintained smoothly despite the relief of endoplasmic reticulum stress through the unfolded protein response, cells enter the apoptosis pathway. Cell death due to increased ER stress or disruption of endoplasmic reticulum homeostasis can lead to viral infectious diseases, metabolic diseases such as obesity and diabetes, neurodegeneration including dementia, and fatty liver disease. It is known as one of the major causes of diseases that induce the development of liver cirrhosis and cancer.

Reactive oxygen species (ROS) generated during the energy production and metabolism of mitochondria are produced through the lipid peroxidation (LPO) reaction to generate nonenal (HNE), malondialdehyde (MDA), and acetaldehyde (acetaldehyde). , Ach) and other aldehyde-like toxic substances cause intracellular accumulation.

Through the secondary metabolism of these substances, chain reactions such as malondialdehyde-acetaldehyde adduct (MAA) and malondialdehyde-lysine adducts (M-lys) are produced. Accumulation of various modified proteins in vivo results in increased oxidative stress.

Increased oxidative stress affects the energy metabolism process of mitochondria and increases aldehyde substances such as methylglyoxal (MG) and advanced glycation end products (AGEs) in cells, disrupting cellular energy metabolism further aggravates

As such, reactive aldehydes such as HNE and MDA, which are the result of lipid peroxidation (LPO) reaction caused by increased active oxygen and oxidative stress, and glyceraldehyde-3-phosphate (GA3P), which is an intermediate in glycolysis, When aldehyde substances such as aldehydes are overproduced and accumulated in cells, cytotoxicity occurs.

Accumulation of reactive oxygen species or reactive aldehydes weakens intracellular antioxidant defense systems such as glutathione, leading to an interaction that ultimately causes an increase in ER stress through disturbance of energy metabolism and accumulation of unfolded proteins.

When stress occurs in the endoplasmic reticulum of hepatocytes, smooth endoplasmic reticulum is activated and fat accumulates in the liver, forming fatty liver and developing steatohepatitis. However, there is a demand for the development of drugs that inhibit endoplasmic reticulum stress to inhibit fat accumulation in the liver and prevent steatohepatitis by inhibiting acute liver damage.

United States Patent Publication No. US-2021-0254023 (USSN 17/176,365), published Aug. 19, 2021

Lin et al. “Endoplasmic reticulum Stress in Disease Pathogenesis”, Annu Rev Pathol. (2008);3:399-425 Sicari et al. “A guide to assesing endoplasmic reticulum homeostasis and stress in mammalian systems”, The FEBS Journal 287. (2020);27-42. Ozcan and Tabas et al. “Role of Endoplasmic Reticulum Stress in Metabolic Disease and Other Disorders”, Annu Rev Med. (2012);63:317-328. Malhi et al. “Endoplasmic reticulum stress in liver disease”, Journal of hepatology (2011);vol 54:795-809. Bhattaral et al. “The aftermath of the interplay between the endoplasmic reticulum stress response and redox signaling”, Experimental & Molecular Medicine (2021);53:151-167. Maamoun et al. “Crosstalk Between Oxidative Stress and Endoplasmic reticulum (ER) Stress in Endothelial Dysfunction and Aberrant Angiogenesis Associated With Diabetes: A Focus on the Protective Roles of Heme Oxygenase (HO)-1”, Frontiers in Physiology Vol 10,(2019);Article 70 . Liao et al. “aldehyde dehydrogenase-2 Deficiency Aggravates Cardiac Dysfunction Elicited by Endoplasmic Reticulum Stress Induction”, Mol Med. (2012);18:785-793. Ramana et al., “Lipid Peroxidation: Production, Metaboism, and Signaling Mechanisms of Malondialdehyde and 4-Hydroxy-2-Nonenal”, Oxidative Medicine and Cellular Longevity Vol 2014; Article ID 360438:31. Console et al., “The Link Between the Mitochondrial Fatty Acid Oxidation Derangement and Kidney Injury”, Front. Physiol. (2020);11:794.

Despite these many prior studies, a food composition or pharmaceutical composition for suppressing or treating fatty liver or steatohepatitis by suppressing stress applied to the endoplasmic reticulum of liver cells has not yet been developed.

Therefore, a basic object of the present invention is to contain aldehyde dehydrogenase (ALDH), which rapidly decomposes endogenous aldehydes that cause endoplasmic reticulum stress, thereby removing endoplasmic reticulum stress factors in advance to block the occurrence of endoplasmic reticulum stress, mutant yeast. It is to provide a derived composition.

Another object of the present invention is to provide a food composition or pharmaceutical composition containing mutant yeast, which reduces the possibility of endoplasmic reticulum stress and inhibits or prevents the occurrence of fatty liver or steatohepatitis.

Another object of the present invention is to Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, KCTC14985BP containing any one or mixtures thereof selected from the group consisting of, fatty liver inhibitory food composition and fatty liver prevention or To provide a therapeutic composition.

The objects of the present invention as described above are a mutant yeast-derived composition that blocks the occurrence of endoplasmic reticulum stress by removing the endoplasmic reticulum stress in advance by containing aldehyde dehydrogenase (ALDH) that rapidly decomposes endogenous aldehydes that cause endoplasmic reticulum stress. is achieved by providing

In addition, the objects of the present invention as described above are Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP, KCTC14985BP Powder or melt of any one or a mixture thereof (hereinafter referred to as KARC) Abbreviated) is achieved by providing.

The present invention is any one selected from microorganisms consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, KCTC14985BP in Saccharomyces cerevisiae of the present invention, in which fatty liver symptoms have occurred (human or animal) due to endoplasmic reticulum stress of hepatocytes Or, after administration of KARC, which is a dry powder or a lysate of a mixture thereof, the accumulation of fat in the liver and the inflammatory response of the liver were alleviated after 24 hours and 48 hours.

Saccharomyces cerevisiae of the present invention was administered with KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP, KCTC14985BP dry powder or lysate of any one selected from the group consisting of, or mixtures thereof. It was confirmed that the accumulation of was reduced, and the expression of inflammatory response factors in the liver was reduced.

1 and 2 show that 2mg/kg tunicamycin (Tm) was administered to a general mouse model to induce endoplasmic reticulum stress in liver tissue, and then the composition of the present invention was administered and in mouse liver tissue at 24 and 48 hour intervals. This is a diagram analyzed to verify the efficacy of fatty liver (n = 5).
1 shows the morphology of the liver of mice 24 hours and 48 hours after administration of tunicamycin and KARC 10unit/kg, 20unit/kg. Fatty liver was induced by tunicamycin and the color of the liver became pale due to the fat of the liver, but the liver of the KARC-administered group recovered to a reddish color.
Figure 2 analyzed the morphology of fat in the liver tissue through hematoxylin-eosin staining (Hmatoxylin-Eosin, H&E staining). Fat in hepatocytes was enlarged with tunicamycin, but fat in hepatocytes was reduced in the KARC-administered group.
3 and 4 are schematic diagrams of the change in lipid content in liver tissue at 24 and 48 hour intervals after administration of tunicamycin and KARC to a normal mouse model (n = 5).
Figure 3 shows that the content of triglyceride (TG) in liver tissue increased by tunicamycin was significantly decreased in all KARC-administered groups.
Figure 4 shows that the content of total cholesterol (T-chol) in liver tissue increased by tunicamycin decreased in a concentration-dependent manner in the KARC-administered group.
5, 6, 7, 8, and 9 are quantitative real-time by isolating mRNA from liver tissue after 24 hours and 48 hours by administering 2mg/kg tunicamycin, 10unit/kg, and 20unit/kg of KARC to a general mouse model. It is a diagram analyzing the expression of genes related to endoplasmic reticulum stress through reverse transcription quantitative real-time PCR (RT-qPCR) (*p<0.05, **p<0.01) (n=5).
Chop , an endoplasmic reticulum stress marker in FIG. 5, represents a C/EBP homologous protein, and regulates the expression of proteins involved in inducing apoptosis, such as BCL-2 reduction and BIM increase due to intracellular endoplasmic reticulum stress. It relieves endoplasmic reticulum stress, and the decrease in Chop mRNA expression by KARC shows that endoplasmic reticulum stress due to abnormal protein removal is reduced through promotion of cell death in the endoplasmic reticulum.
Grp78 in FIG. 6 represents glucose-regulated protein 78, and as one of the representative chaperone proteins, functions to maintain and regulate endoplasmic reticulum homeostasis.
The decrease in the mRNA expression level of Grp78 by KARC administration means that ATF6, IRE1, etc. are regulated to reduce the accumulation of unfolded protein, and as a result, endoplasmic reticulum stress is alleviated.
Ire-1α in FIG. 7 represents inositol-requiring enzyme 1 alpha (inositol-requiring enzyme 1 alpha) and is a representative unfolded protein response (UPR) indicator. Ire-1α induces abnormally generated and accumulated unfolded proteins to be reformed into normal structures through the secretion of chaperone proteins, or removes proteins that will enter the endoplasmic reticulum at the mRNA level (RIDD, regulated IRE-1 dependent decay )do.
In addition, Ire-1α activates JNK and NF-κB proteins to degrade unfolded proteins by increasing autophagy or inducing apoptosis, alleviating endoplasmic reticulum stress and regulating homeostasis.
The decrease in the mRNA expression level of Ire-1α by KARC administration means that the unfolded protein acting as a cytotoxin was reduced and the endoplasmic reticulum stress was alleviated.
Gadd34 in FIG. 8 represents a growth arrest and DNA damage inducible protein (34). Gadd34 is a protein that induces eLF2α dephosphorylation in conjunction with ATF4, a transcription factor. As eLF2α dephosphorylation occurs and apoptosis is promoted, Gadd34 promotes apoptosis due to eLF2α dephosphorylation deficiency or promotes the translation of abnormal proteins It acts to remove the unfolded protein.
9 shows Atf4 . Atf4 means transcriptional activating factor 4 (activating transcriptional factor 4), and the decrease in Atf4 expression level indicates that endoplasmic reticulum stress is alleviated.
Accordingly, the decrease in mRNA expression level of Atf4 or Gadd34 by KARC administration in FIGS. 8 and 9 shows that endoplasmic reticulum stress and the number of apoptotic cells were reduced as a result.
10, 11, 12, and 13 are treated with 2mg/kg tunicamycin in a general mouse model and then administered with 10unit/kg and 20unit/kg of KARC, 24 and 48 hours later, isolating mRNA from liver tissue It is a schematic diagram of analyzing the expression of inflammatory genes through RT-qPCR (* p <0.05, ** p <0.01) (n = 5).
F4/80 in FIG. 10 is a unique marker of macrophages, and exists in macrophages that perform phagocytosis to express inflammatory genes. Reduction of F4/80 mRNA expression by KARC administration means that intracellular inflammation was reduced.
Mcp1 in FIG. 11 represents monocyte chemoattracted protein-1. This is a kind of chemokine, which is expressed in various cell groups, and is an inflammation-linked substance that acts so that T-cells, monocytes, etc. gather around inflammation and move easily.
The decrease in the mRNA expression level of Mcp-1 by KARC administration means a decrease in inflammation and inflammatory response.
Tnf-α in FIG. 12 represents tumor necrosis factor-α, and is a kind of cytokine caused by increased inflammation and inflammatory response, and shows a phenomenon of proliferation with increased phagocytosis. Therefore, the decrease in the mRNA expression level of Tnf-α by KARC administration means that inflammatory substances are reduced or the inflammatory response is attenuated .
13 Il-6 represents interleukin-6, which is known to induce inflammation by expressing inflammatory genes. A reduction in the mRNA expression level of Il-6 by KARC administration meant that inflammation was reduced.
14, 15, 16, and 17 show that a general mouse model was treated with 2mg/kg tunicamycin, and then 10unit/kg and 20unit/kg of KARC were administered, and 24 and 48 hours later, mRNA was isolated from liver tissue and RT. -This is a diagram of analyzing the expression of genes related to fatty acid oxidation through qPCR (*p<0.05, **p<0.01) (n = 5).
Ppar-α in FIG. 14 represents peroxisome proliferator-activated receptor alpha, and maintains homeostasis of lipid metabolism by improving β-Oxidation of fatty acids in muscle and liver. get involved in
The increase in the mRNA expression level of Ppar-α by KARC administration improves fat metabolism, lowers low-density triglyceride, and increases HDL-Cholesterol (high density lipoprotein cholesterol), showing that the lipid metabolism control function of the liver is restored. .
15, Pgc-1α represents peroxisome proliferator-activated receptor gamma coactivator 1-alpha, and is involved in mitochondrial biogenesis and gluconeogenesis in the liver. As a protein, the increase in Pgc-1α mRNA expression level by KARC shows that the amount of energy production and energy metabolism in the liver is increased.
Cpt-1α in FIG. 16 represents carnitine palmitoyltransferase 1 alpha, is located in the mitochondrial lipid membrane, is an essential protein involved in β-oxidation, and has an acyl group in the fatty acid chain. ) into acylcarnitine, which induces long-chain fatty acids to pass through the mitochondrial membrane.
The increase in the mRNA expression level of Cpt-1α by KARC administration shows that energy generation and metabolism through β-oxidation in the liver are improved.
Fgf21 in FIG. 17 represents Fibroblast growth factor 21, which is a fibroblast growth factor synthesized in excess in liver, pancreas and adipose tissue. The expression level of Fgf21 is regulated by peroxisome proliferator-activated receptor gamma (Ppar-γ) and eroxisome proliferator-activated receptor alpha (Ppar-α), and when stress rises, it is activated to adapt to or defend against stress.
Therefore, the decrease in the mRNA expression level of Fgf21 by KARC administration shows that the stress state of hepatocytes is reduced.
18 is a general mouse model treated with 2mg/kg tunicamycin, followed by administration of 10unit/kg and 20unit/kg of KARC, and after 24 and 48 hours, protein isolated from liver tissue was analyzed through western blot to determine endoplasmic reticulum stress. , It is a schematic of measuring the expression of proteins related to lipid metabolism and fatty acid oxidation. The expression levels of CHOP, IRE1α, p-eIF2α, eIF2α, FAS, ACC1, Scd-1, PPARα, and CPT1 proteins were measured (n=5).
In FIG. 18, the endoplasmic reticulum stress markers CHOP and IRE1α were increased by tunicamycin as shown in the gene expression results of [Figs. 5 and 7], but in the group administered with 20 units/kg of KARC, protein expression decreased, showing that endoplasmic reticulum stress was reduced are giving
18, p-eIF2α represents phosphorylation of eukaryotic initiation factor-2α, and eIF2α is one of four protein kinases, PERK, PKR, GCN2, and HRI. As a gene that is activated when abnormalities are phosphorylated, it is known to regulate protein synthesis and affect memory ability.
p-eIF2α activates kinase when cells are subjected to endoplasmic reticulum stress by accumulation of unfolded protein (UP) or misfolded protein (MP) in the endoplasmic reticulum caused by heat, ultraviolet rays, external infection, etc. do.
Overexpression of the eIF2 protein induces a decrease in intracellular endoplasmic reticulum stress by reducing the total amount of the protein. Thus, reduction of eIF2 for KARC administration showed that endoplasmic reticulum stress was reduced.
FAS in FIG. 18 represents fatty acid synthase, which is an energy required for cell growth and an essential enzyme system involved in de novo lipogenesis necessary for cell signal transmission.
18 shows that FAS protein decreased by endoplasmic reticulum stress decreased energy metabolism by fatty acid oxidation, and increased FAS protein by KARC indicates that energy metabolism by fatty acid oxidation was restored.
ACC1 in Figure 18 represents acetyl-CoA carboxylase 1 (ACC1, acetyl-CoA Carboxylase 1), an enzyme that produces a metabolite called malonyl-coenzyme A related to the formation of fatty acids or fat burning Expression is regulated by AMPK (AMP-activated protein kinase), which regulates energy metabolism of carbohydrates and fats.
The protein expression of ACC1, which was decreased due to endoplasmic reticulum stress, was restored by KARC administration, showing that the liver's ability to convert fat into energy through oxidation was improved.
Scd-1 in FIG. 18 represents stearoyl-CoA desaturase-1 (Scd-1, stearoyl-CoA desaturase-1), and Scd-1, an enzyme mainly present in liver endoplasmic reticulum, is a saturated fatty acid CoA such as stearic acid. It is an enzyme that converts oleic acid into unsaturated fatty acids, which are one of the main constituents of cell lipid membranes, and its expression is regulated by AMPK (AMP-activated protein kinase).
Scd-1 protein expression decreased due to endoplasmic reticulum stress was restored by KARC, indicating improved lipid metabolism regulation and recovery of hepatocytes.
In FIG. 18, the protein expression of PPARα and CTP1, which are fatty acid oxidation markers, was decreased by tunicamycin as described in [Figs. 14 and 16], but increased by KARC, resulting in energy generation and metabolism through oxidation of fatty acids in the liver. shows improvement.
In Figure 18, GAPDH represents 'glyceraldehyde-3-phosphate dehydrogenase (GAPDH, glyceraldehyde-3-phosphate dehydrogenase)', and since it is stably expressed in cells and the expression level does not change well depending on cell conditions, housekeeping It is used as a house keeping gene. It was used as a reference marker for protein quantification, and it shows that the same amount of protein was used.
19 shows changes in enzyme activity when KwonP-1 strain was orally administered.
20 is a change in enzyme activity when the KwonP-2 strain is orally administered.
21 shows changes in enzyme activity when the KwonP-3 strain was orally administered.
22 is a change in enzyme activity when the PicoYP strain is orally administered.
23 shows changes in enzyme activity when the PicoYP-01 strain was orally administered.
24 is a change in enzyme activity when the PicoYP-02 strain was orally administered.
25 is a growth curve and enzyme activity when the KwonP-1 strain was cultured in a 5L fermentor.
Figure 26 is a growth curve and enzyme activity when the KwonP-2 strain was cultured in a 5L fermentor.
27 is a growth curve and enzyme activity when the KwonP-3 strain was cultured in a 5L fermentor.
28 is a growth curve and enzyme activity when the PicoYP strain is cultured in a 5L fermentor
29 is a growth curve and enzyme activity when the PicoYP-01 strain was cultured in a 5L fermenter.
30 is a growth curve and enzyme activity when the PicoYP-02 strain was cultured in a 5L fermenter.
31 is a graph showing the degradation of acetaldehyde in the human body by KARC.
32 is a graph showing the degradation of malondialdehyde in the body by KARC.
33 is a graph showing the stabilization of malondialdehyde in humans by KARC.

Hereinafter, a method for preparing Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP, KCTC14985BP dry powder or lysate will be described in detail.

However, these examples are for illustration of compositions achieving the object of the present invention, and the scope of the present invention is not limited to only the compositions described in the following examples.

[Example 1]

Selection of wild yeast parent strain to undergo mutation

In the present invention, a yeast parent strain was obtained by mixing domestic makgeolli, a traditional Korean liquor, with a 0.9% NaCl solution and stirring at 200 rpm for 1 hour, and the supernatant was YPD (yeast extract peptone dextrose broth) medium Diluted with 10 ^-6 to prepare a dilution solution.

120 μl of the diluted solution was dispensed into YPD solid medium and cultured at 30 ° C. for 1 week under aerobic conditions, and Saccharomyces cerevisiae was isolated through morphological characteristics of colonies, culture in YM medium, and microscopic observation.

Aldehyde dehydrogenase and glutathione contents were measured in the isolated Saccharomyces cerevisiae yeast, and the parent strain to proceed with the mutation was selected based on the high production of aldehyde dehydrogenase and glutathione in the results. .

1-1: Measurement of aldehyde dehydrogenase

The inventors added a certain amount of Dinitrophenylhydrazine (DNPH) to acetaldehyde and reacted at a certain concentration to form a compound of acetaldehyde-hydrazone (Ach-DNPH), which was formed by using the mobile phase Acetonitrile and water as a solvent in a C18 column for HPLC. According to the reference (Guan et al. , 2012) that it can be detected and quantified at 360 nm by development, the amount of aldehyde reduced by the reaction of aldehyde dehydrogenase (ALDH) was analyzed.

For specific aldehyde dehydrogenase activity measurement, 50 mM Potassium phosphate buffer (pH 8.0), 1 mM acetaldehyde, and 1 mM NADP+ enzyme cofactor were added to 10 μL of the measured microorganism lysate as the enzyme reaction solution, and then reacted at 30 ° C. After adding 50 μl of 10 mM DNPH thereto, acetaldehyde-hydrazone (Ach-DNPH) labeling was performed at 22° C. for 1 hour.

For labeling, the reaction was terminated by adding 3 M sodium acetate (pH 9), and the layer in which the acetaldehyde-DNPH compound was dissolved was separated by adding a double volume of Acetonitrile, and then injected into HPLC for analysis.

The concentration of the labeled aldehyde was analyzed using the material standard curve of Aldehyde-DNPH (Sigma-Aldrich), and the HPLC analysis condition was a solvent (Acetonitrile, water) flowed at a flow rate of 1 ml / min using a C18 column and an ultraviolet detector 360 were obtained and analyzed at nm wavelengths. At this time, 1 unit of aldehyde dehydrogenase was set to 1 mM of reduced acetaldehyde-DNPH concentration per minute, and the activity of aldehyde dehydrogenase was expressed as a unit per mg protein.

1-2: Method for measuring glutathione

To analyze the concentration of glutathione, 1 mL of the suspension was centrifuged in cultured Saccharomyces cerevisiae to obtain precipitated cells, and 1 mL of water was added to the precipitated cells, followed by stirring at 85 ° C and 1,000 rpm for 2 hours for extraction. did After extraction, cells were removed using a centrifugal separator, and the supernatant was collected by filtering with a 0.22 μm filter.

Through HPLC (Shimazu LC-20AD) analysis, the concentration of glutathione contained in the recovered filtrate was measured. On the other hand, glutathione concentration was analyzed using a glutathione standard curve, and HPLC analysis conditions were analyzed using a C18 column. A mobile phase solvent (2.02 g/L Sodium 1-heptanesulfonate monohydrate, 6.8 g/L Potassium dihydrogen phosphate, pH 3.0, methanol mixture) was flowed at a flow rate of 1 ml/min, and the concentration of glutathione detected at a wavelength of 210 nm with a UV detector was measured.

As a result of analyzing the aldehyde dehydrogenase and glutathione contents of a total of 200 different yeast strains obtained from Korean makgeolli, 10 yeast strains showed high aldehyde dehydrogenase or glutathione production ability as shown in Table 1.

Yeast #97, which has the second highest aldehyde dehydrogenase content at 0.10 Unit/protein (mg) and has the highest glutathione at 0.42%, was selected as the wild-type final parent strain, and the mutants of the present invention process was performed.

strain name ALDH2 activity
(Unit/mg-protein) Glutathione content (%) Screening Yeast #6 0.06 0.38 Yeast #18 0.11 0.14 Yeast #22 0.08 0.38 Yeast #41 0.14 0.22 Yeast #97 0.10 0.42 Selected parent strain (Wild-Type) Yeast #109 0.10 0.36 Yeast #112 0.09 0.40 Yeast #126 0.10 0.28 Yeast #168 0.11 0.38 Yeast #197 0.08 0.41

[Example 2]

Identification of the parental strain used in the mutation process of the present invention

Prior to performing the mutation process of the present invention, identification was performed to confirm the exact strain of the wild-type parent strain (Yeast #97, Wild-Type Yeast). Pure colonies were smeared on the entire solid medium to secure enough bacteria for DNA extraction, and DNA was extracted using the HiGene™ Genomic DNA prep kit (BIOFACT Co., Ltd., Daejeon, Korea) according to the manufacturer's instructions.

To amplify the ITS region rRNA gene, polymerase chain reaction (PCR) was performed using ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 (5'-TCCTCCGCTTATTGATTGC-3') as primers, and sequencing was performed.

The DNA sequence is separated through the Bioedit program (http://www.mbio.ncsu.edu/BioEdit/Bioedit.html), and the DNA sequence amplified using ITS4 is complementary to the reverse complete process. got it

The matching of the nucleotide sequences was confirmed through the Clustal X program (http://www.clustal.org/clustal2), and the matching sequences were separated and BLAST (Basic Local Alignment Search Tool) of the National Center for Biotechnology Information (NCBI) was used. The bacteria were identified using [SEQ ID NO: 5]. As a result of the identification, it was confirmed that the isolated yeast (Yeast W.T.) was 100% identical to Saccharomyces cerevisiae.

[Example 3]

Selection of mutant candidate strains with excellent aldehyde dehydrogenase production ability

The process of inducing mutation of the parent strain of wild-type Saccharomyces cerevisiae is described in US Patent Application No. 17/176,365 (US Publication No. US-202 1-0254023-Aug. 19, 2021). Al) was carried out according to the method described.

In the mutagenesis method of the present invention, 1) the first step of inducing mutant yeast by treating a wild yeast strain producing both aldehyde dehydrogenase and glutathione with ethyl methanesulfonate (EMS) or nitrosoguanidine (NGD), 2) A second step of selecting a mutant yeast strain having excellent methylglyoxal adaptability from the mutant yeast strains obtained in the first step, 3) Adaptability to lysine among mutant yeast strains showing methylglyoxal adaptability selected in the second step Through the third step of selecting mutant yeasts showing , 30 mutant strains were secured, and strains were selected based on their growth curves, ALDH and ADH enzyme activities, coenzyme content, and glutathione content, and subculture for each strain proceeded.

3-1: Growth characterisitics

Saccharomyces cerevisiae is a representative microorganism that is positive for the Crabtree effect, and produces ethanol at the same time as it grows under aerobic conditions. In order to grow bacteria with a high yield, it is necessary that the yeast in culture has high resistance to ethanol.

The mutant strain was inoculated with 1 ml of the culture medium to Saccharomyces cerevisiae, the cell concentration of which was adjusted so that the OD _{660 nm} value was 1, in 99 ml of YPD medium containing 0, 5, 7, and 10% of different concentrations of ethanol. It was cultured under conditions of ℃ and 200 rpm. The growth curve was measured at 3-hour intervals for 48 hours, and the time of the lag phase, the specific growth rate (OD _660nm / hr) of the exponential phase, and the final growth degree (OD _660nm ) were measured. Evaluated based on

As the ethanol concentration increased, the time of the induction phase increased, and the final growth rate and specific growth rate decreased. When comparing the final growth degree, the 9 mutant strains showed that about 50% growth was maintained in high concentration ethanol (10%) compared to low concentration ethanol (5%). These strains showed a shorter induction period and higher specific growth rate than other strains in high concentration ethanol.

# 5% ethanol 7% ethanol 10% ethanol Selection hr OD _660nm /hr OD _{660 nm} hr OD _660nm /hr OD _660nm hr OD _660nm /hr OD _660nm One 9 0.6477 22.2 15 0.5210 16.5 24 0.1968 4.81 2 12 0.3675 12.34 24 0.1835 4.11 36 0.0140 0.212 3 9 0.4285 15.8 15 0.2888 9.12 24 0.0880 2.16 4 15 0.9683 25.3 15 0.8815 25.3 15 0.4085 12.4 K-1 5 12 0.7368 14.12 15 0.7337 12.23 21 0.2205 5.68 6 12 0.9683 9 15 0.8815 5.44 33 0.6269 0.448 7 24 0.9664 22.3 27 0.8467 16.8 30 0.2673 5.17 8 6 0.7268 23 9 0.6222 21.5 15 0.3778 12.11 K-2 9 6 0.8433 22.12 9 0.7484 25.34 15 0.3005 9.41 10 3 0.4880 14.5 18 0.2013 6.12 24 0.0808 2.14 11 3 0.2766 11.15 9 0.2223 8.22 24 0.0988 2.41 12 6 0.7149 21.68 9 0.5969 20.52 17 0.3317 11.4 K-3 13 9 0.6106 22.4 12 0.4906 16.4 15 0.1813 5.68 14 12 0.6136 20.6 24 0.3060 6.85 36 0.0278 0.41 15 12 0.4759 15.45 18 0.1751 5.41 33 0.0707 0.896 16 9 0.8533 23.8 15 0.8065 20.9 21 0.4953 12.1 K-4 17 21 0.6016 14.85 24 0.3955 8.45 24 0.0547 1.26 18 12 0.7766 19.25 18 0.4437 12.4 24 0.1219 2.85 19 3 0.5050 14.75 9 0.4463 14.6 21 0.2521 6.23 20 27 0.0666 1.41 36 0.0278 0.36 - - - 21 3 0.6044 22.14 9 0.6051 20.64 12 0.3247 11.1 K-5 22 24 0.5798 13.4 27 0.5080 10.1 30 0.1604 3.1 23 15 0.6455 16.9 15 0.5877 16.9 15 0.2003 6.3 24 3 0.7269 20.4 6 0.6375 18.6 12 0.3522 10.5 K-6 25 9 0.4858 16.7 15 0.3908 12.4 24 0.1476 3.6 26 6 0.6559 17.2 9 0.5821 19.7 15 0.3177 9.6 K-7 27 9 0.2857 10.5 15 0.1925 6.1 24 0.0587 1.4 28 12 0.6315 12.1 15 0.6289 10.5 21 0.1890 4.9 29 6 0.5451 17.3 9 0.4667 16.1 15 0.2834 9.1 K-8 30 9 0.7826 21.8 9 0.6614 19.9 21 0.3267 10.6 K-9

3-2: Measurement of alcohol dehydrogenase and aldehyde dehydrogenase activity

The activity of alcohol dehydrogenase (ADH) was measured by adding 10 μl of yeast lysate to 990 μl reaction mixture [50 mM potassium phosphate buffer (pH 8.0), 2 mM NAD ⁺ , 1% ethanol]. The activity of aldehyde dehydrogenase (ALDH) was measured by adding 10 μl of the yeast lysate of the present invention to 990 μl reaction mixture [50 mM potassium phosphate buffer (pH 8.0), 3 mM NADP ⁺ , 1.5 mM acetaldehyde]. The enzymatic reaction proceeded at 30° C. for 5 minutes, and the concentration of NAD(P)H produced was measured by measuring the absorbance at 340 nm.

As a result of measuring the enzyme activity of 9 selected mutant strains (K-1 to K-9), the ADH enzyme activity was the lowest of 382.69 unit/g and the highest of 975.29 unit/g. -Strain ( Saccharomyces cerevisiae KCTC 7296) increased by 5.1 to 13.1 times, respectively. The activity of aldehyde dehydrogenase (ALDH) was as low as 15.23 unit/g and as high as 72.16 unit/g, which increased by 5.3 to 24.9 times, respectively, compared to the control yeast type-strain.

When comparing the increase in enzyme activity compared to the control standard yeast type-strain (KCTC7296), unlike six mutant strains (K-1, 4, 6, 7, 8, 9) with similar ADH and ALDH enzyme activity increase rates, , The three new strains (K-2, 3, and 5) of the present invention showed an increase in ALDH enzyme activity of 18.3, 23.2, and 24.9 times, respectively, about twice as much as the ADH enzyme activity increase rate of 9.7, 11.6, and 13.1 times, which is very high. showed an increase.

The present inventors named three new mutant strains (K-2, K-3, K-5) of the present invention, which are specialized for increasing aldehyde dehydrogenase (ALDH) activity, as PicoYP, PicoYP-01, and PicoYP-02, respectively. , and deposited them in the Korean Collection for Type Cultures of the Korea Research Institute of Bioscience and Biotechnology, and were assigned accession numbers KCTC14983BP, KCTC14984BP, and KCTC14985BP.

3-3: Comparison of coenzyme (NAD, NADP) production abilities of 9 mutant yeasts

Using the NADH/NAD quantification kit (MAK037, Sigma-Aldrich) and the NADPH/NADP quantification kit (MAK038, Sigma-Aldrich), NAD _total and NADP _total present in lysates of 9 mutant yeasts were measured. . After converting NAD(P) ⁺ in the sample to NAD(P)H using NAD(P) cycling buffer and NAD(P) cycling enzyme mix, a color reaction was induced using NAD(P) developer. The degree of color development was measured in the form of absorbance at 450 nm, and the concentration was calculated by converting to concentration through a standard curve.

As a result of measurement for 9 mutant strains (K-1 to K-9), NAD _total was 7.3 and 10.8 times higher than Type-strain, respectively, with a minimum of 126 μmole / g and a maximum of 195 μmole / g. NADP _total was a minimum of 2.4 μmole / g and a maximum of 5.8 μmole / g, which increased by 11.4 and 27.6 times, respectively, compared to the control strain, Type-strain (KCTC7296).

When comparing the increase in coenzyme content compared to the control reference strain Type-strain (KCTC7296), the increase rate of _total NADP content was less than twice the increase rate of _total NAD content. , 7, 8, 9), the three new mutant strains of the present invention (K-2: KCTC14983BP, K-3: KCTC14984BP, K-5: KCTC14985BP) showed an increase in NADP _total of 25.7, 22.9, It was 27.6 times, showing a very high increase rate, more than twice the increase rate of NAD _total , which were 10.8, 9.9, and 11.3 times, respectively.

3-4: Comparison of glutathione productivity of 9 mutant yeasts

Measurement of the glutathione content of the lysate of the mutant strain of the present invention was performed in the same manner as in Example 1-2. As a result of measuring the glutathione content of 9 mutant strains, the lowest was 0.85% and the highest was 1.05%, which increased by 2.7 and 3.3 times, respectively, compared to the control strain type strain (KCTC7296).

The three mutant strains (K-2, 3, and 5) of the present invention have an increased rate of aldehyde dehydrogenase (ALDH) activity and coenzyme The content increase rate of was high.

[Example 4]

Comparison of carbon source utilization measurement (API test) among 7 mutant strains

“Three mutant strains for simultaneous production of ALDH and glutathione” K-1 (KCTC13925BP), K-4 (KCTC14122BP), K-9 (14123BP) and The control standard yeast strain (KCTC7296) and the carbon source used for growth of the three new mutant yeast strains K-2 (KCTC14983BP), K-3 (KCTC14984BP), and K-5 (KCTC14985BP) to maximize the ALDH production capacity of the present invention was measured.

In order to confirm the sugar utilization characteristics and novelty of each strain, various carbon sources used for growth of each strain were analyzed according to the manufacturer's instructions of the API 50 CHL kit (API systems, BioMreux, SA, France).

8ml YPD medium was dispensed into a 15ml conical tube, a control strain (KCTC7296), three conventional strains deposited by the present inventors (KCTC13925BP, KCTC14122BP, KCTC14123BP, and three mutant new strains of the present invention (KCTC14983BP, KCTC14984BP, KCTC14985BP) ) was inoculated.

The strains in the logarithmic growth phase were secured by culturing at 30 ° C. and 200 rpm for 24 hours, and the strains were washed three times with physiological saline using a centrifuge to remove the influence of the medium composition. An inoculum of 2 McFarland suspension was prepared using API 50 CHL medium and filled into a tube of a strip. The results were analyzed after incubation at 30°C for 24 hours.

API 50 CHL medium used for the API test basically shows a purple color, but when acid is produced by the energy metabolism of bacteria, it changes to yellow through blue and green. Based on this, it was judged whether or not the carbon source was used, and the degree of growth of the fungus was recorded as the number of + (purple: growth X, blue: +, green: ++, yellow: +++).

19 carbon sources (L-arabinose, ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, N-acetyl-glucosamine, arbutin) of 7 strains used in the experiment , salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, raffinose, and gentiobiose) were measured.

Rhamnose was used by 3 strains (KwonP-1, PicoYP-01, PicoYP-02), and sorbitol was used by 4 strains (KwonP-2, KwonP-3, PicoYP-01, PicoYP-02). α-methyl-D-mannoside was used by 4 strains (T.S., KwonP-1, KwonP-2, PicoYP-02), and amygdalin was used by 6 strains (KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, PicoYP-02) were used, D-turanose was used by 4 strains (T.S., KwonP-1, KwonP-3, PicoYP-02), and D-tagatose was used by 3 strains ( T.S., KwonP-3, PicoYP-02) were used, and gluconate was used as a carbon source only for the control strain Type-strain.

However, mannitol and sorbitol, which are alcoholic carbon sources, produced a particularly significant difference in the growth of yeast (Table 3). API test results of three novel mutant yeast strains of the present invention (PicoYP:KCTC14983BP, PicoYP-01:KCTC14984BP, PicoYP-02:KCTC14985BP, control reference strain Type strain (KCTC7296) and the present inventors in the past Unlike other mutant yeast strains (KCTC13925BP, KCTC14122BP, KCTC14123BP) for simultaneous production of glutathione and ALDH deposited, as shown in Table 3 below, the type of sugar used for growth is different, and even among the three new mutant strains There was a difference in the type of sugar mainly used for growth.

reference strain for control KwonP-1 KwonP-2 KwonP-3 Pico
YP Pico
YP-01 Pico
YP-02 L-Arabinose ++ +++ ++ +++ ++ ++ +++ Ribose +++ +++ +++ +++ +++ +++ +++ D-Xylose + ++ + + ++ ++ + D-Galactose + +++ ++ ++ +++ ++ ++ D-Glucose ++ +++ +++ ++ +++ ++ ++ D-Fructose ++ +++ ++ ++ +++ ++ ++ D-Mannose ++ +++ +++ ++ +++ ++ ++ Rhamnose + ++ ++ Mannitol + + + + ++ +++ +++ Sorbitol + + +++ +++ α-Methyl-D-Mannoside +++ + + +++ N-Acetyl-Glucosamine +++ +++ +++ +++ +++ +++ +++ Amygdalin + + + ++ ++ ++ Arbutin +++ +++ +++ +++ +++ +++ +++ Salicin +++ +++ +++ +++ +++ +++ +++ Cellobiose +++ +++ +++ +++ +++ +++ +++ Maltose ++ +++ +++ +++ +++ +++ +++ Lactose ++ ++ ++ ++ ++ ++ ++ Melibiose ++ + +++ +++ ++ ++ +++ Sucrose ++ +++ ++ ++ +++ +++ ++ Trehalose ++ + ++ ++ ++ ++ ++ Raffinose ++ + + ++ ++ ++ +++ Gentiobiose ++ ++ ++ ++ ++ ++ ++ D-Turanose + + + + D-Tagatose ++ + ++ Gluconate +

The three new mutant yeasts of the present invention, KCTC14983 (PicoYP), KCTC14984BP (PicoYP-01), and KCTC14985BP (PicoYP-02), which were selected as strains most suitable for ALDH enzyme production ability and subcultured, were the present inventors in terms of glutathione production ability. It was similar to the existing deposited strains KCTC13925BP (Kwon P-1), KCTC14122BP (Kwon P-2), and KCTC14123BP (Kwon P-3), but the rate of increase in ADH and ALDH enzyme activity and coenzyme content increased remarkably. did

Strain Enzyme activity ^* Coenzyme concentration ^** GSH (%) Name ADH ALDH NAD _total NADP _total Type-strain 74.6 2.9 17.2 0.21 0.32 control strain KCTC7296 K-1 542.26 23.11 169.8 4.1 1.00 KwonP-1 KCTC13925BP K-2 725.11 53.1 185 5.4 0.86 PicoYP KCTC14983BP K-3 866.41 67.4 171 4.8 0.85 PicoYP-01 KCTC14984BP K-4 625.11 31.4 176 5.1 0.98 KwonP-2 KCTC14122BP K-5 975.29 72.16 195 5.8 0.89 PicoYP-02 KCTC14985BP K-6 458.88 16.21 154 3.1 1.05 - K-7 382.69 15.23 126 2.4 1.00 - K-8 422.17 16.19 142 2.9 0.99 - K-9 533.54 20.68 167 3.2 1.00 KwonP-3 KCTC14123BP

The increase rate (fold) compared to the control strain (Type-strain) was calculated and shown in [Table 5] below.

Strain Enzyme activity ^* Coenzyme concentration ^** GSH (%) Name ADH ALDH NAD _total NADP _total Type-strain One One One One One - KCTC7296 K-1 7.3 8.0 9.9 19.5 3.1 KwonP-1 KCTC13925BP K-2 9.7 18.3 10.8 25.7 2.7 PicoYP KCTC14983BP K-3 11.6 23.2 9.9 22.9 2.7 PicoYP-01 KCTC14984BP K-4 8.4 10.8 10.2 17.1 3.1 KwonP-2 KCTC14122BP K-5 13.1 24.9 11.3 27.6 2.8 PicoYP-02 KCTC14985BP K-6 6.2 5.6 9.0 14.8 3.3 - K-7 5.1 5.3 7.3 11.4 3.1 - K-8 5.7 5.6 8.3 13.8 3.1 - K-9 7.2 7.1 9.7 15.2 3.1 KwonP-3 KCTC14123BP

[Example 5]

Changes in aldehyde dehydrogenase activity of mutant strains in gastric acid

In order for the yeast composition containing aldehyde dehydrogenase (ALDH) to be administered orally and active into the intestines (small intestine, large intestine) to live and act, low pH and strong proteolytic enzymes such as pepsin are secreted by the stomach It must pass safely without destroying the enzymatic activity.

Artificial gastric juice (pH 1.17) was adjusted to pH 3 and pH 5 similar to the conditions in the stomach using NaOH solution to create an environment in the stomach when food was ingested. 7 ml of artificial gastric juice solution (pH 1.17, 3, 5) was added to 1 g of mutant strain yeast of the present invention, adjusted to 36.5 ° C., the same as body temperature, and then reacted for 5 minutes, 30 minutes, 60 minutes, and 90 minutes. After completion of the reaction, the mixture was neutralized to pH 7 using NaOH solution to prepare a mixture having a final volume of 10 ml, and then the aldehyde dehydrogenase (ALDH) activity of the mutant strain yeast of the present invention was measured.

As a result of reacting gastric juice (pH 1.17) with the mutant yeast of the present invention, the aldehyde dehydrogenase (ALDH) activity decreased by more than 92.88% on average compared to the control group after 5 minutes of reaction, and the longer the reaction time, the lower the aldehyde dehydrogenase (ALDH) activity. After 90 minutes of reaction, it decreased by 98.89% on average compared to the control group. At pH 3 and pH 5, higher enzyme activity was maintained, and after 90 minutes of reaction, it decreased by 96.66% on average compared to the control group at pH 3 and by 56.83% at pH 5.

More specifically, as a result of reacting gastric juice (pH 1.17) with KwonP-1 (KCTC13925BP), the aldehyde dehydrogenase (ALDH) activity decreased by 90.94% compared to the control group after 5 minutes of reaction, and was measured as 5.57 unit / g, and the reaction time The longer the reaction, the lower the enzyme activity, and after 90 minutes of reaction, it was measured as 0.88 unit / g, which was reduced by 98.57% compared to the control group [FIG. 19]. At pH 3 and pH 5, higher enzyme activity was maintained, and after 90 minutes of reaction, it was measured at 2.05 unit / g, a decrease of 96.66% compared to the control group at pH 3, and at 32.12 unit / g, decreased by 47.76% at pH 5.

As a result of reacting gastric juice (pH 1.17) with KwonP-2:KCTC14122BP, ALDH activity decreased by 91.18% compared to the control group after 5 minutes of reaction, and was measured as 5.43 units/g. After that, it decreased by 98.81% compared to the control group and was measured as 0.73 unit/g [FIG. 20]. At pH 3 and pH 5, higher enzyme activity was maintained, and after 90 minutes of reaction, pH 3 was measured at 1.47 unit / g, which is a decrease of 97.62% compared to the control group, and 26.99 unit / g, which is 56.11% decrease at pH 5.

As a result of reacting gastric juice (pH 1.17) with KwonP-3:KCTC14123BP, ALDH activity decreased by 89.99% compared to the control group after 5 minutes of reaction, and was measured as 6.16 units/g. After that, it decreased by 97.85% compared to the control group and was measured as 1.32 units/g [FIG. 21]. At pH 3 and pH 5, higher enzyme activity was maintained, and after 90 minutes of reaction, pH 3 was measured at 4.55 units / g, a decrease of 92.61% compared to the control group, and 23.18 unit / g, a decrease of 62.31% at pH 5.

As a result of reacting gastric juice (pH 1.17) with PicoYP:KCTC14983BP, ALDH activity decreased by 92.84% compared to the control group after 5 minutes of reaction, and was measured as 4.40 units/g. It was measured as 1.03 unit/g, decreasing by 98.33% compared to the control group [FIG. 22]. At pH 3 and pH 5, higher enzyme activity was maintained, and after 90 minutes of reaction, it was measured at 2.05 unit / g, which is a decrease of 96.66% compared to the control group at pH 3, and at 28.31 unit / g, which is decreased by 53.97% at pH 5.

As a result of reacting gastric juice (pH 1.17) with PicoYP-01:KCTC14984BP, ALDH activity decreased by 95.71% compared to the control group after 5 minutes of reaction, and was measured as 2.64 units/g. After that, it decreased by 99.76% compared to the control group and was measured as 0.15 unit/g [FIG. 23]. Higher enzyme activity was maintained at pH 3 and pH 5, and after 90 minutes of reaction, pH 3 was measured at 1.10 unit / g, which is a decrease of 98.21% compared to the control group, and 25.38 unit / g, which is 58.74% decrease at pH 5.

As a result of reacting gastric juice (pH 1.17) with PicoYP-02:KCTC14985BP, ALDH activity decreased by 96.66% compared to the control group after 5 minutes of reaction, and was measured as 2.05 unit/g. After that, no enzyme activity was observed [FIG. 24]. At pH 3 and pH 5, higher enzyme activity was maintained, and after 90 minutes of reaction, pH 3 was measured at 1.10 unit / g, which is a decrease of 98.21% compared to the control group, and 23.32 unit / g, which is 62.08% decrease at pH 5.

Extreme pH 1.17 is the pH of the gastric juice secreted when food is ingested, and since the pH rises from 3 to 5 when food is mixed in the stomach, it is unlikely that pH 1.17 will be reached, but the remaining enzyme activity is maintained even under extreme conditions of pH 1.17. measured.

From the above results, the novel mutant yeast strains of the present invention (PicoYP, PicoYP-01, PicoYP-02) were severely destroyed by about 92-97% of enzyme activity when reacted at extreme pH of 1.17 for 5 minutes, but the remaining It was concluded that about 2-5 units of the enzyme, which can sufficiently act in the intestine, remained, and a significant amount of the enzyme survived at pH 3 and 5, so that it could be used for oral administration.

[Example 6]

5L fermenter culture test

YPD liquid medium (2% peptone, 1% yeast extract, 2% glucose) was inoculated with 6 mutant yeast strains, and primary seed culture was performed at 30°C and 200 rpm for 18 hours. In the case of 5L Jar culture, 20 ml of seed culture was inoculated into 1980 ml of YPD liquid medium, and the fermentation conditions were the same as for seed culture and cultured for 48 hours. During the culture, 10 ml of the culture medium was collected, and the absorbance (OD _{660 nm} ) and enzyme activity were measured.

The final growth (OD _660nm ) of KwonP-1:KCTC13925BP was 134.4, 4.35% higher than the type-strain, and the specific growth rate (OD _660nm /hr) was similar to that of the type-strain (KCTC7296). A similar pattern was shown in the growth curve, but the ALDH activity was 33.6 units / g, which was 11.96 times higher than the type-strain [FIG. 25].

The final growth (OD _660nm ) of KwonP-2 (KCTC14122BP) was 133.8, 3.88% higher than the type-strain, and the specific growth rate (OD _660nm /hr) was 14.8% higher than the type-strain. Growth of KwonP-2 was terminated earlier than Type-strain (KCTC7296), and ALDH activity was 31.5 units/g, 11.21 times higher than Type-strain [FIG. 26].

The final growth (OD _660nm ) of KwonP-3 (KCTC14123BP) was 134.1, 4.12% higher than the type-strain, and the specific growth rate (OD _660nm /hr) was 6.08% lower than the type-strain. The growth of KwonP-3 was terminated earlier than the type-strain, and the ALDH activity was 29.5 units/g, 10.5 times higher than the type-strain [FIG. 27].

The final growth (OD _660nm ) of PicoYP (KCTC14983BP) was 123.8, 3.88% lower than the type-strain, and the specific growth rate (OD _660nm /hr) was 6.22% higher than the type-strain (KCTC7296). The growth curve showed a similar pattern to the type-strain, but the ALDH activity was 44.2 units/g, 15.73 times higher than the type-strain [FIG. 28].

The final growth (OD _660nm ) of PicoYP-01 (KCTC14984BP) was 126.9, 1.47% lower than the type-strain, and the specific growth rate (OD _660nm /hr) was 2.14% higher than the type-strain (KCTC7296). The growth curve showed a similar pattern to the type-strain, but the ALDH activity was 47.1 units/g, 16.76 times higher than the type-strain [FIG. 29].

The final growth (OD _660nm ) of PicoYP-02 (KCTC14985BP) was 148.1, 14.99% higher than the type-strain, and the specific growth rate (OD _660nm /hr) was 9.64% lower than the type-strain (KCTC7296). The growth curve was located above the type-strain, and the ALDH activity was 52.68 units/g, which was 18.75 times higher than the type-strain [FIG. 30].

[Example 7]

Preparation of lysate KARC of 6 mutant strains of the present invention

In order to remove and inhibit proteases for preservation of enzymes (ALDH, ADH) included in the mutant enzyme lysate of the present invention, and to remove cell debris such as cell walls, mutant yeasts are dried Alternatively, a lysate or a KARC (Kwon Aldehyde Reducing Composition) composition was prepared by mixing them in a free ratio.

In the mutant yeast of the present invention and the medium in which it was cultured, various substances such as yeast metabolites such as ethanol and proteolytic enzymes secreted by the yeast were present. In order to extract and preserve aldehyde dehydratase, coenzyme, and glutathione present in yeast, it is necessary to sufficiently remove substances outside the yeast cell, and a washing process was performed for this purpose. Washing of the mutant strain was carried out by dispensing 40 ml of the culture solution into a 50 ml conical tube, centrifuging at 13,000 rpm for 15 minutes, and removing the supernatant.

As a result of centrifugation, since residual medium remains inside the pellet, which is produced by the union of yeast, 30ml purified water is added, the pellet is sufficiently released through vortexing, and the previous process is repeated 3 times to sufficiently remove the residual medium did

Ethanol resistance of yeast is known to be up to 13%, and yeast is killed when exposed to high concentrations of ethanol. To induce the death of yeast, the washed pellet was sufficiently released using 10 ml of 20% ethanol solution, and the yeast was killed by stirring at 100 rpm for 30 minutes. When the reaction time was over, 30 ml of purified water was added to lower the ethanol concentration to 5%, and the previous washing process was repeated three times to sufficiently remove ethanol.

To preserve ALDH and ADH from proteolytic enzymes present in yeast cells, 2 tablets of protease inhibitor (Pierce protease inhibitor mini tablets, EDTA-free, Thermo Scientific) were added to 10ml of 1X PBS, and washing was completed. The pellet was sufficiently released.

In order to prepare the lysate of the mutant yeast prepared in the present invention, 4 g of glass beads were administered and stirred to break the yeast cell wall, and 30 Second vortexing and 30 second ice incubation were repeated 6 times.

After the destruction of the cell wall of the yeast is complete, add 10ml of 100mM potassium phosphate buffer and mix sufficiently by simple vortexing for 3 to 5 seconds. Cell structures such as yeast cell walls and glass beads were removed by centrifugation at 13,000 rpm for 15 minutes, and the supernatant was filtered through a 0.2 μm filter (Minisart ^ⓡ Syringe Filter, Sartorius, Goettingen, Germany) to prepare a KARC composition.

In order to preserve the enzymes (ALDH, ADH) included in the mutant enzyme lysate (lysate) of the present invention, intracellular proteases are removed and inhibited, and cell debris such as cell walls are removed, KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, KCTC14985BP A lysate selected from six mutant yeasts or a KARC composition in which they were mixed in a free ratio was prepared (Table 6).

The ADH and ALDH enzyme activities of KARC1 prepared from KwonP-1 were 461.4 unit/g and 28.6 unit/g, respectively, and the coenzyme NADtotal and NADPtotal contents were 176.2 μmole/g and 5.1 μmole/g, respectively, GSH was found to be 0.98 wt%.

The ADH and ALDH enzyme activities of KARC2 prepared from KwonP-2 were 482.1 unit/g and 29.8 unit/g, respectively, and the coenzyme NADtotal and NADPtotal contents were 175.4 μmole/g and 5.2 μmole/g, respectively, GSH was found to be 0.96 wt%.

The ADH and ALDH enzyme activities of KARC3 prepared from KwonP-3 were 477.5 unit/g and 28.1 unit/g, respectively, and the coenzyme NADtotal and NADPtotal contents were 177.2 μmole/g and 5.1 μmole/g, respectively, GSH was found to be 1.00 wt%.

The ADH and ALDH enzyme activities of KARC4 prepared from PicoYP were 586.8 unit/g and 33.8 unit/g, respectively, and the coenzyme NADtotal and NADPtotal contents were 184.3 μmole/g and 5.7 μmole/g, respectively. It was found to be 0.84 wt%.

The ADH and ALDH enzyme activities of KARC5 prepared from PicoYP-01 were 621.6 unit/g and 38.2 unit/g, respectively, and the coenzyme NADtotal and NADPtotal contents were 186.9 μmole/g and 5.6 μmole/g, respectively, GSH was found to be 0.83 wt%.

The ADH and ALDH enzyme activities of KARC6 prepared from PicoYP-02 were 664.1 unit/g and 41.6 unit/g, respectively, and the coenzyme NADtotal and NADPtotal contents were 195.0 μmole/g and 5.8 μmole/g, respectively, GSH was found to be 0.88 wt%.

KARC is a mixture in any free ratio between the lysates of the six mutant strains deposited by the present inventors, and the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities are 547.6 units, respectively. / g, 33.1 unit / g, the contents of NAD _total and NADP _total , which are coenzymes, were 180.4 μmole / g and 5.4 μmole / g, respectively, and the glutathione (GSH) content was 0.84 wt%.

Through this result, it was confirmed that the aldehyde decomposition ability of KARC was maintained during the lysate manufacturing process, and the ability to promote high aldehyde dehydrogenation reaction, HNE, MDA, 3,4-dihydroxyphenylacetaldehyde (DOPAL ) showed the ability to remove toxic aldehydes such as

designation strain ADH
(Unit/g) ALDH
(Unit/g) NADtotal
(µmole/g) NADPtotal
(µmole/g) GSH
(wt %) KARC1 KwonP-1 461.4 28.6 176.2 5.1 0.98 KARC2 KwonP-2 482.1 29.8 175.4 5.2 0.96 KARC3 KwonP-3 477.5 28.1 177.2 5.1 1.00 KARC4 PicoYP 586.8 33.8 184.3 5.7 0.84 KARC5 PicoYP-01 621.6 38.2 186.9 5.6 0.83 KARC6 PicoYP-02 664.1 41.6 195.0 5.8 0.88 KARC overall average 547.6 33.1 180.4 5.4 0.91

[Example 8]

Base sequence analysis of aldehyde dehydrogenase contained in the mutant yeast of the present invention

In order to confirm the difference in the gene sequence of the aldehyde dehydrogenase of the mutant yeast of the present invention and the aldehyde dehydrogenase of the parent strain before the mutation, six mutant yeasts of the parent strain and KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP Whole genome sequencing was performed. Pure colonies of the yeast strains were smeared on the entire solid medium to secure the bacteria, and gene sequences were analyzed.

Among the yeast aldehyde dehydrogenases (ALD) enzymes produced by yeast strains, ALD2 (SEQ ID NO: 3) and ALD3 (SEQ ID NO: 4) were located continuously linked to chromosome 13, There was a non-coding region consisting of 689 base sequences in between.

ALD2 and ALD3 of yeast strains were continuously present in the same genome, but each encoded an aldehyde dehydrogenase. ALD2 and ALD3 are aldehyde dehydrogenases that differ from each other in 125 base sequences (8.2%), although they have similarities, such as each consisting of 1,521 nucleotides and encoding 506 amino acids. confirmed with

However, in the six mutant yeast strains deposited by the present inventors (KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, KCTC14985BP), there is no stop codon at the end of the ALD2 sequence, so proteins are synthesized continuously. Results [SEQ ID NO: 1].

More specifically, the aldehyde dehydrogenase (ALD2) of the existing control yeast strain (KCTC7296) has 30 base sequences (5') encoding 9 amino acids (N-VHINLSLDN-C) excluding the stop codon at the 3 end. -GTTCACATAAATCTCTCTTTGGACAACTAA-3') was present [SEQ ID NO: 3].

However, in the aldehyde dehydrogenases of the six mutant yeasts of the present invention, there is a 42 base sequence (5'-AGATATAGATTATACACATTTAGAAATTAGCCAAAAGAAAA-3') encoding 14 amino acids (N-RYRLYTFRKLAKRK-C) between the 5 ends of ALD2 and ALD3. A characteristic nucleotide sequence appeared [SEQ ID NO: 2]

As such, in the six mutant strains of the present invention, from the 1,492nd base of ALD2 to the 647th base of the non-coding region, the stop codon of ALD2 disappeared, resulting in a new gene consisting of a total of 3,054 bases, It encoded the novel aldehyde dehydrogenase of the present invention. [SEQ ID NO: 1]

[Example 8]

Test to confirm liver fat reduction effect induced by endoplasmic reticulum stress of mutant yeast strain lysate (KARC) of the present invention

When tunicamycin (Tm) is administered to animals, N-glycosylation of proteins is inhibited, resulting in endoplasmic reticulum stress as unfolded proteins (UP) are produced. Tunicamycin acts as an inhibitor of N-acetylglucosamine transferase in eukaryotic cells, preventing the formation of N-acetylglucosamine lipid metabolites and glycosylation of synthesized proteins.

In the endoplasmic reticulum, the proteins synthesized in the ribosome are subjected to higher order structure and post-translational modification, and the folding and assembly of glycoproteins are performed. When glycoproteins are not properly produced due to tunicamycin, proteins are not folded and assembled and accumulated in the endoplasmic reticulum, resulting in endoplasmic reticulum stress. Eventually, endoplasmic reticulum stress induces cell death.

In order to investigate the efficacy of the KARC component of the present invention to inhibit acute fatty liver mediated by endoplasmic reticulum stress, the present inventors analyzed the endoplasmic reticulum stress effect by KARC and the fat-related morphology of liver tissue using model animals in which endoplasmic reticulum stress was induced.

More specifically, C57BL/6J male mice (Central Experimental Animals, Korea), which are endoplasmic reticulum stress induction model animals, were subjected to 12-hour (day) and 12-hour (night) light/dark cycles at 23°C and 60-70% humidity (6 a.m.- 6 p.m. light, 6 p.m.- 6 a.m. dark) with free access to water, and housed in a specific pathogen-free mouse facility for testing.

Liver sample sizes from experimental animals were estimated according to known assay variability. After inducing endoplasmic reticulum stress by administering 2 mg/kg tunicamycin intraperitoneally to mice, the efficacy of KARC was verified at 24 and 48 hour intervals.

In the present specification, KARC, which is a dry powder or lysate of KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP, KCTC14985BP in Saccharomyces cerevisiae, contains not only glutathione but also aldehyde dehydrogenase (ALDH) that promotes aldehyde dehydrogenase ), coenzymes NAD, NADP, and alcohol dehydrogenase (ADH).

Mice were anesthetized with isoflurane (Hana Pharmaceutical, Gyeonggi-do) and sacrificed at 9 AM. In order to analyze the morphology of liver tissue, its structure was confirmed by H&E staining, and H&E tissue staining was performed through an experimental process known in the art.

Liver tissue lipids were extracted according to Folch's method using methanol and chloroform, and liver tissue total cholesterol levels were quantified using a total cholesterol assay kit (AM 202-K, Asan Pharmaceutical Co., Ltd.), and absorbance of samples and standards at 500 nm was measured.

Liver tissue triglyceride levels were quantified using a triglyceride assay kit (AM 157S-K, Asan Pharmaceutical Co., Ltd., Seoul, Korea) and absorbance of samples and standards at 550 nm using Infinite　 200 PRO (Tecan Trading AG, Switzerland) was measured.

As a result, as shown in [Figure 1], it was confirmed that the form of fatty liver appeared in liver tissue by tunicamycin.

In the group administered with tunicamycin, the color remained unchanged at 24 and 48 hours due to fatty liver, but when KARC was administered at 10 unit/kg and 20 unit/kg, the higher the concentration, the higher the concentration. The recovery rate of liver color was found to be good, and it was confirmed that KARC was effective in recovering fatty liver.

In the morphological analysis of tissue through H&E staining, as shown in [Fig. 2], the form of fat in the liver tissue was less in the drug-administered group.

As a result of measuring the contents of triglyceride [Fig. 3] and total cholesterol [Fig. 4] in the liver tissue after the induction of antifoam stress, they increased in the liver of the mouse to which the antifoam stress was induced, but were significantly different in the liver of the mouse administered with KARC. decreased. In this liver biopsy, it was shown that KARC, a lysate of Saccharomyces cerevisiae of the present invention, restores fatty liver formation.

[Example 9] Changes in endoplasmic reticulum stress-related factors according to KARC administration

Tunicamycin blocks the transfer of N-acetylglucosamine 1-phosphate to the site of dolichol phosphate in proteins, thereby inhibiting the process of attaching sugar residues to the NH ₃ residues of asparagine in proteins. .

It also inhibits DNA synthesis by inhibiting entry into the S phase of the cell cycle and slowing down the G1 phase to inhibit the synthesis of glycosylated proteins. In the present invention, after treating a general mouse model with tunicamycin, 20 units/kg of the mutant yeast lysate KARC of the present invention was administered to verify the efficacy of the mutant yeast composition KARC of the present invention at 24 and 48 hour intervals.

The present inventors analyzed acute fatty liver-related factors in liver tissues of mice after administration of tunicamycin and administration of the mutant yeast composition KARC.

Specifically, factors related to ER stress, inflammation, lipid synthesis, and fatty acid oxidation were analyzed in the liver tissues of mice after administration of tunicamycin and KARC to investigate the efficacy of inhibiting acute fatty liver formation.

mRNAs and proteins of endoplasmic reticulum stress-related genes in the liver tissue of mice in which endoplasmic reticulum stress was induced: Chop (Fig. 3A), Grp78 (Fig. 3B), Ire-1α (Fig. 3C), Gadd34 (Fig. 3D), Atf4 (Fig. 3E) , F4/80 of inflammatory genes (Fig. 4A), Mcp1 (Fig. 4B), Tnf-α (Fig. 4C), Il-6 (Fig. 4D) and Pparα of genes related to fatty acid oxidation (Fig. 5A), Pgc-1α ( Fig. 5B), and the expression level of Cpt-1α (Fig. 5C) was measured.

Total RNA was prepared from liver tissue of mice in which endoplasmic reticulum stress was induced by the TriZol procedure (Invitrogen), and cDNA was synthesized using the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA) did.

To measure the mRNA expression levels of various genes, qPCR was performed with the CFX96TM real-time PCR system (Bio-Rad Laboratories).

mRNA levels were normalized to the expression of the ribosomal protein L32 by calculating the delta-delta threshold cycle method. The primer sequences of the genes used for qPCR are listed in Table 5. Western blot analysis was performed by extracting protein samples from mouse liver tissue induced by endoplasmic reticulum stress.

Liver tissue lysates (extracts or lysates) were extracted using T-PER™ (Tissue Protein Extraction Reagent; Thermo Scientific, Rockford, IL, USA) supplemented with protease and phosphatase inhibitors (Thermo Scientific). The protein was diluted with 5-fold sample buffer (EBA-1052, ELPIS BIOTECH, Seoul, Korea) and heated at 95°C for 5 minutes. Then, the proteins were separated by 5-15% Tris-HCl SDS/PAGE gel electrophoresis and transferred to a nitrocellulose membrane (GE Healthcare, Uppsala, Sweden).

All immunoblots were measured by HRP-conjugated secondary antibodies with enhanced chemiluminescence (Clarity ™ Western ECL Substrate, Bio-Rad Laboratories). Then, protein bands were detected with a chemiluminescence imaging system (Fusion Fx, Vilber Lourmat, Eberhardzell, Germany).

As a result, as shown in [Figs. 5, 6, 7, 8, 9], the expression of endoplasmic reticulum stress-related genes was analyzed in drug-treated liver tissue after inducing endoplasmic reticulum stress through QPCR, and the mutant yeast composition KARC lysate In all cases, the genes related to the endoplasmic reticulum stress were improved.

When endoplasmic reticulum stress is induced, it is known that the expression of inflammatory genes in liver tissue also increases, and as a result of confirming the expression of related marker genes, as shown in [Figs. expression was reduced by

As a result of confirming the expression of genes related to fatty acid oxidation, as shown in [Figs. 14, 15, 16, and 17], it was found that fatty acid oxidation by reducing endoplasmic reticulum stress was greatly improved in the group administered with KARC.

As a result of analyzing the expression of proteins related to endoplasmic reticulum stress, CHOP, IRE1α, and p-eIF2α expression decreased in all cases where KARC was administered, and as a result of analyzing the expression of proteins related to lipid metabolism and fatty acid oxidation, they were involved in lipid metabolism In all of the KARC-administered groups, it was confirmed that the endoplasmic reticulum stress was improved after treatment and the expression of related proteins was reduced [FIG. 18].

[Example 10]

Oxidative stress reduction effect test by oral administration of KARC.

Active oxygen or oxidative stress increases when alcohol is consumed by producing excess acetaldehyde (Ach) by alcohol dehydrogenase (ADH). In the case of aldehyde dehydrogenase gene mutation or excessive aldehyde due to alcohol excess, fat peroxidation is induced.

As a result, acetaldehyde and malondialdehyde exacerbate oxidative stress, interfere with mitochondrial energy metabolism, and induce endoplasmic reticulum stress through accumulation of denatured proteins in cells, resulting in cell death.

As a result of measuring the concentration of acetaldehyde in blood according to time after drinking in [Figure 31], the area under the curve (AUC) of acetaldehyde in blood was 13.02 ± 1.18 mg h/dL when alcohol was consumed alone, and 10 units/ When taking kg KARC, it was 9.39 ± 1.07 mg h/dL, a statistically significant decrease of 26.13% compared to when alcohol was consumed alone (P=0.005),

When taking 20unit/kg KARC, it was 5.22 ± 0.99 mg h/dL, a statistically significant 55.71% decrease compared to when alcohol was consumed alone (P<0.001). Also, when comparing the 10unit/kg KARC group and the 20unit/kg KARC group, the 20unit/kg KARC group significantly decreased (P=0.034), reducing the total amount of acetaldehyde in the blood over time in a dose-dependent manner. confirmed.

The blood concentration of acetaldehyde, known as the cause of oxidative stress, was reduced after taking KARC, and the effect of reducing oxidative stress in humans was observed by oral administration of KARC.

In [Figure 32], as a result of measuring the concentration of MDA in blood during the chemotherapy period, the group treated without taking KARC (control group) was 0.607 ± 0.161 μM, and the group treated while taking KARC (experimental group ) was 0.223 ± 0.033 μM, which was statistically significantly reduced by 63.3% compared to the control group (P<0.001).

In addition, in the control group, the deviation of the MDA concentration in the blood was very large, with a minimum of 0.427 μM and a maximum of 0.885 μM, but in the experimental group, the deviation was greatly reduced to a minimum of 0.158 μM and a maximum of 0.269 μM. The effect was confirmed [FIG. 33].

The increase in active oxygen (ROS) in vivo due to various causes such as drug use, stress, and intense exercise leads to nonenal (HNE), malondialdehyde (MDA), acetaldehyde (Ach), etc. through the lipid peroxidation (LPO) reaction. Reactive aldehyde substances accumulate in cells to increase oxidative stress.

In addition, aldehydes generated in this way react with surrounding proteins or accumulate as stabilized end products (Advanced Lipidperoxidation End Products) of reactive aldehydes such as malondialdehyde-acetaldehyde adduct (MAA) and malondialdehyde lysine adducts (M-lys adducts) through secondary metabolic processes. It acts as a toxin to various cells, causing interactions that further exacerbate oxidative stress.

This accumulation of oxidative stress disturbs the energy metabolism process in the mitochondria, leading to the accumulation of aldehyde-based sugar metabolic intermediates such as methylglyoxal (MG) and glycerylaldehyde-3-phosphate (GA3P) in cells, or the chain reaction of aldehydes. Accumulation of advanced glycation end products (AGEs) stabilized by AGEs weakens the cellular antioxidant defense system, such as glutathione, and consequently increases endoplasmic reticulum stress and apoptosis.

The increase in reactive oxygen species and oxidative stress causes an increase in ER stress through an increase in reactive aldehydes such as HNE and MDA, and an increase in modified proteins such as advanced glycemic oxides (AGEs) and advanced lipid peroxidation end products (ALEs). It is known to have a mutual circulation and amplification effect.

The possibility of reducing oxidative stress and improving homeostasis of endoplasmic reticulum stress through the control of malondialdehyde, a marker for measuring active oxygen and oxidative stress by the administration of KARC, the mutant yeast composition of the present invention, was confirmed.

KARC, a mutant yeast composition, showed an effect of reducing active oxygen and oxidative stress by reducing the concentration of malondialdehyde in blood, and by reducing the concentration of acetaldehyde and malondialdehyde in human blood, thereby reducing active oxygen and oxidative stress It showed the effect of preventing and curing endoplasmic reticulum stress.

[Example 11] Short-term administration toxicity test of the KARC composition of the present invention

Example 11-1. Preparation of experimental animals

As experimental animals, female and male ICR mice (7 weeks old) were distributed and acclimatized for 7 days. During the acclimatization period, general symptoms were observed and only healthy animals were used for the test. Feed and water were freely consumed, and group separation was carried out to make a total of 10 animals, 5 females and 5 males in each group based on an average body weight of about 20 g the day before oral administration.

Example 11-2 Administration of test substance

Test substances were prepared by dissolving them in physiological saline so that the doses of the experimental animals were 0, 750, 3000, and 5000 mg/Kg, respectively, based on the content of the mutant yeast lysate KARC of the present invention.

The standard for the administered dose was in accordance with the Korea national Toxicology Program (KNTP) toxicity test manual of the Ministry of Food and Drug Safety. The maximum applied dose of 5000 mg/Kg, guided by the KNTP manual, was applied as the maximum concentration in this experiment. Samples prepared for each group were administered orally once each to the test animals, and physiological saline was administered to the normal group (G1).

Example 11-3. observation and autopsy

Symptoms were observed at least once a day for animals in all test groups from the day of acquisition to the day of necropsy, and symptoms were observed for 7 days after oral administration. After symptom observation, autopsy was performed, and changes in each organ were observed with the naked eye during autopsy.

As a result of a single dose toxicity test of the ALDH-containing KARC composition of the present invention using mice, no deaths were observed for 7 days at concentrations up to 5000 mg / kg of mutant yeast KARC, and peculiarities such as weight gain and feed intake were observed. Couldn't find it. In addition, no unusual findings were found in the autopsy performed after the observation was completed.

Through the above examples, using the mutant yeast composition containing aldehyde dehydrogenase (ALDH) of the present invention, a method for preparing a therapeutic agent for fatty liver and steatohepatitis, pharmacological effect, administration method, therapeutically effective amount for disease model, short-term administration acute toxicity Representative examples for have been described in detail through each drawing and test results, but these are only one example of the present invention.

Therefore, 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.

Therefore, even food or treatment containing a modified form of aldehyde dehydrogenase embodying the technical gist of the present invention described in the following claims falls within the legal protection scope of the present invention.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC13925BP 20190822 Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14122BP 20200130 Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14123BP 20200130 Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14983BP 20220527 Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14984BP 20220527 Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14985BP 20220527

Claims (5)

Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP, KCTC14985BP An endoplasmic reticulum stress reliever containing any one selected from the group consisting of, or a mixture thereof.
Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP, KCTC14985BP A fatty liver inhibitory food composition containing any one or a mixture thereof selected from the group consisting of.
Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP, KCTC14985BP A composition for preventing or treating fatty liver symptoms containing any one selected from the group consisting of or a mixture thereof.
Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP, KCTC14985BP Hepatitis preventive food composition containing any one or mixtures thereof selected from the group consisting of. Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP, KCTC14985BP Hepatitis treatment composition containing any one selected from the group consisting of, or a mixture thereof.

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