Food and pharmaceutical compositions for treating fatty liver and inflammation by reducing endoplasmic reticulum stress

Abstract

The present invention relates to food and pharmaceutical compositions that alleviate endoplasmic reticulum stress and thereby suppress or prevent various diseases caused by endoplasmic reticulum stress. More specifically, the present invention relates to food and pharmaceutical compositions for preventing or treating symptoms of fatty liver or hepatitis, which contain a lysate of any one selected from the group consisting of Saccharomyces celloviciae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP, or a mixture thereof.

Description

[Technical Field]

[0001] The present invention relates to foods and pharmaceutical compositions that alleviate endoplasmic reticulum stress, thereby suppressing or preventing various diseases caused by endoplasmic reticulum stress.The present invention is directed to foods and pharmaceutical compositions that inhibit or prevent fatty liver or hepatitis.

[0002] More specifically, the present invention relates to a food or pharmaceutical composition derived from mutant yeast that inhibits or prevents hepatitis, alcoholic fatty liver disease (AFLD), and non-alcoholic fatty liver disease (NAFLD) by alleviating endoplasmic reticulum stress.

[0003] The present invention is directed to food and pharmaceutical compositions for preventing or treating symptoms of fatty liver or hepatitis, which contain 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 technology]

[0004] The causes of fatty liver disease include alcohol consumption and obesity. Fatty liver symptoms can also appear in patients with hyperlipidemia or diabetes who have high blood lipid levels. Fatty liver symptoms can be caused by oxidative stress, as well as an increase in toxic aldehydes due to drugs that cause endoplasmic reticulum stress, corticosteroids, and female hormones.

[0005] When alcohol is consumed, fat synthesis is promoted due to increased endoplasmic reticulum stress (ER stress) in the liver, which leads to the accumulation of fat in liver tissue and the prevention of normal energy metabolism.

[0006] Symptoms of nonalcoholic fatty liver disease can be caused by factors other than alcohol consumption. Various drugs have been developed to alleviate the symptoms of fatty liver, but to date, no drug has been clearly shown to be effective in treating fatty liver disease.

[0007] Steatohepatitis refers to inflammation accompanied by lipid accumulation and hepatocyte necrosis in liver tissue due to insulin resistance and obesity, which can progress to chronic hepatitis and cirrhosis.

[0008] The endoplasmic reticulum (ER) is a membrane-bound structure present in the cytoplasm of eukaryotic cells, connected to the nuclear membrane. The space within the ER occupies 10% of the cell volume and more than 50% of the total cell membrane.

[0009] There are two types of endoplasmic reticulum: the rough endoplasmic reticulum (rER), which has a rough surface due to the presence of many ribosomes that synthesize proteins, and the smooth endoplasmic reticulum (sER), which has a smooth surface without ribosomes.

[0010] The smooth endoplasmic reticulum (sER) is where membrane lipids and steroid hormones are synthesized, detoxification occurs, and lipids, phospholipids, and steroids are synthesized. Within the sER, glucose 6-phosphate is dephosphorylated and split into glucose for excretion, and it also plays a role in the detoxification of organic substances, including alcohol, by oxidizing them. Smooth endoplasmic reticulum is abundant in the liver, muscle cells, steroid-, and fat-producing cells.

[0011] Ribosomes are attached to the surface of the rough endoplasmic reticulum (rER) and are responsible for mRNA translation during protein synthesis. The membrane-enclosed lumen of the ER is where protein folding, which creates secondary structure, and protein maturation and modification processes occur. Chaperone proteins and enzymes that mature and modify proteins are distributed in the rough endoplasmic reticulum.

[0012] Among proteins synthesized by ribosomes, proteins with an N-terminal ER signal peptide enter the endoplasmic reticulum. At this time, lipid-soluble membrane proteins remain in the endoplasmic reticulum membrane, while water-soluble proteins enter the lumen of the endoplasmic reticulum.

[0013] Among proteins synthesized by ribosomes, unfolded proteins (UP), which are not transformed into their proper secondary structure, accumulate in the endoplasmic reticulum (ER), resulting in increased endoplasmic reticulum stress (ER stress). In addition, when abnormally folded proteins (MP) are not removed and accumulate in the ER, ER stress increases.

[0014] The response phenomenon in which unfolded, abnormal proteins that cause endoplasmic reticulum stress are repaired or degraded and removed is called the unfolded protein response (UPR).

[0015] Cells respond by activating the unfolded protein response (UPR) to alleviate ER stress or maintain ER homeostasis. Proteins involved in activating the UPR include Grp78, IRE-1α, ATF6, and PERK.

[0016] When ER stress is alleviated via the unfolded protein response, but homeostasis is not maintained smoothly, cells enter the apoptotic pathway. Cell death caused by increased ER stress or disruption of ER homeostasis can lead to metabolic diseases such as viral infections, obesity, diabetes mellitus, neurodegenerative diseases including dementia, and can cause fatty liver, liver cirrhosis, and cancer.

[0017] Reactive oxygen species (ROS) generated during mitochondrial energy production and metabolic processes produce toxic substances such as nonenal (HNE), malondialdehyde (MDA), and acetaldehyde within cells.

[0018] Through secondary metabolism of these substances, malondialdehyde-acetaldehyde adducts (MAA) and malondialdehyde-lysine adducts (M-lys) are produced, and through this chain reaction, various modified proteins accumulate in the body, further increasing oxidative stress.

[0019] This increase in oxidative stress affects the mitochondrial energy metabolism process, increasing aldehydes such as methylglyoxal and advanced glycation end products (AGEs) in cells, resulting in further disruption of cellular energy metabolism.

[0020] In this way, reactive aldehydes such as HNE and MDA, which are the result of lipid peroxidation due to increased oxygen and oxidative stress, and aldehydes such as glyceraldehyde-3-phosphate, an intermediate product in glycolysis, accumulate in cells in excess, causing cytotoxicity.

[0021] Accumulation of reactive oxygen or reactive aldehydes in cells weakens cellular antioxidant defense systems such as glutathione, ultimately causing increased endoplasmic reticulum stress (ER stress) through disruption of energy metabolism and accumulation of unfolded proteins.

[0022] When stress occurs within the endoplasmic reticulum of hepatic parenchymal cells, the smooth endoplasmic reticulum is activated, causing fat to accumulate in the liver, causing symptoms of fatty liver and progressing to steatohepatitis.

[0023] There is an urgent need for the development of food or pharmaceutical compositions that inhibit endoplasmic reticulum stress and fat accumulation in the liver, thereby preventing acute liver damage and steatohepatitis. [Prior art documents] [Patent documents]

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

[0025] TIFF2026500116000002.tif142151 Summary of the Invention [Problem to be solved by the invention]

[0026] Disclosure of the Invention Technical issues Despite these many previous studies, to date, no food or pharmaceutical composition has been developed that inhibits or treats fatty liver or steatohepatitis by inhibiting the stress applied to the endoplasmic reticulum of hepatocytes.

[0027] The basic object of the present invention is to provide a mutant yeast-derived composition that contains an aldehyde dehydrogenase enzyme that rapidly decomposes endogenous aldehydes that cause endoplasmic reticulum stress, thereby blocking the occurrence of endoplasmic reticulum stress by eliminating factors that cause endoplasmic reticulum stress in advance.

[0028] Another object of the present invention is to provide food and pharmaceutical compositions containing mutant yeast that reduce the possibility of endoplasmic reticulum stress and inhibit or prevent the development of fatty liver or steatohepatitis.

[0029] Yet another object of the present invention is to provide a food composition and a pharmaceutical composition for preventing and treating fatty liver, which contain any one selected from the group consisting of Saccharomyces celloviciae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP, or a mixture thereof. [Means for solving the problem]

[0030] Solutions to problems The first object of the present invention described above can be achieved by providing a composition derived from a mutant yeast containing an aldehyde dehydrogenase that can rapidly decompose endogenous aldehydes before endoplasmic reticulum stress occurs.

[0031] Another object of the present invention can also be achieved by providing a food composition or pharmaceutical composition containing a lysate of any one selected from the group consisting of Saccharomyces cellovisiee KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, or KCTC14985BP, or a mixture thereof (hereinafter abbreviated as KARC). [Effects of the Invention]

[0032] Advantageous Effects of the Invention In animals with symptoms of fatty liver caused by endoplasmic reticulum stress in hepatocytes, alleviation of symptoms of fatty liver and hepatitis was observed 24 and 48 hours after administration of the KARC of the present invention.

[0033] It was confirmed that animals administered with the KARC of the present invention had reduced endoplasmic reticulum stress in the liver, reduced accumulation of fatty liver, and reduced expression of inflammatory factors in the liver. [Brief explanation of the drawings]

[0034] Detailed Description of the Drawings Administration of 2 mg / kg of tunicamycin (Tm) to mice induced endoplasmic reticulum stress in liver tissue. Figures 1 and 2 show the effect of the composition of the present invention on reducing fatty liver in mouse liver tissue 24 and 48 hours after administration.

[0035] [Figure 1]Figure 1 shows the color of mouse livers 24 and 48 hours after administration of tunicamycin and 10 and 20 units / kg of KARC. Livers in which fatty liver disease was induced by tunicamycin became lighter in color due to fat. The livers of mice administered KARC returned to red.

[0036] [Figure 2] Figure 2 shows the morphology of fat in liver tissue stained with hematoxylin and eosin (H&E). Tunicamycin administration increased fat in hepatocytes, whereas KARC administration decreased fat in hepatocytes.

[0037] [Figure 3-4] [Figures 3 and 4] are graphs showing changes in lipid content in liver tissue at intervals of 24 and 48 hours after administration of tunicamycin and KARC to mice (n=5).

[0038] *Triglyceride (TG) content in liver tissue, which was increased by tunicamycin administration, was significantly reduced by KARC administration [Figure 3]. Total cholesterol (T-chol) content in liver tissue, which was increased by tunicamycin administration, was reduced by KARC administration in a concentration-dependent manner [Figure 4].

[0039] [Figure 5-9] Figures 5, 6, 7, 8, and 9 show the expression of genes related to endoplasmic reticulum stress (*p<0.05, **p<0.01) (n=5). Mice were administered 2 mg / kg of tunicamycin and 10 and 20 units / kg of KARC. After 24 and 48 hours, mRNA was isolated from liver tissue. Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) was performed on the isolated mRNA.

[0040] Chop (an endoplasmic reticulum stress marker) in Figure 5 represents a C / EBP homologous protein. ER stress changes through the regulation of the expression levels of proteins involved in apoptosis induction, such as a decrease in BCL-2 and an increase in BIM, which are caused by increased ER stress in cells. The decrease in Chop mRNA expression levels by KARC administration indicates that ER stress is reduced by the removal of abnormal proteins in the ER that promote cell death [Figure 5].

[0041] Grp78 stands for glucose-regulated protein 78. It is one of the representative chaperone proteins and functions to maintain and regulate endoplasmic reticulum homeostasis. The decrease in Grp78 mRNA expression level caused by KARC administration implies that the accumulation of unfolded proteins was reduced by regulating ATF6 and IRE-1α, resulting in the alleviation of endoplasmic reticulum stress [Figure 6].

[0042] IRE-1α stands for inositol-requiring enzyme 1 alpha. It is a typical indicator of the unfolded protein response (UPR). IRE-1α induces abnormally produced and accumulated unfolded proteins to reassemble into their normal structure via the secretion of chaperone proteins or removes proteins that enter the endoplasmic reticulum at the mRNA stage (RIDD, regulated IRE-1α-dependent decay). In addition, IRE-1α activates JNK and NF-κβ proteins to degrade unfolded proteins by increasing autophagy or inducing apoptosis, thereby alleviating endoplasmic reticulum stress and maintaining homeostasis.

[0043] The decrease in IRE-1α mRNA expression levels due to KARC administration means that unfolded proteins, which act as cytotoxic agents, are reduced and endoplasmic reticulum stress is alleviated [Figure 7].

[0044] [Figure 8]Gadd34 in Figure 8 represents growth arrest and DNA damage-induced protein (Growth Arrest and DNA Damage-Inducible 34). Gadd34, together with the transcription factor ATF4, induces the dephosphorylation of eLF2α. Dephosphorylation of eLF2α occurs, promoting apoptosis. Gadd34 acts to remove unfolded proteins by promoting apoptosis due to the lack of dephosphorylation of eLF2α or by promoting the translation of abnormal proteins.

[0045] [Figure 9] [Figure 9] shows ATF4. ATF4 stands for activating transcription factor 4. A decrease in ATF4 expression level indicates that endoplasmic reticulum stress is alleviated. In Figures 8 and 9, a decrease in the mRNA expression level of ATF4 or Gadd34 due to KARC administration indicates that endoplasmic reticulum stress is reduced and the number of dying cells is reduced.

[0046] Mice were administered 2 mg / kg of tunicamycin followed by 10 units / kg and 20 units / kg of KARC. 24 and 48 hours later, mRNA was isolated from the liver tissue of the mice. Figures 10, 11, 12, and 13 show the expression of inflammatory genes via RT-qPCR of the isolated mRNA ( * p<0.05, ** p<0.01) (n=5).

[0047] [Figure 10] F4 / 80 in Figure 10 is a specific marker for macrophages. It is present in macrophages that perform phagocytosis and acts to express inflammatory genes. The decrease in F4 / 80 mRNA expression level due to KARC administration indicates a reduction in cellular inflammation.

[0048] [Figure 11]McP1 in Figure 11 stands for monocyte chemotactic protein 1. It is a type of chemokine that is expressed in various cells. McP1 is a substance associated with inflammation, which allows T cells, monocytes, etc. to gather around inflammation and easily migrate.

[0049] The decrease in Mcp-1 mRNA expression levels by KARC administration indicates a decrease in inflammation and inflammatory responses.

[0050] [Figure 12] TNF-α in Figure 12 represents tumor necrosis factor α, a type of cytokine that appears due to increased inflammation and inflammatory responses. This phenomenon is associated with increased phagocytosis. The decrease in TNF-α mRNA expression level due to KARC administration indicates a reduction in inflammatory substances or a weakening of the inflammatory response.

[0051] [Figure 13] II-6 in Figure 13 represents interleukin 6, which causes inflammation by expressing inflammatory genes. The decrease in II-6 mRNA expression level due to KARC administration indicates a reduction in inflammation.

[0052] Mice were administered 2 mg / kg of tunicamycin followed by 10 and 20 units / kg of KARC. 24 and 48 hours later, mRNA was isolated from liver tissue. RT-qPCR of the isolated mRNA was performed. The expression of genes related to fatty acid oxidation is shown in Figures 14, 15, 16, and 17. * p<0.05, ** p<0.01) (n=5).

[0053] [Figure 14]Ppar-α in Figure 14 stands for peroxisome proliferator-activated receptor alpha. It is involved in maintaining lipid metabolism homeostasis by improving fatty acid oxidation (β-oxidation) in muscle and liver. The mRNA expression level of Ppar-α increased with KARC administration. Fat metabolism was improved, low-density triglycerides were reduced, and high-density lipoprotein cholesterol (HDL-cholesterol (high-density lipoprotein cholesterol)) was increased. This indicates that the liver's lipid metabolism regulation function is restored by KARC administration.

[0054] [Figure 15] Pgc-1α in Figure 15 stands for peroxisome proliferator-activated receptor gamma coactivator 1-alpha. This protein is involved in intracellular mitochondrial biogenesis and hepatic gluconeogenesis. The increase in Pgc-1α mRNA expression levels by KARC administration indicates increased energy production and energy metabolism in the liver.

[0055] [Figure 16] Cpt-1α in Figure 16 represents carnitine palmitoyltransferase 1 alpha. This is an essential protein located in the mitochondrial lipid membrane and involved in the oxidation of fatty acids (beta-oxidation). This protein converts the acyl group of fatty acid chains into acylcarnitines, facilitating the passage of long-chain fatty acids through the mitochondrial membrane. The increase in Cpt-1α mRNA expression levels due to KARC administration indicates improved energy production and metabolism via fatty acid oxidation (beta-oxidation) in the liver.

[0056] [Figure 17]Fgf21 in Figure 17 stands for fibroblast growth factor 21. It is a fibroblast growth factor that is oversynthesized in the liver, pancreas, and adipose tissue. The expression level of Fgf21 is regulated by Ppar-γ (peroxisome proliferator-activated receptor gamma) and Ppar-α (peroxisome proliferator-activated receptor alpha). When stress increases, it is activated to adapt to or defend against stress. The decrease in Fgf21 mRNA expression level by KARC administration indicates a reduction in the stress state of hepatic parenchymal cells.

[0057] A mouse model was administered 2 mg / kg of tunicamycin followed by 10 and 20 units / kg of KARC. After 24 and 48 hours, Western blot analysis was performed on proteins isolated from liver tissue. Figure 18 shows the expression of proteins related to endoplasmic reticulum stress, lipid metabolism, and fatty acid oxidation. The expression levels of CHOP, IRE-1α, p-eIF2α, eIF2α, FAS, ACC1, Scd-1, PPARα, and CPT1 proteins were measured (n=5).

[0058] [Figure 18] As shown in Figure 18, the endoplasmic reticulum stress markers CHOP and IRE-1α were increased by tunicamycin administration, as shown in Figures 5 and 7. In the group administered 20 units / kg of KARC, protein expression was decreased, indicating a reduction in endoplasmic reticulum stress.

[0059] In Figure 18, p-eIF2α represents the phosphorylation of eukaryotic initiation factor-2α. eIF2α is a gene that is activated when one or more of four protein kinases (PERK, PKR, GCN2, and HRI) are phosphorylated. This regulates protein synthesis and also affects memory function.

[0060] When cells experience ER stress due to the accumulation of unfolded proteins (UP) or misfolded proteins (MP) in the ER, such as those caused by heat, UV radiation, or external infection, the p-eIF2α phosphatase is activated.

[0061] Overexpression of this eIF2 protein reduces the total amount of protein, thereby reducing intracellular endoplasmic reticulum stress. The decrease in eIF2 following KARC administration indicates reduced endoplasmic reticulum stress.

[0062] FAS in Figure 18 is fatty acid synthase. It is an essential enzyme system involved in lipid biosynthesis (de novo lipogenesis), which is necessary for the energy required for cell growth and cell signaling. The decrease in FAS protein due to endoplasmic reticulum stress indicates a decrease in energy metabolism through fatty acid oxidation. The increase in FAS protein due to KARC administration indicates that energy metabolism has been restored through fatty acid oxidation.

[0063] ACC1 in [Figure 18] is acetyl-CoA carboxylase 1 (ACC1). It is an enzyme that produces a metabolite called malonyl-coenzyme A, which is involved in the formation of fatty acids and the burning of fat. The expression of ACC1 is regulated by AMPK (AMP-activated protein kinase), which regulates carbohydrate and fat energy metabolism.

[0064] The decreased ACC1 protein expression caused by endoplasmic reticulum stress was restored by KARC administration, indicating that the liver's ability to convert energy through fat oxidation was improved.

[0065] In Figure 18, Scd-1 represents stearoyl-CoA desaturase-1 (Scd-1 (stearoyl-CoA desaturase-1)). Scd-1, an enzyme primarily present in the endoplasmic reticulum of the liver, converts saturated fatty acids, such as stearic acid, and CoA into unsaturated fatty acids, such as oleic acid, which are major components of cellular lipid membranes. Its expression is regulated by AMPK (AMP-activated protein kinase). The decrease in Scd-1 protein expression caused by endoplasmic reticulum stress is restored by KARC administration. This indicates improved control of lipid metabolism and hepatocyte recovery. The expression of fatty acid oxidation markers PPARα and CTP1 in Figure 18 is reduced by tunicamycin, as described in Figures 14 and 16. The expression of these proteins increases with KARC administration. This indicates improved energy production and metabolism via fatty acid oxidation in the liver.

[0066] In Figure 18, GAPDH stands for "glyceraldehyde-3-phosphate dehydrogenase (GAPDH)." It is used as a housekeeping gene because it is stably expressed in cells and its expression level does not easily change depending on cellular conditions. It is used as a reference marker for protein quantification, indicating that the same amount of protein is used.

[0067] [Figure 19] [Figure 19] shows the change in enzyme activity when the KwonP-1 strain was orally administered.

[0068] [Figure 20] [Figure 20] shows the changes in enzyme activity when the KwonP-2 strain is orally administered.

[0069] [Figure 21] [Figure 21] shows the change in enzyme activity when the KwonP-3 strain was orally administered.

[0070] [Figure 22][Figure 22] shows the changes in enzyme activity when the PicoYP strain is orally administered.

[0071] [Figure 23] [Figure 23] shows the changes in enzyme activity when the PicoYP-01 strain is orally administered.

[0072] [Figure 24] [Figure 24] shows the changes in enzyme activity when the PicoYP-02 strain is orally administered.

[0073] [Figures 19, 20, 21, 22, 23, 24], Kwon P-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02 were orally administered for 90 minutes under conditions similar to the human gastric digestion process (1 < pH < 5). Changes in ALDH enzyme activity were measured. The ALDH enzyme activity was maintained at pH = 5, with a minimum of 37.29 units / g and a maximum of 52.24% (similar to the conditions observed during food intake). It was confirmed that the enzyme activity was maintained when KARC was orally administered.

[0074] [Figure 25] [Figure 25] shows the growth curve and enzyme activity when the KwonP-1 strain is cultured in a 5 L fermenter.

[0075] [Figure 26] [Figure 26] shows the growth curve and enzyme activity when the KwonP-2 strain is cultured in a 5 L fermenter.

[0076] [Figure 27] [Figure 27] shows the growth curve and enzyme activity when the KwonP-3 strain is cultured in a 5 L fermenter.

[0077] [Figure 28] [Figure 28] shows the growth curve and enzyme activity when the PicoYP strain is cultured in a 5 L fermenter.

[0078] [Figure 29] [Figure 29] shows the growth curve and enzyme activity when the PicoYP-01 strain was cultured in a 5 L fermenter.

[0079] [Figure 30] [Figure 30] shows the growth curve and enzyme activity when the PicoYP-02 strain was cultured in a 5 L fermenter.

[0080] In Figures 25, 26, 27, 28, 29, and 30, the novel mutant strains KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-01 were cultured in 5-L fermenters using YPD medium under the same conditions. The cultures were run at 30°C and 200 rpm for 48 hours. When comparing the growth curves (OD660nm) and ALDH enzyme activity of each strain with those of the type strain, the ALDH enzyme activity was at least 10.5-fold higher and up to 18.75-fold higher. PicoYP-01 had the highest ALDH activity of 52.68 units / g, compared with KwonP-3, which had the lowest ALDH activity of 29.5 units / g.

[0081] [Figure 31] [Figure 31] is a graph showing the decomposition of acetaldehyde in the human body by KARC.

[0082] [Figure 32] [Figure 32] is a graph showing the decomposition of malondialdehyde in the human body by KARC.

[0083] [Figure 33] [Figure 33] is a graph showing the stabilization of malondialdehyde in the human body by KARC.

[0084] In studies to confirm the reduction of endogenous blood acetaldehyde [Figure 31] and blood malondialdehyde [Figure 32] in humans, the effects of KARC administration appear to be reducing acetaldehyde and malondialdehyde, which are biomarkers for hangovers, fatigue, and cardiovascular disease. [Figure 33] shows the effect of KARC administration in a state where oxidative stress has increased due to medication, etc., by reducing malondialdehyde, a biomarker for oxidative stress and reactive oxygen species. DETAILED DESCRIPTION OF THE INVENTION

[0085] BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a method for producing a dry powder of the KARC of the present invention, which is a lysate of Saccharomyces celloviciae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, or KCTC14985BP, will be described in more detail.

[0086] These examples are for illustrative purposes only of compositions that can achieve the objectives of the present invention, and therefore the scope of the present invention is not limited to only the compositions illustrated in the following examples. [Example]

[0087] Mode of Invention [Example 1] Screening of wild yeast parent strains to promote mutations. In this study, various types of makgeolli (traditional Korean wine) were mixed with a 0.9% NaCl solution to prepare makgeolli suspensions. The makgeolli suspensions were stirred at 200 rpm for 1 hour. The supernatant containing the wild-type yeast strain was diluted with YPD (yeast extract peptone dextrose broth) medium. The diluted solutions were prepared to a concentration 106 times the original solution. The diluted solutions were then spread onto YPD agar medium. The agar medium was statically cultured at 30°C under aerobic conditions for 1 week. Saccharomyces celloviciae was initially screened based on colony morphology, growth characteristics on YPD medium, and microscopic observation.

[0088] The ALDH activity and glutathione content of the screened Saccharomyces celloviciae strains were measured. The parent strains were selected based on ALDH activity and glutathione production.

[0089] 1-1: Measurement of aldehyde dehydrogenase Acetaldehyde reacted with dinitrophenylhydrazine (DNPH) to form acetaldehyde hydrazone (Ach-DNPH) compounds. The Ach-DNPH compounds were detected at 360 nm by HPLC equipped with a C18 column. The amount of aldehyde reduced by the degradation reaction catalyzed by aldehyde dehydrogenase (ALDH) was quantified via the amount of detected Ach-DNPH compounds.

[0090] The enzymatic reaction was carried out at 30°C by adding 10 μl of yeast lysate to 990 μl of reaction mixture (50 mM potassium phosphate buffer (pH 8.0), 1.5 mM acetaldehyde, and 3 mM NADP+). After the enzymatic reaction was completed, 50 μl of 10 mM DNPH was added to induce the formation of Ach-DNPH. The formation of Ach-DNPH was allowed to proceed for 1 hour at 22°C.

[0091] The formation of Ach-DNPH was stopped by adding 3 M sodium acetate (pH 9). The formed Ach-DNPH compound was separated by adding two volumes of acetonitrile. The separated Ach-DNPH compound (in ACN) was analyzed by injection into HPLC.

[0092] The concentration of Ach-DNPH compounds was analyzed by HPLC under conditions of 360 nm wavelength, 1 ml / min flow rate, and acetonitrile / water mobile phase development on a C18 column. The area values ​​of the resulting chromatograms were converted using a standard curve of aldehyde-DNPH (Sigma-Aldrich) to quantify the concentration of Ach-DNPH compounds. A reduction of 1 mM Ach-DNPH per minute was calculated as 1 unit of ALDH. ALDH activity was standardized as units / mg of protein.

[0093] 1-2: Measurement of glutathione Yeast cells were harvested by centrifuging 1 ml of Saccharomyces celloviciae culture medium. A suspension was prepared by adding 1 ml of water to the harvested yeast cells. Glutathione was extracted by stirring the suspension at 1,000 rpm for 2 hours at 85°C. The suspension was centrifuged to remove the yeast cells, and the supernatant was filtered through a 0.22 μm filter to obtain a sample containing glutathione.

[0094] The glutathione concentration of the samples was analyzed by HPLC (Shimazu LC-20AD) equipped with a C18 column. The glutathione concentration was analyzed at a wavelength of 210 nm using a mobile phase (2.02 g / L sodium 1-heptanesulfonate monohydrate, 6.8 g / L potassium dihydrogen phosphate, pH 3.0, methanol mixture) developed at a rate of 1 ml / min. The area values ​​of the resulting chromatograms were analyzed using a glutathione standard curve.

[0095] Two hundred different types of yeast obtained from Korean makgeolli were analyzed for ALDH activity and glutathione content. The 10 types of yeast listed in Table 1 had higher ALDH activity or glutathione production capacity than the other yeasts.

[0096] The ALDH activity of yeast #97 was 0.10 units / mg protein, the second highest overall. The glutathione content of yeast #97 was 0.42%, the highest of all. Yeast #97 was selected as the parent strain and the mutagenesis procedure was performed.

[0097] [Table 1]

[0098] [Example 2] Identification of parent strains used in the mutagenesis process Identification was performed to confirm the exact species of the wild-type parent strain (yeast #97, wild-type yeast). To ensure sufficient yeast cells for DNA extraction, only single yeast colonies were plated on YPD agar medium. DNA was extracted using a Genomic DNA prep kit (HiGene™, BIOFACT Co., Ltd., Daejeon, Korea) according to the manufacturer's instructions.

[0099] To amplify the yeast ITS region of the rRNA gene, polymerase chain reaction (PCR) was performed on yeast chromosomal DNA using ITS5 (forward) and ITS4 (reverse) primers. PCR results were analyzed by DNA sequencing.

[0100] The DNA sequence of the parent strain was isolated using the Bioedit program. The reverse strands of the PCR results were converted into paired sequences through a reverse completion process.

[0101] The Cluster X program confirmed that the sequence of the forward strand matched the sequence of the reverse strand. Parent strains that matched the sequence information confirmed through the above experimental process were identified using the BLAST database provided by the US National Center for Biotechnology Information (NCBI). As a result of the identification, it was found that the rRNA of the parent strain ITS was 100% identical to that of Saccharomyces celloviciae.

[0102] [Example 3] Selection of mutant strains with improved aldehyde dehydrogenase production The mutagenesis process was carried out on the wild-type Saccharomyces celloviciae parent strain according to the method described in US Patent Application No. 17 / 176,365.

[0103] To induce mutations in the parent yeast strain, a wild-type yeast strain that produces both ALDH and glutathione was treated with ethyl methanesulfonate (EMS) or nitrosoguanidine (NGD). The mutated yeast strains were exposed to various concentrations of methylglyoxal. Mutants with superior adaptation to methylglyoxal were selected. The selected yeast strains were then exposed to various concentrations of lysine. Mutants with superior adaptation to lysine were selected. Thirty mutant strains with superior adaptation to methylglyoxal and lysine were obtained. Each of the 30 yeast strains was evaluated via five characteristics: growth curve, ALDH activity, ADH activity, coenzyme content, and glutathione content.

[0104] 3-1: Growth characteristics Saccharomyces celloviciae is a crab tree-positive microorganism that grows under aerobic conditions and simultaneously produces ethanol. To cultivate yeast with high yields, Saccharomyces celloviciae with high ethanol tolerance is required.

[0105] YPD media with different ethanol concentrations were prepared (no ethanol, 5%, 7%, and 10%). Culture media for Saccharomyces celloviciae (yeast) were adjusted to an OD of 1 at 660 nm. Each mixture of prepared YPD medium and yeast culture medium was diluted 99:1. Finally, four YPD media containing yeast with different alcohol concentrations were prepared. Each YPD medium mixed with yeast was cultured at 30°C with shaking at 200 rpm. Growth curves of the mutant strains were measured every 3 hours for 48 hours. The growth curves of each mutant strain were evaluated based on three characteristics: the time (or duration) of the lag phase, the specific growth rate (OD / h) of the exponential phase, and the maximum density (OD).

[0106] The higher the ethanol concentration in YPD medium, the longer the lag phase. The maximum density and specific growth rate decreased. Comparing the maximum densities of the mutants at low (5%) and high (10%) ethanol concentrations, it was found that in the case of nine mutants, growth was maintained at 50% of the growth rate at the high concentration compared to the growth at the low concentration. The growth characteristics of the nine mutants that distinguished them from the other strains were their short lag phase and high specific growth rate.

[0107] [Table 2] TIFF2026500116000005.tif961543-2: Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities

[0108] Alcohol dehydrogenase (ADH) activity was measured by adding 10 μl of yeast lysate to a 990 μl reaction mixture containing 50 mM potassium phosphate buffer (pH 8.0), 2 mM NAD+, and 1% ethanol. Aldehyde dehydrogenase (ALDH) activity was measured by adding 10 μl of yeast lysate to a 990 μl reaction mixture containing 50 mM potassium phosphate buffer (pH 8.0), 3 mM NAD+, and 1.5% acetaldehyde. The enzymatic reactions of ADH and ALDH were carried out at 30°C for 5 minutes, and the concentration of NAD(P)H produced as a result of the enzymatic reactions was measured via absorbance at 340 nm.

[0109] The enzyme activities of nine mutant strains (K-1 to K-9) selected in this study were measured. The ADH activity of the mutant strains was a minimum of 382.69 units / g and a maximum of 975.29 units / g. The ADH activity of the mutant strains was increased by at least 5.1 times and a maximum of 13.1 times compared to the type strain (reference yeast, Saccharomyces celloviciae KCTC7296). The ALDH activity of the mutant strains was a minimum of 15.23 units / g and a maximum of 72.16 units / g. The ALDH activity of the mutant strains was increased by at least 5.3 times and a maximum of 24.9 times compared to the enzyme activity of the type strain.

[0110] The six mutant strains (K-1, 4, 6, 7, 8, and 9) showed similar rates of increase in ADH and ALDH enzyme activities compared with the type strain. The ALDH enzyme activity in three mutant strains (K-2, 3, and 5) was 18.3, 23.2, and 24.9 times higher, respectively, than that of the type strain. The ADH enzyme activity in three mutant strains (K-2, 3, and 5) was 9.7, 11.6, and 13.1 times higher, respectively, than that of the type strain. The rate of increase in ALDH enzyme activity in three mutant strains (K-2, 3, and 5) was twofold higher than that of ADH.

[0111] We have identified three novel mutant strains (K-2, 3, and 5) that have been adapted to increase aldehyde dehydrogenase (ALDH) activity and named them PicoYP, PicoYP-01, and PicoYP-02, respectively. The three novel mutant strains were deposited at the Biological Resources Center of the Korea Research Institute of Bioscience and Biotechnology and assigned the accession numbers KCTC14983BP, KCTC14984BP, and KCTC14985BP, respectively.

[0112] 3-3: Coenzyme (NAD and NADP) content NAD in lysates extracted from mutant strains 合計 and NADP 合計 was measured with the NADH / NAD+ assay kit and the NADPH / NADP+ assay kit, respectively. NAD(P) in the samples was converted to NAD(P)H using the NAD(P) cycling buffer and the NAD(P) cycling enzyme mix. The colorimetric test reaction was induced with the NAD(P) developer and measured as the absorbance at 450 nm. The colorimetric test reaction was measured as the absorbance at 450 nm. The absorbance of the samples was substituted into the equation corresponding to the standard curve to determine the NAD(P) of the yeast lysate. 合計 was calculated.

[0113] The coenzyme contents of the nine mutants (K-1 to K-9) selected in this study were measured. 合計 The NAD of the mutant strains was 126 nmole / g at the minimum and 195 nmole / g at the maximum. 合計 The NADP of the mutant strain increased by at least 7.3 times and up to 10.8 times compared to the type strain. 合計 The minimum and maximum NADP contents were 2.4 nmole / g and 5.8 nmole / g, respectively. 合計 The content increased by at least 11.4-fold and up to 27.6-fold compared to the type strain.

[0114] In six mutants (K-1, 4, 6, 7, 8, and 9), NADP 合計 The rate of increase in NAD 合計 The NADP of the three novel mutants (PicoYP, PicoYP-01, and PicoYP-02) increased less than twofold. 合計 The increase rates of NAD content were 25.7, 22.9, and 27.6 times, respectively. 合計 The increase rates of NADP content were 10.8, 9.9, and 11.3 times, respectively. 合計 The increase rate is NAD 合計 The rate of increase was more than double.

[0115] 3-4: Glutathione (GSH) content The glutathione contents of the nine mutant strains were measured in the same manner as in Examples 1 and 2. The glutathione contents of the mutant strains ranged from a minimum of 0.85% to a maximum of 1.05%. The glutathione contents of the mutant strains increased by at least 2.7-fold and up to 3.3-fold compared to the type strain. The three novel mutant strains (PicoYP, PicoYP-01, and PicoYP-02) showed higher increases in ALDH activity and coenzyme content than the others.

[0116] The three novel mutant yeast strains (PicoYP, PicoYP-01, and PicoYP-02) had glutathione production capabilities similar to those of the existing deposited strains (Kwon P-1, Kwon P-2, and Kwon P-3). The three novel mutant yeast strains had significantly increased ADH and ALDH enzyme activities and coenzyme contents compared to the existing deposited strains.

[0117] [Table 3]

[0118] [Table 4]

[0119] [Example 4] Comparison of carbon source selection A domestic patent application for this study was filed on February 18, 2020. We investigated the carbon source selection for the growth of three mutant strains (KwonP-1, KwonP-2, and KwonP-3) with high ALDH and glutathione levels. Various carbon sources used for growth by the reference yeast strain (KCTC7296) were measured. To determine the maximum capacity for ALDH production, we investigated the carbon source selection for the growth of three new mutant strains (PicoYP, PicoYP-01, and PicoYP-02).

[0120] The characteristics and novelty of the carbon source selection of the strains were analyzed by API 50 CHL kit (API systems, BIOMERIEUX, SA, France).

[0121] A 15 ml conical tube was prepared containing 8 ml of YPD medium. Each of the seven mutant strains was inoculated into the prepared conical tube.

[0122] After culturing the inoculated conical tubes at 30°C and 200 rpm for 24 hours, each of the seven mutant strains was isolated from the exponential growth phase and extracted. To eliminate the influence of the carbon source contained in the residual YPD medium, the yeast was washed three times using a centrifuge. A yeast suspension with a 2 McFarland concentration was prepared using API 50 CHL medium. The prepared yeast suspension was then filled into strip tubes. The strips containing the suspension were cultured at 30°C for 24 hours.

[0123] The API 50 CHL medium used for API testing was purple in color. As acid was produced via energy metabolism, the medium turned blue, green, and finally yellow. Finally, the type of carbon source used by the mutant strain was recorded based on the color change: purple x, blue +, green ++, and yellow +++.

[0124] All seven mutants tested used 19 different carbon sources for energy production and growth: L-arabinose, ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, N-acetyl-glucosamine, arbutin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, raffinose, and gentiobiose.

[0125] Rhamnose was used by only three mutants: KwonP-1, PicoYP-01, and PicoYP-02. Sorbitol was used by four mutants: KwonP-1, KwonP-3, PicoYP-01, and PicoYP-02. α-Methyl-D-mannoside was used by four mutants: the type strain, KwonP-1, KwonP-2, and PicoYP-02. Amygdalin was used by six mutants: KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02. D-Turanose was used by four mutants: the type strain, KwonP-1, KwonP-3, and PicoYP-02. D-tagatose was used by three mutant strains: the type strain, KwonP-3, and PicoYP-3. Gluconate was used only by the type strain.

[0126] Mannitol and sorbitol, which correspond to alcohol carbon sources, had significant effects on yeast growth. The three new mutant strains differed from the other four yeast strains in the type of sugars they used for growth. The preferred alcohol carbon source usage differed slightly among the three new mutant strains (PicoYP, PicoYP-01, and PicoYP-02) [Table 5].

[0127] [Table 5]

[0128] [Example 5] Changes in ALDH activity of mutant strains in gastric juice When KARC is administered orally, to maintain enzymatic activity in the intestine, it must pass safely without being destroyed by stomach acid, which secretes potent proteolytic enzymes such as pepsin.

[0129] NaOH solution was added to artificial gastric juice at pH 1.17 to artificially generate two simulated solutions at pH 3 and pH 5, which mimic the human gastric environment during food digestion. 1 g of KARC was added to 7 ml of artificial gastric juice and 7 ml of the two simulated solutions and mixed at 36.5°C for 5, 30, 60, and 90 minutes, respectively. NaOH solution was added to the reaction mixture to adjust the acidity to pH 7. 10 ml samples were taken from each of the solutions adjusted to pH 7 for analysis. ALDH activity was analyzed from each sample.

[0130] Under pH = 1.17 conditions, the ALDH activity of the samples decreased by more than 92.88% compared to the control group during a 5-minute reaction. Under pH = 1.17 conditions, the ALDH activity of the samples decreased by an average of 98.89% over 90 minutes. Over 90 minutes, the ALDH activity of the samples decreased by an average of 96.66% at pH = 3 and 56.83% at pH = 5. Ultimately, over the 90-minute reaction, the ALDH activity at pH = 3 and 5 remained relatively higher than that at pH = 1.17.

[0131] Specifically, the ALDH activity of KwonP-1 (KCTC13925BP) at pH = 1.17 decreased by 90.94% to 5.57 units / g compared to the control after 5 minutes of incubation. The ALDH activity of KwonP-1 decreased by 98.57% to 0.88 units / g after 90 minutes (Figure 19). The enzyme activity at pH = 3 and 5 remained relatively higher than that at pH = 1.17. After 90 minutes of incubation, the ALDH activity of KwonP-1 decreased by 96.66% to 5.57 units / g at pH = 3 and by 98.57% to 0.88 units / g at pH = 5.

[0132] The ALDH activity of KwonP-2 (KCTC14122BP) at pH 1.17 decreased by 91.18% to 5.43 units / g after 5 minutes of reaction. The ALDH activity of KwonP-2 decreased by 98.81% to 0.73 units / g after 90 minutes (Figure 20). At pH 3 and pH 5, higher enzyme activity was maintained than at pH 1.17. After 90 minutes of reaction, the ALDH activity decreased by 97.62% to 1.47 units / g at pH 3 and by 56.11% to 26.99 units / g at pH 5.

[0133] The ALDH activity of KwonP-3 (KCTC14123BP) at pH = 1.17 decreased by 89.99% to 6.16 units / g after 5 minutes of reaction. The ALDH activity of KwonP-3 decreased by 97.85% to 1.32 units / g after 90 minutes (Figure 21). At pH 3 and pH 5, higher enzyme activity was maintained than at pH 1.17. After 90 minutes of reaction, the ALDH activity decreased by 92.61% to 4.55 units / g at pH = 3 and by 62.31% to 22.18 units / g at pH = 5.

[0134] The ALDH activity of PicoYP (KCTC14983BP) at pH = 1.17 decreased by 92.84% to 4.40 units / g after 5 minutes of reaction. The ALDH activity of PicoYP decreased by 98.33% to 1.03 units / g after 90 minutes of reaction [Figure 22]. Higher enzyme activity was maintained at pH 3 and pH 5. After 90 minutes of reaction, the ALDH activity decreased by 96.66% to 2.05 units / g at pH = 3 and by 53.97% to 28.31 units / g at pH = 5.

[0135] The ALDH activity of PicoYP-01 (KCTC14984BP) at pH = 1.17 decreased by 95.71% to 2.64 units / g after 5 minutes of incubation. The ALDH activity of PicoYP-01 decreased by 99.76% to 0.15 units / g after 90 minutes (Figure 23). At pH 3 and pH 5, the enzyme activity remained higher than that of gastric juice. After 90 minutes of incubation, the ALDH activity decreased by 98.21% to 1.10 units / g at pH = 3 and by 58.74% to 25.38 units / g at pH = 5.

[0136] The ALDH activity of PicoYP-02 (KCTC14985BP) at pH = 1.17 decreased by 96.66% to 2.05 units / g after 5 minutes of reaction. The ALDH activity of PicoYP-02 decreased by 99.76% to 0.15 units / g after 90 minutes (Figure 24). At pH 3 and pH 5, higher enzyme activity was maintained than in gastric juice. After 90 minutes of reaction, the ALDH activity decreased by 98.21% to 1.10 units / g at pH = 3 and by 62.08% to 23.32 units / g at pH = 5.

[0137] pH 1.17 is the pH secreted by unprocessed gastric juice. When humans eat food, the unprocessed gastric juice and food mix in the stomach, raising the pH from 3 to 5, making it unlikely that a pH of 1.17 will be reached. Nevertheless, ALDH activity in the mutant strain was retained even at the extreme condition of pH 1.17.

[0138] Finally, the ALDH enzyme activity of the new mutants (PicoYP, PicoYP-01, and PicoYP-02) decreased from 92% to 97% under strongly acidic conditions at pH 1.17, but remained at 2 to 5 units / g. This means that 2 to 5 units of enzyme activity remained, sufficient for function in the intestine. It remained higher even at pH 3 and 5 compared to pH 1.17. This led to the conclusion that the new mutants (PicoYP, PicoYP-01, and PicoYP-02) could be administered orally.

[0139] [Example 6] Growth characteristics of 5L fermenter culture Each was inoculated into YPD medium (2% peptone, 1% yeast extract, 2% glucose), and the primary seed culture was carried out at 30°C and 200 rpm for 18 hours. 20 ml of the cultured seed was inoculated into 1980 ml of YPD medium and cultured again in 5 L. Cultivation in a 5 L culture tank was carried out at 30°C and 200 rpm for 48 hours. Growth curves at OD660nm and enzyme activities were analyzed using 10 ml samples collected from the secondary culture.

[0140] The maximum density (OD660nm) of KwonP-1 (KCTC13925BP) was 134.4. The maximum density of KwonP-1 was 4.35% higher than that of the type strain (KCTC7296). The growth curve characteristics and specific growth rate (OD660nm / h) of KwonP-1 were similar to those of the type strain. The ALDH activity of KwonP-1 was 33.6 units / g. The ALDH activity of KwonP-1 was 11.96-fold higher than that of the type strain (Figure 25).

[0141] The maximum density (OD660nm) of KwonP-2 (KCTC14122BP) was 133.8. The maximum density of KwonP-2 was 3.88% higher than that of the type strain. Growth of KwonP-2 terminated earlier than that of the type strain. The specific growth rate (OD660nm / h) of KwonP-2 was 14.8% higher than that of the type strain. The ALDH activity of KwonP-2 was 31.5 units / g. The ALDH activity of KwonP-2 was 11.21-fold higher than that of the type strain (Figure 26).

[0142] The maximum density (OD660nm) of KwonP-3 (KCTC14123BP) was 134.1. The maximum density of KwonP-3 was 4.12% higher than that of the type strain. Growth of KwonP-3 terminated earlier than that of the type strain. The specific growth rate (OD660nm / h) of KwonP-3 was 6.08% higher than that of the type strain. The ALDH activity of KwonP-3 was 29.5 units / g. The ALDH activity of KwonP-3 was 10.5-fold higher than that of the type strain (Figure 27).

[0143] The maximum density (OD660nm) of PicoYP (KCTC14983BP) was 123.8. The maximum density of PicoYP was 3.88% higher than that of the type strain. The growth curve characteristics of PicoYP were similar to those of the type strain. The specific growth rate (OD660nm / h) of PicoYP was 6.22% higher than that of the type strain. The ALDH activity of PicoYP was 44.2 units / g. The ALDH activity of PicoYP was 15.73-fold higher than that of the type strain [Figure 28].

[0144] The maximum density (OD660nm) of PicoYP-01 (KCTC14984BP) was 126.9. The maximum density of PicoYP-01 was 1.47% higher than that of the type strain. The growth curve characteristics of PicoYP-01 were similar to those of the type strain. The specific growth rate (OD660nm / h) of PicoYP-01 was 2.14% higher than that of the type strain. The ALDH activity of PicoYP-01 was 47.1 units / g. The ALDH activity of PicoYP-01 was 16.76-fold higher than that of the type strain [Figure 29].

[0145] The maximum density (OD660nm) of PicoYP-02 (KCTC14985BP) was 148.1. The maximum density of PicoYP-02 was 14.99% higher than that of the type strain. The growth curve of PicoYP-02 was located at the top compared to the type strain. The specific growth rate (OD660nm / h) of PicoYP-02 was 9.64% lower than that of the type strain. The ALDH activity of PicoYP-02 was 52.68 units / g. The ALDH activity of PicoYP-02 was 18.75-fold higher than that of the type strain (Figure 30).

[0146] [Example 7] Preparation of mutant strain lysate (KARC) To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysate, proteases were removed and inhibited. To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysate, cell debris was removed. The dried products or lysates of the mutant strains were mixed to prepare the KARC composition.

[0147] The mutant strain and the culture medium contained various substances, such as yeast metabolites and proteolytic enzymes secreted by the yeast. To extract and preserve the ALDH, coenzymes, and glutathione present in the yeast, it was necessary to thoroughly remove substances external to the yeast. A washing process was performed to wash the mutant strain by dispensing 40 ml of culture medium into a 50 ml conical tube, centrifuging it at 13,000 rpm for 15 minutes, and removing the supernatant.

[0148] As a result of centrifugation, residual medium remained inside the pellet produced by the aggregated yeast bacteria. After adding 30 ml of purified water, the pellet was thoroughly loosened by vortexing, and the previous process was repeated three times to thoroughly remove the residual medium.

[0149] The ethanol resistance of yeast is known to be up to 13%, and yeast bacteria die when exposed to high concentrations of ethanol. The washed pellet was thoroughly dissolved using 10 ml of 20% ethanol solution to induce yeast bacterial death. The ethanol-dissolved pellet was stirred at 100 rpm for 30 minutes to allow the yeast to die. When the reaction time was complete, 30 ml of purified water was added to reduce the ethanol concentration to 5%. The previous washing process was repeated three times to thoroughly remove the ethanol.

[0150] To protect ALDH and ADH from the degradative effects of proteases present in yeast cells, 10 ml of 1X PBS was prepared by dissolving two protease inhibitor tablets (Pierce protease inhibitor mini-tablets, EDTA-free, Thermo Scientific). The above solution was added to the washed yeast pellet and allowed to fully release.

[0151] To prepare the lysates of the mutant strains prepared in this invention, 4 g of glass beads were added and stirred to disrupt the yeast cell walls. To prevent enzyme denaturation due to the heat generated during the yeast disruption process, vortexing for 30 seconds and incubating for 30 seconds were repeated six times.

[0152] After the yeast cell wall was completely broken down, 10 ml of 100 mM potassium phosphate buffer was added and mixed by vortexing for 3-5 seconds. The mixture was centrifuged at 13,000 rpm for 15 minutes to remove cell structures such as yeast cell walls and glass beads. The supernatant was filtered through a 0.2 μm filter (Minisart® Syringe Filter, Sartorius, Goettingen, Germany) to prepare the KARC composition.

[0153] To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysates, intracellular proteases were removed and inhibited, and cell debris such as cell walls were removed. KARC compositions were prepared at free ratios with lysates selected from six mutant strains (KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02) or their mixtures (Table 6).

[0154] KARC1 was produced from KwonP-1. The enzyme activities of ADH and ALDH of KARC1 were 461.4 units / g and 28.6 units / g, respectively. 合計 and NADP 合計 The coenzyme contents of KARC1 and KARC2 were 176.2 nmole / g and 5.1 nmole / g, respectively. The GSH content of KARC1 was 0.98 wt%.

[0155] KARC2 was produced from KwonP-2. The enzyme activities of ADH and ALDH of KARC2 were 482.1 units / g and 29.8 units / g, respectively. 合計 and NADP 合計 The coenzyme contents of KARC2 and KARC3 were 175.4 nmole / g and 5.2 nmole / g, respectively. The GSH content of KARC2 was 0.96 wt%. KARC3 was produced from KwonP-3. The enzyme activities of ADH and ALDH in KARC2 were 477.5 units / g and 28.1 units / g, respectively. 合計 and NADP 合計 The coenzyme contents of KARC3 and KARC4 were 177.2 nmole / g and 5.1 nmole / g, respectively. The GSH content of KARC3 was 1.00 wt%.

[0156] KARC4 was produced from PicoYP. The enzyme activities of ADH and ALDH of KARC2 were 586.8 units / g and 33.8 units / g, respectively. 合計 and NADP 合計The coenzyme contents of KARC4 and KARC5 were 184.3 nmole / g and 5.7 nmole / g, respectively. The GSH content of KARC4 was 0.84 wt%.

[0157] KARC5 was produced from PicoYP-01. The enzyme activities of ADH and ALDH of KARC5 were 621.6 units / g and 38.2 units / g, respectively. 合計 and NADP 合計 The coenzyme contents of KARC5 and KARC6 were 186.9 nmole / g and 5.6 nmole / g, respectively. The GSH content of KARC5 was 0.84 wt%.

[0158] KARC6 was produced from PicoYP-02. The enzyme activities of ADH and ALDH in KARC5 were 664.1 units / g and 41.6 units / g, respectively. 合計 and NADP 合計 The coenzyme contents of the two were 195.0 nmole / g and 5.8 nmole / g, respectively. The GSH content of KARC6 was 0.88 wt%.

[0159] KARC was produced by free mixing of dry powder with lysates prepared from the six deposited strains. The mean enzyme activities of ADH and ALDH in the KARC composition were 547.6 units / g and 33.1 units / g, respectively. The coenzyme NAD in the KARC composition 合計 and the coenzyme NADP 合計 The average contents of glutathione in the KARC compositions were 180.4 nmole / g and 5.4 nmole / g, respectively. The average content of glutathione in the KARC compositions was 0.84 wt%.

[0160] The aldehyde degradation ability of KARC continued throughout the lysate production process. KARC demonstrated the ability to remove endogenous aldehydes such as HNE, MDA, and 3,4-dihydroxyphenylacetaldehyde (DOPAL).

[0161] [Table 6]

[0162] [Example 8] Analysis of ALDH sequences contained in mutant strains Differences between the ALD (yeast aldehyde dehydrogenase) of both mutant and parent strains were investigated. Whole genome sequencing was performed on the parent strain and mutant strains KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02. Mutant cells were obtained by culturing pure strains on solid medium. The genome sequences of the obtained mutant strains were analyzed.

[0163] Among the ALDs (yeast aldehyde dehydrogenases) in the novel mutant strain, ALD2 (SEQ ID NO: 3) was found to be condensed with ALD3 (SEQ ID NO: 4) on chromosome 13. A non-coding region of 689 nucleotides was located between the genes encoding ALD2 and ALD3.

[0164] ALD2 and ALD3 existed contiguously in the same genome. ALD2 and ALD3 encoded aldehyde dehydrogenases, respectively. The gene encoding ALD2 consisted of 1,521 nucleotides and 506 amino acids and was nearly identical to ALD3, but had an 8.2% sequence difference. ALD2 and ALD3 were identified as distinct aldehyde dehydrogenases, differing from each other by 125 base pairs (8.2%).

[0165] In six mutant strains (KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02), the protein is synthesized continuously because there is no stop codon at the end of the ALD2 sequence. As a result, a new, larger ALDH enzyme is created by linking parts of ALD2 and ALD3 (SEQ ID NO: 1).

[0166] ALD2 (SEQ ID NO: 3) of the type strain (KCTC7296) consisted of a 30-nucleotide sequence (5-GTTCACATAAATCTCTCTTTGGACAACTAA-3) encoding nine amino acids (N-VHINLSLDN-C) at the terminal, excluding the stop codon.

[0167] The ALD2 of the six mutant strains consisted of a specific 42-nucleotide sequence (5-AGATATAGATTATACACATTTAGAAAATTAGCCAAAAGAAAA-3) [SEQ ID NO: 2] encoding 14 amino acids (N-RYRLYTFRKLAKRK-C) between the 5′ end of ALD2 and ALD3.

[0168] The deletion of ALD2 from nucleotide 1492 to nucleotide 647 of the non-coding region resulted in no termination codon at the end of the gene sequence encoding ALD2. Finally, the six deposited mutant strains contained a new mutant gene consisting of a total of 3,054 bases encoding a novel ALD [SEQ ID NO: 1].

[0169] [Example 9] Efficacy of KARC in reducing endoplasmic reticulum (ER stress)-induced liver fat Administration of tunicamycin (Tm) to animals inhibits protein N-glycosylation, leading to the formation of unfolded proteins (UP). Administration of tunicamycin to animals causes ER stress. Tunicamycin inhibits the N-acetylglucosamine transferase enzyme in eukaryotic cells. Inhibition of N-acetylglucosamine transferase inhibits the formation of N-acetylglucosamine lipid metabolites and the glycosylation of newly synthesized proteins.

[0170] In the endoplasmic reticulum (ER), proteins synthesized on ribosomes undergo higher-order conformation and post-translational modifications, leading to glycoprotein folding and assembly. Inhibition of glycoprotein production by tunicamycin prevents protein maturation. The maturation of disrupted proteins results in the production of misfolded or unfolded proteins. Misfolded or unfolded proteins fail to assemble properly and accumulate in the ER, causing ER stress. ER stress leads to cell death.

[0171] To investigate the efficacy of KARC in inhibiting endoplasmic reticulum (ER) stress-mediated acute hepatic steatosis, we analyzed the effects of KARC and the fat-related morphology of liver tissue in an animal model in which ER stress was induced.

[0172] Specifically, C57BL / 6J male mice (Jungang Laboratories, Korea), a model animal for ER stress induction, were housed in a facility at 23°C and 60-70% humidity under a 12-hour (light) and 12-hour (dark) light-dark cycle (light from 6:00 AM to 6:00 PM, dark from 6:00 PM to 6:00 AM). Mice were allowed free access to water in a germ-free facility.

[0173] The liver sample size of the experimental animals was estimated based on the analysis. ER stress was induced by intraperitoneally administering 2 mg / kg of tunicamycin to mice, and the effect of KARC was tested at intervals of 24 and 48 hours.

[0174] KARC was prepared from the dried powder or lysate of Saccharomyces celloviciae KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, or PicoYP-02. The prepared KARC contains glutathione, aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), and the coenzymes NAD and NADP.

[0175] The mice were anesthetized with isoflurane (Gyeonggi Hana Pharmaceutical) and sacrificed at 9:00 a.m. To analyze the morphology of the liver tissue, H&E staining was used to confirm its structure. H&E tissue staining was performed.

[0176] Lipids from liver tissue were extracted using methanol and chloroform according to the Folch method. Total cholesterol levels in liver tissue were quantified using a total cholesterol assay kit (AM 202-K, Asan Pharmaceuticals), and the absorbance of samples and standards was measured at 500 nm.

[0177] Triglyceride levels in liver tissue were quantified using a triglyceride assay kit (AM 157S-K, Asan Pharmaceuticals, Seoul, Korea). Quantified liver tissue triglyceride levels were measured by measuring the absorbance of samples and standards at 550 nm using an Infinite 200 PRO (Tecan Trading AG, Switzerland).

[0178] Tunicamycin induced the appearance of fatty liver in liver tissue [Figure 1].

[0179] In the tunicamycin-injected group, the disappearance of fatty liver persisted, and the liver remained discolored 24 and 48 hours after injection. In the groups administered 10 units / kg and 20 units / kg of KARC, the higher the KARC concentration, the higher the rate of recovery from fatty liver.

[0180] Tissue morphology analysis by H&E staining also showed that the amount of fat in liver tissue was lower in the KARC-treated group compared with the vehicle group [Figure 2].

[0181] The triglyceride [Figure 3] and total cholesterol [Figure 4] contents in liver tissue were determined after ER stress induction. In the livers of mice with ER stress induction, triglycerides and total cholesterol in liver tissue increased. In the livers of mice administered KARC, both triglycerides and total cholesterol in liver tissue decreased. KARC, a lysate of Saccharomyces celloviciae, was effective in treating fatty liver.

[0182] [Example 10] Changes in ER stress-related factors following KARC administration Tunicamycin prevents the transfer of N-acetylglucosamine 1-phosphate to the dolichol phosphate site of proteins, thereby inhibiting sugar binding to the NH3 of asparagine, a protein building block.

[0183] Tunicamycin inhibits the synthesis of glycosylated proteins by preventing cell cycle entry into the S phase and inhibits DNA synthesis by prolonging the G1 phase. ER stress-induced model animals were prepared by injecting tunicamycin into normal mouse models. The effects of KARC, a mutant yeast lysate, were investigated by administering 20 units / kg of KARC to the ER stress-induced model animals for 24 or 48 hours.

[0184] Immediately after tunicamycin or KARC administration, factors associated with acute hepatic steatosis were analyzed in the liver tissue of mice.

[0185] To investigate the effect of inhibiting acute fatty liver formation, we performed a detailed analysis of factors related to endoplasmic reticulum stress, inflammation, lipid synthesis, and fatty acid oxidation in the liver tissue of mice injected with tunicamycin or KARC.

[0186] The mRNA and protein expression levels of ER stress-related genes Chop [Figure 5], Grp78 [Figure 6], Ire-1α [Figure 7], Gadd34 [Figure 8], and Atf4 [Figure 9] were measured in the liver tissue of ER stress-induced mice. The expression levels of inflammatory genes F4 / 80 [Figure 10], McP1 [Figure 11], TNF-α [Figure 12], and Il-6 [Figure 13] were measured in the liver tissue of ER stress-induced mice. The expression levels of fatty acid oxidation-related genes Ppar-α [Figure 14], Pgc-1α [Figure 15], Cpt-1α [Figure 16], and Fgf21 [Figure 17] were measured.

[0187] Total RNA was prepared from liver tissue of mice in which ER stress had been induced by the TriZol procedure (Invitrogen). Using the total RNA as a template, cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA).

[0188] To measure the mRNA expression levels of various genes, qPCR was performed on a CFX96™ Real-Time PCR System (Bio-Rad Laboratories).

[0189] The mRNA levels were normalized to the expression of ribosomal protein L32 by calculating the delta-delta threshold cycle method. The primer sequences of the genes used for qPCR are listed in Table 7. Western blot analysis was performed by extracting protein samples from the liver tissues of ER stress-induced mice.

[0190] Liver tissue lysates (extracts or digests) were extracted using Tissue Protein Extraction Reagent (T-PER; Thermo Scientific, Rockford, IL, USA) supplemented with protease and phosphatase inhibitors (Thermo Scientific). Proteins in the liver tissue lysates were diluted with 5X sample buffer (EBA-1052, ELPIS BIOTECH, Seoul, Korea) and heated at 95°C for 5 min. Proteins were separated by 5-15% Tris-HCl SDS / PAGE gel electrophoresis and transferred to a nitrocellulose membrane (GE Healthcare, Uppsala, Sweden).

[0191] All immunoblots were probed with enhanced chemiluminescence HRP-conjugated secondary antibodies (Clarity Western ECL Substrate, Bio-Rad Laboratories), and protein bands were detected with a chemiluminescence imaging system (Fusion Fx, Vilber Lourmat, Eberhardzell, Germany).

[0192] The expression of ER stress-related genes was analyzed by qPCR in the liver tissue of KARC-treated mice after ER stress induction. As shown in Figures 5, 6, 7, 8, and 9, all KARC-treated groups showed improved expression of ER stress-related genes, and their expression increased upon ER stress induction. It is known that mice with ER stress induced increased the expression of inflammatory genes in the liver tissue.

[0193] The expression of marker genes related to inflammatory genes was analyzed. As shown in Figures 10, 11, 12, and 13, the expression of all gene markers was reduced by KARC.

[0194] The expression of genes related to fatty acid oxidation was analyzed. As shown in Figures 14, 15, 16, and 17, in the KARC-treated group, fatty acid oxidation was improved due to the reduction in ER stress.

[0195] When analyzing the expression of proteins related to ER stress, the expression of CHOP, Ire-1α, and p-eIF2α was reduced in all cases of KARC administration. The expression of proteins related to lipid metabolism and fatty acid oxidation was also analyzed. In all cases, KARC administration improved ER stress and reduced the expression of proteins involved in lipid metabolism and fatty acid oxidation [Figure 18].

[0196] [Table 7] TIFF2026500116000011.tif67152

[0197] [Example 11] Effect of reducing oxidative stress Reactive oxygen species, or oxidative stress, increases during alcohol consumption due to excess acetaldehyde (Ach) produced by alcohol dehydrogenase (ADH). Aldehyde dehydrogenase (ALDH) converts it to acetic acid, which is then excreted from the body. Genetic mutations in aldehyde dehydrogenase, or excess aldehydes resulting from excessive alcohol consumption, can lead to fat peroxidation.

[0198] The resulting acetaldehyde and malondialdehyde exacerbate oxidative stress and disrupt mitochondrial energy metabolism. Endoplasmic reticulum stress is induced through the accumulation of unfolded proteins in cells, leading to cell death.

[0199] Blood acetaldehyde concentrations were measured over time following alcohol consumption (Figure 31). The area under the curve (AUC) of blood acetaldehyde (Ach) was 13.02 ± 1.18 mg-h / dL following alcohol consumption alone. When administered at a dose of 10 units / kg KARC, the area under the curve (AUC) of blood acetaldehyde (Ach) was significantly reduced by 26.13% to 9.39 ± 1.07 mg-h / dL compared to alcohol consumption alone (P = 0.005).

[0200] At a dose of 20 units / kg, the AUC of blood acetaldehyde (Ach) was significantly reduced by 55.71% to 5.22 ± 0.99 mg-h / dL compared with alcohol consumption alone (P < 0.001). When comparing the 10 unit / kg KARC group with the 20 unit / kg KARC group, blood acetaldehyde (Ach) was significantly reduced in the 20 unit / kg KARC group (P = 0.034). KARC demonstrated a dose-dependent reduction in total blood acetaldehyde (Ach) over time.

[0201] The reduction in blood acetaldehyde (Ach) levels resulting from KARC administration has a positive impact on reducing oxidative stress and promoting health.

[0202] The blood malondialdehyde (MDA) concentration was measured during chemotherapy [Figure 32]. The blood MDA concentration in the control group was 0.607±0.161μM. The group treated with KARC showed a significant 63.3% reduction in blood MDA concentration compared to the control group, measuring 0.223±0.033μM (P<0.001).

[0203] In the control group, blood MDA levels varied considerably, ranging from 0.427 μM to 0.885 μM. In the KARC-treated group, this range was significantly reduced, ranging from 0.158 μM to 0.269 μM. This confirmed the effect of not only reducing but also stabilizing blood MDA levels, as demonstrated in Figure 33.

[0204] Various factors, such as drug intake, stress, and strenuous exercise, lead to an increase in intracellular reactive oxygen species, which trigger lipid peroxidation and oxidation processes in endogenous amines such as dopamine, norepinephrine, serotonin, and histamine. Reactive aldehyde compounds, including 4-hydroxynonenal (HNE), malondialdehyde (MDA), acetaldehyde (Ach), and dopamine-derived aldehydes, accumulate intracellularly and exacerbate oxidative stress.

[0205] These aldehydes then react with surrounding proteins and undergo secondary metabolic processes to form stable end products, such as malondialdehyde-acetaldehyde adducts (MAA) and malondialdehyde-lysine adducts (M-lys adducts), known as advanced lipid peroxidation end products. The accumulation of these products exerts toxic effects on various cells, further enhancing oxidative stress.

[0206] This cumulative oxidative stress disrupts intracellular mitochondrial energy metabolism, leading to the accumulation of aldehyde intermediates in aldehyde-based glucose metabolism, including methylglyoxal (MG) and glyceraldehyde-3-phosphate (GA3P). Aldehyde-related chain reactions result in the accumulation of stable end glycoxidation products, known as advanced glycation end products (AGEs), which weaken intracellular antioxidant defense systems such as glutathione (GSH). These processes increase endoplasmic reticulum (ER) stress, leading to increased cell apoptosis in neuronal cells.

[0207] Increased reactive oxygen species and oxidative stress are associated with increased levels of reactive aldehydes, such as HNE and MDA, as well as modified proteins, such as advanced glycation end products (AGEs) and advanced lipid peroxidation end products (ALEs). This chain of events, which reinforces and amplifies each other, is known to increase endoplasmic reticulum stress (ER stress).

[0208] KARC administration effectively modulated malondialdehyde, a marker of reactive oxygen species and oxidative stress, demonstrating its potential to reduce oxidative stress and improve endoplasmic reticulum (ER) stress homeostasis. KARC significantly reduced malondialdehyde levels in the bloodstream, demonstrating its ability to reduce reactive oxygen species and oxidative stress.

[0209] By reducing the levels of acetaldehyde and malondialdehyde in human blood, KARC demonstrated its potential to prevent and correct ER stress via reducing ROS and oxidative stress.

[0210] [Example 12] Acute oral administration test 12-1. Preparation of experimental animals The experimental animals were female and male ICR mice (7 weeks old). The received ICR mice were acclimated for 7 days. During the acclimation period, the adopted mice were observed for general symptoms, and only healthy animals were used for the short-term administration toxicity test. Food and water were consumed ad libitum. Based on the average body weight of approximately 20 g on the day before oral administration, the groups were divided into 10 groups, with 5 mice in each group and 5 mice in each group.

[0211] 12-2. Administration of test substance The test substances were dissolved in physiological saline so that the doses administered to the experimental animals were 0, 750, 3,000, and 5,000 mg / kg, respectively, based on the content of KARC, the mutant yeast lysate of the present invention.

[0212] The dosage standards for the administration were in accordance with the toxicity test manual of the Korea National Toxicology Program (KNTP) of the Ministry of Food and Drug Safety. The maximum application dose of 5,000 mg / kg as instructed by the KNTP manual was set as the maximum concentration for this experiment. The samples prepared for each group were orally administered once to each test animal. Physiological saline was administered to the normal group (G1).

[0213] 12-3. Observation and autopsy All animals in the test groups were observed for symptoms at least once a day from the day of acquisition until the day of autopsy. Symptoms were observed for 7 days after oral administration. After observing the symptoms of the rats, autopsies were performed. During autopsy, changes in each organ were observed with the naked eye.

[0214] A single-dose toxicity test of the KARC composition of the present invention containing ALDH was conducted using mice. As a result, no cases of mouse death were observed at concentrations of up to 5,000 mg / kg of mutant yeast KARC for 7 days. No abnormal characteristics such as weight gain or changes in food intake were found in the mice. No abnormal findings were found in the autopsy results performed after the end of the observation period.

[0215] [Example 13] Preparation of food and pharmaceutical compositions for preventing and improving fatty liver disease and liver dysfunction by detoxifying endogenous acetaldehyde

[0216] [1] Food and pharmaceutical compositions containing KARC as an active ingredient for preventing and improving fatty liver disease and liver dysfunction have been prepared. It is possible to prepare food or pharmaceutical compositions containing KARC powder with various composition ratios. For example, the powder composition according to the present invention has the function of preventing liver dysfunction and improving liver function by taking 13 g of the composition twice a day. The weight ratios of the components and phases of the food or pharmaceutical composition containing the powder composition are shown in Table 8.

[0217] [Table 8] [Industrial Applicability]

[0218] Industrial Applicability In food and pharmaceutical compositions, KARC dry powder, excipients, and natural sweeteners such as fructooligosaccharides, enzyme-treated stevia (Stevia), anhydrous citric acid, iso-isotodextrin (Isomalt), and xylitol, citrus juice powder, and citrus flavor powder are added. The processing and testing of the raw materials and final products of the food or pharmaceutical compositions were performed in accordance with the general testing methods and the Health Functional Food Act as described in the Korean Food Standards.

[0219] A food or pharmaceutical composition containing KARC can prevent and improve the deterioration of liver function.

[0220] The above examples have provided a detailed description of KARC, a mutant yeast composition containing aldehyde dehydrogenase, including: therapeutically effective doses in disease models, methods of manufacture, pharmacological effects, acute oral toxicity, and representative examples of food or pharmaceutical compositions. The efficacy of KARC has been described in detail through the above examples, but these are merely illustrative of the present invention.

[0221] Those skilled in the art can easily derive various modifications and other aspects equivalent to the present invention from the above-described aspects of the present invention.

[0222] Even foods or therapeutic agents containing modified forms of aldehyde dehydrogenase that embody the technical idea of ​​the present invention as described in the claims fall within the scope of legal protection of the present invention.

[0223] TIFF2026500116000013.tif186124

[0224] TIFF2026500116000014.tif185123

[0225] TIFF2026500116000015.tif188125

[0226] TIFF2026500116000016.tif185127

[0227] TIFF2026500116000017.tif185123

[0228] TIFF2026500116000018.tif188122

Claims

1. A composition for alleviating endoplasmic reticulum stress, comprising any one selected from the group consisting of Saccharomyces cellovisiee KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP, or a mixture thereof.

2. A food composition for inhibiting fatty liver disease, comprising any one selected from the group consisting of Saccharomyces cellovisiee KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP, or a mixture thereof.

3. A pharmaceutical composition for preventing or treating symptoms of fatty liver, comprising any one selected from the group consisting of Saccharomyces cellovisiee KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP, or a mixture thereof.

4. A food composition for preventing hepatitis, comprising any one selected from the group consisting of Saccharomyces cellovisie KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP, or a mixture thereof.

5. A composition for treating hepatitis, comprising any one selected from the group consisting of Saccharomyces cellovisiee KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP, or a mixture thereof.

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