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

Abstract

The present invention relates to food and pharmaceutical compositions for relieving endoplasmic reticulum stress to suppress or prevent various diseases caused by endoplasmic reticulum stress. More specifically, the present invention relates to a food and pharmaceutical composition for preventing or treating fatty liver symptoms or hepatitis, comprising a lysate of any one selected from the group consisting of Saccharomyces cerevisiae KCTC 13925BP, KCTC 14122BP, KCTC 14123BP, KCTC 14983BP, KCTC 14984BP, and KCTC 14985BP, or a mixture thereof.

Description

Technical Field

[0001] The present invention relates to a food and a pharmaceutical composition for relieving endoplasmic reticulum stress, thereby suppressing or preventing various diseases caused by endoplasmic reticulum stress. The present invention relates to a food and a pharmaceutical composition for inhibiting or preventing fatty liver or hepatitis.

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

[0003] The present invention relates to a food and a pharmaceutical composition for preventing or treating fatty liver symptoms or hepatitis, comprising a lysate of any one selected from the group consisting of saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, or a mixture thereof. Background Art

[0004] Causes of fatty liver disease include alcohol consumption and obesity. Symptoms of fatty liver disease may also occur in patients with hyperlipidemia or diabetes, who have high blood lipid levels. Symptoms of fatty liver disease may be caused by an increase in toxic aldehydes due to oxidative stress, as well as medications that cause endoplasmic reticulum stress, corticosteroids, and estrogen.

[0005] When drinking alcohol, endoplasmic reticulum stress (ER stress) in the liver increases, which promotes fat synthesis, causing fat accumulation in liver tissue and hindering normal energy metabolism.

[0006] Symptoms of non-alcoholic fatty liver disease may occur due to factors other than alcohol consumption. Various medications are being developed to alleviate the symptoms of fatty liver disease, but to date, no medication has been definitively proven to be effective in treating fatty liver disease.

[0007] Steatohepatitis is an inflammatory condition associated with lipid accumulation in the liver and hepatocellular necrosis caused by insulin resistance and obesity. Steatohepatitis can progress to chronic hepatitis and cirrhosis.

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

[0009] There are two types of ER: the rough ER (rER), which has a rough surface due to the presence of many ribosomes that synthesize proteins, and the smooth ER (sER), which has a smooth surface due to the absence of ribosomes.

[0010] The smooth endoplasmic reticulum (sER) is where membrane lipids and steroid hormones are synthesized, detoxification occurs, and lipid, phospholipid, and steroid synthesis occurs. In the sER, glucose-6-phosphate is dephosphorylated and separated into glucose for secretion. It also plays a role in detoxifying organic substances, including alcohols, by oxidizing them. The sER is abundant in liver cells, muscle cells, and cells that produce steroids and fat.

[0011] Ribosomes are attached to the surface of the rough endoplasmic reticulum (rER) and are responsible for the translation of mRNA during protein synthesis. Within the membrane-enclosed compartments of the ER, protein folding and maturation, as well as modifications that create protein secondary structure, occur. The ER is home to chaperone proteins and enzymes that facilitate protein maturation and modification.

[0012] Among proteins synthesized by ribosomes, those with ER signal peptides at their N-termini enter the endoplasmic reticulum. At this time, lipid-soluble membrane proteins remain in the endoplasmic reticulum membrane, while water-soluble proteins enter the endoplasmic reticulum lumen.

[0013] Among proteins synthesized by ribosomes, unfolded proteins (UPs), which remain unconverted into their unique secondary structures, accumulate in the endoplasmic reticulum, leading to increased endoplasmic reticulum stress (ER stress). Furthermore, if abnormally folded proteins (MPs) are not removed and accumulate in the endoplasmic reticulum, ER stress increases.

[0014] The phenomenon of restoring or decomposing and removing unfolded and abnormal proteins that cause ER stress is called the unfolded protein response (UPR).

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

[0016] Although ER stress is alleviated by the unfolded protein response, when homeostasis cannot be maintained smoothly, cells enter the apoptotic pathway. Cell death due to increased ER stress or disruption of ER homeostasis may lead to metabolic diseases such as viral infections, obesity, diabetes, neurodegenerative diseases (including dementia), and cause fatty liver, cirrhosis, and cancer.

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

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

[0019] This increase in oxidative stress affects the energy metabolism process in mitochondria, increasing the amount of aldehydes such as methylglyoxal and advanced glycation end products (AGEs) in the cells. As a result, the disorder of cellular energy metabolism becomes more serious.

[0020] As a result, increased oxygen and oxidative stress lead to excessive accumulation of reactive aldehydes (such as HNE and MDA) generated by lipid peroxidation, as well as aldehyde substances such as the glycolytic intermediate glyceraldehyde-3-phosphate, in cells. The accumulation of these substances leads to cytotoxicity.

[0021] The accumulation of reactive oxygen species or reactive aldehydes in cells weakens the cell's antioxidant defense system (such as glutathione), ultimately leading to increased endoplasmic reticulum stress (ER stress) through energy metabolism disorders and accumulation of unfolded proteins.

[0022] When stress occurs in the ER of hepatocytes, the smooth ER becomes activated and fat accumulates in the liver, causing symptoms of fatty liver and the development of steatohepatitis.

[0023] There is an urgent need to develop food or pharmaceutical compositions that suppress endoplasmic reticulum stress and fat accumulation in the liver, thereby preventing acute liver injury and steatohepatitis.

[0024] [Prior art literature]

[0025] [Patent Document]

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

[0027] [Non-patent literature]

[0028] Lin et al. "Endoplasmic reticulum Stress in Disease Pathogenesis". AnnuRev Pathol. (2008); 3: 399-425

[0029] Sicari et al. "A guide to assessing endoplasmic reticulum homeostasis and stress in mammalian systems". The FEBS Journal 287. (2020); 27-42.

[0030] Ozcan and Tabas et al.″Role of Endoplasmic Reticulum Stress inMetabolic Disease and Other Disorders".Annu Rev Med.(2012);63:317-328.

[0031] Malhi et al.″Endoplasmic reticulum stress in liver disease".Jounal ofhepatology(2011);vol 54:795-809.

[0032] Bhattaral et al.″The aftermath of the interplay between thcendoplasmic reticulum stress response and redox signaling",Experimental&Molecular Medicine(2021);53:151-167.

[0033] Maamoun et al.″Crosstalk Between Oxidative Stress and Endoplasmicreticulum(ER)Stress in Endothelial Dysfunction and Aberrant AngiogenesisAssociated With Diabetes:A Focus on the Protective Roles of Heme Oxygenase(HO)-1″.Frontiers in Physiology Vol10,(2019);Article70.

[0034] Liao et al.″Aldehyde Dehydrogenase-2Deficiency Aggravates CardiacDysfunction Elicited by Endoplasmic Reticulum Stress Induction″.Mol Med.(2012);18:785-793.

[0035] Ramana et al., "Lipid Peroxidation: Production. Metabolism, and Signaling Mechanisms of Malondialdehyde and 4-Hydroxy-2-Nonenal". Oxidative Medicine and Cellular Longevity Vol 2014; Article ID 360438: 31.

[0036] Console et al.. "The Link Between the Mitochondrial Fatty AcidOxidation De-rangement and Kidney Injury", Front. Physiol. (2020); 11: 794. Summary of the Invention Technical issues

[0037] Despite many of these previous studies, to date, no food composition or pharmaceutical composition has been developed that inhibits or treats fatty liver or steatohepatitis by suppressing the stress applied to the endoplasmic reticulum of hepatocytes.

[0038] The basic object of the present invention is to provide a composition derived from mutant yeast, which blocks the occurrence of endoplasmic reticulum stress by pre-eliminating factors causing endoplasmic reticulum stress, and the composition comprises aldehyde dehydrogenase, which rapidly decomposes endogenous aldehydes that cause endoplasmic reticulum stress.

[0039] Another object of the present invention is to provide a food composition and a pharmaceutical composition comprising the mutant yeast, which reduce the likelihood of endoplasmic reticulum stress and inhibit or prevent the occurrence of fatty liver or steatohepatitis.

[0040] Another object of the present invention is to provide a food composition and a pharmaceutical composition for preventing and treating fatty liver, which comprises any one selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, KCTC14985BP or a mixture thereof.

[0041] Solution to the problem

[0042] The main object of the present invention as described above can be achieved by providing a composition derived from a mutant yeast comprising an aldehyde dehydrogenase that can rapidly decompose endogenous aldehydes in advance before the occurrence of endoplasmic reticulum stress.

[0043] Another object of the present invention can also be achieved by providing a food composition or a pharmaceutical composition, which comprises a lysate of any one selected from the group consisting of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP or KCTC14985BP, or a mixture thereof (hereinafter referred to as KARC).

[0044] Beneficial effects of the present invention

[0045] When the KARC of the present invention was administered to animals having fatty liver symptoms due to endoplasmic reticulum stress in hepatocytes, alleviation of fatty liver symptoms and liver inflammation was observed 24 and 48 hours after administration.

[0046] It has been confirmed that endoplasmic reticulum stress is reduced, fatty liver accumulation is reduced, and the expression of hepatic inflammatory factors is reduced in the liver of animals administered with the KARC of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Endoplasmic reticulum stress was induced in liver tissue by administering 2 mg / kg tunicamycin (Tm) to mice. Figure 1 and Figure 2 The effect of reducing fatty liver in mouse liver tissue 24 hours and 48 hours after administration of the composition of the present invention was shown.

[0048] [ Figure 1 ] shows the liver color of mice 24 and 48 hours after administration of tunicamycin and KARC 10 and 20 units / kg. The livers of mice induced with fatty liver disease by tunicamycin became lighter in color due to fat. The livers of mice administered KARC regained their red color.

[0049] [ Figure 2 Hematoxylin-eosin (H&E) staining showed the morphology of fat in liver tissue. Tunicamycin administration increased fat in hepatocytes, but KARC administration decreased fat in hepatocytes.

[0050] [ Figure 3 、 Figure 4 ] is a graph showing changes in lipid content in liver tissue at intervals of 24 hours and 48 hours after administration of tunicamycin and KARC to mice (n=5).

[0051] * Tunicamycin administration increased triglyceride (TG) content in liver tissue, which was significantly reduced by KARC administration [ Figure 3 Tunicamycin administration increased the total cholesterol (T-chol) content in liver tissue, which was decreased in a concentration-dependent manner by KARC administration [ Figure 4 ].

[0052] [ Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 and Figure 9 ] is a graph showing the expression of genes associated with endoplasmic reticulum stress (*p<0.05, **p<0.01) (n=5). Mice were administered tunicamycin (2 mg / kg) and KARC (10 units / kg and 20 units / kg), and mRNA was isolated from liver tissue 24 and 48 hours later. The isolated mRNA was subjected to quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR).

[0053] Figure 5 The ER stress marker Chop shown in the figure represents a C / EBP homolog. ER stress is altered by regulating the expression levels of proteins involved in inducing apoptosis, such as decreased BCL-2 and increased BIM levels due to increased ER stress in cells. The decreased Chop mRNA expression level caused by KARC administration suggests that ER stress is reduced by removing abnormal proteins in the ER that promote cell death. Figure 5 ].

[0054] Grp78 stands for glucose-regulated protein 78. This is one of the representative chaperone proteins that has the function of maintaining and regulating endoplasmic reticulum homeostasis. The reduction in Grp78 mRNA expression levels caused by KARC administration means that by regulating ATF6 and IRE-1α, the accumulation of unfolded proteins is reduced, resulting in the alleviation of endoplasmic reticulum stress. Figure 6 ].

[0055] IRE-1α stands for inositol-requiring enzyme 1α. It is a representative indicator of the unfolded protein response (UPR). IRE-1α induces abnormally produced and accumulated unfolded proteins to reform into normal structures through the secretion of chaperone proteins, or removes proteins destined for the endoplasmic reticulum at the mRNA stage (RIDD, regulated IRE-1α-dependent degradation). Furthermore, IRE-1α activates JNK and NF-κB proteins to degrade unfolded proteins by increasing autophagy or inducing apoptosis, thereby alleviating ER stress and maintaining homeostasis.

[0056] The reduction in IRE-1α mRNA expression levels caused by KARC administration means that unfolded proteins, which act as cytotoxins, are reduced and ER stress is alleviated [ Figure 7 ].

[0057] [ Figure 8Gadd34 in [ ] stands for growth arrest and DNA damage inducible 34. Gadd34 is a protein that, in combination with the transcription factor ATF4, induces dephosphorylation of eLF2α. Dephosphorylation of eLF2α occurs and promotes apoptosis. Gadd34 removes unfolded proteins by promoting apoptosis caused by insufficient eLF2α dephosphorylation or by promoting the translation of abnormal proteins.

[0058] [ Figure 9 ] shows ATF4. ATF4 stands for activating transcription factor 4. Reduced ATF4 expression levels indicate relief of ER stress. Figure 8 and Figure 9 In the 247 cells, the decreased mRNA expression levels of ATF4 or Gadd34 due to KARC administration indicated reduced ER stress and a decreased number of dying cells.

[0059] After 2 mg / kg of tunicamycin was administered to mice, 10 units / kg and 20 units / kg of KARC were administered, and mRNA was isolated from the liver tissues of the mice 24 and 48 hours later. Figure 10 、 Figure 11 、 Figure 12 and Figure 13 ] Expression of inflammatory genes was shown by RT-qPCR of isolated mRNA (*p<0.05, **p<0.01) (n=5).

[0060] [ Figure 10 F4 / 80 is a unique marker for macrophages. It is present in phagocytic macrophages and plays a role in expressing inflammatory genes. The reduction in F4 / 80 mRNA expression levels after KARC administration indicates a reduction in cellular inflammation.

[0061] [ Figure 11 Mcp1 in the figure stands for monocyte chemoattractant protein-1. This is a type of chemokine expressed in a variety of cells. Mcp1 is an inflammation-related substance that causes T cells, monocytes, and other cells to gather around inflamed areas and move more easily.

[0062] The decreased Mcp-1 mRNA expression level caused by KARC administration indicates a decrease in inflammation and inflammatory response.

[0063] [ Figure 12TNF-α in this study stands for tumor necrosis factor-α, a cytokine produced by inflammation and increased inflammatory responses. This finding confirms an increase in phagocytosis. The decrease in TNF-α mRNA expression following KARC administration suggests a reduction in inflammatory substances or a weakened inflammatory response.

[0064] [ Figure 13 The IL-6 in the molecule stands for interleukin-6, which causes inflammation by expressing inflammatory genes. The reduction in IL-6 mRNA expression levels following KARC administration indicates a reduction in inflammation.

[0065] Mice were administered 2 mg / kg tunicamycin followed by 10 and 20 units / kg of KARC, and mRNA was isolated from liver tissue 24 and 48 hours later. RT-qPCR was performed on the isolated mRNA. Expression of genes related to fatty acid oxidation was Figure 14 、 Figure 15 、 Figure 16 and Figure 17 Shown in (*p<0.05, **p<0.01) (n=5).

[0066] [ Figure 14 ] Ppar-α in [ 1 ] stands for peroxisome proliferator-activated receptor α. It participates in maintaining lipid metabolism homeostasis by improving fatty acid oxidation (β-oxidation) in muscle and liver. KARC administration increased the mRNA expression level of Ppar-α. Fat metabolism improved, low-density triglycerides decreased, and high-density lipoprotein cholesterol (HDL-cholesterol) increased. This suggests that KARC administration restored the liver's lipid metabolism regulatory function.

[0067] [ Figure 15 Pgc-1α in [ ] stands for peroxisome proliferator-activated receptor gamma coactivator 1-α. This protein is involved in intracellular mitochondrial biogenesis and hepatic gluconeogenesis. Increased Pgc-1α mRNA expression levels following KARC administration indicate increased energy production and metabolism in the liver.

[0068] [ Figure 16The Cpt-1α in this study stands for carnitine palmitoyltransferase 1α. It is located in the mitochondrial lipid membrane and is an essential protein involved in fatty acid oxidation (β-oxidation). This protein converts acyl groups in fatty acid chains into acylcarnitines, allowing long-chain fatty acids to cross the mitochondrial membrane. Increased Cpt-1α mRNA expression levels following KARC administration indicate improved energy production and metabolism through fatty acid oxidation (β-oxidation) in the liver.

[0069] [ Figure 17 The Fgf21 in the text stands for fibroblast growth factor 21. It is a fibroblast growth factor that is overproduced in the liver, pancreas, and adipose tissue. Fgf21 expression is regulated by Ppar-γ (peroxisome proliferator-activated receptor gamma) and Ppar-α (peroxisome proliferator-activated receptor alpha). It is activated in response to increased stress to either adapt or defend against it. The reduction in Fgf21 mRNA expression levels after KARC administration indicates a reduction in hepatocyte stress.

[0070] After administering 2 mg / kg of tunicamycin to the mouse model, 10 and 20 units / kg of KARC were administered. 24 and 48 hours later, proteins isolated from liver tissue were subjected to Western blotting. Figure 18 The expression of proteins related to endoplasmic reticulum stress, lipid metabolism and fatty acid oxidation is shown. The expression levels of CHOP, IRE-1α, p-eIF2α, eIF2α, FAS, ACC1, Scd-1, PPARα and CPT1 proteins were measured (n=5).

[0071] Figure 18 The ER stress markers CHOP and IRE-1α were increased by tunicamycin administration, as shown in the gene expression results [ Figure 5 and Figure 7 In the group administered with KARC 20 units / kg, protein expression was decreased, indicating a reduction in endoplasmic reticulum stress.

[0072] [ Figure 18 The p-eIF2α in [ ] stands for 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. It regulates protein synthesis and also affects memory.

[0073] When cells experience ER stress due to accumulation of unfolded proteins (UP) or misfolded proteins (MP) in the ER due to heat, ultraviolet light, external infection, etc., the phosphatase of p-eIF2α is activated.

[0074] Overexpression of this eIF2 protein reduces the total amount of protein, thereby reducing ER stress in cells. The decrease in eIF2 after KARC administration indicates a reduction in ER stress.

[0075] [ Figure 18 FAS in [the context of] is fatty acid synthase. This is an essential enzyme system involved in lipid biosynthesis (de novo lipogenesis), which is required for the energy required for cell growth and cell signaling. The reduction of FAS protein due to endoplasmic reticulum stress indicates a reduction in energy metabolism through fatty acid oxidation. The increase in FAS protein caused by KARC administration indicates that energy metabolism through fatty acid oxidation has been restored.

[0076] [ Figure 18 ACC1 in this context stands for acetyl-CoA carboxylase 1 (ACC1). This is an enzyme that produces a metabolite called malonyl-CoA, which is involved in the formation of fatty acids and the burning of fat. ACC1 expression is regulated by AMPK (AMP-activated protein kinase), which regulates the energy metabolism of carbohydrates and fats.

[0077] KARC administration restored ACC1 protein expression, which was reduced due to ER stress, suggesting that the liver's energy conversion capacity through fat oxidation was improved.

[0078] [ Figure 18 ] stands for stearoyl-CoAdesaturase-1 (Scd-1). Scd-1 is an enzyme that is mainly present in the endoplasmic reticulum of the liver. It is an enzyme that converts saturated fatty acids (such as stearic acid) and CoA into unsaturated fatty acids (such as oleic acid), which are one of the main components of cell lipid membranes. Its expression is regulated by AMPK (AMP-activated protein kinase). The decrease in Scd-1 protein expression caused by endoplasmic reticulum stress was restored by KARC administration. This indicates that lipid metabolism control is improved and liver cells are restored. As shown in [ Figure 14 and Figure 16 ] explained in Figure 18 Tunicamycin reduced the expression of PPARα and CTP1, markers of fatty acid oxidation, in the liver. KARC administration increased the expression of these proteins, suggesting improved energy production and metabolism through fatty acid oxidation in the liver.

[0079] exist[ Figure 18In [text], GAPDH represents "glyceraldehyde-3-phosphate dehydrogenase (GAPDH)". Since it is stably expressed in cells and its expression level is not easily affected by cell state, it is used as a housekeeping gene. This is used as a reference marker for protein quantification and shows that the same amount of protein is used.

[0080] Figure 19 shows the change in enzyme activity when the KwonP-1 strain is administered orally.

[0081] Figure 20 shows the change in enzyme activity when the KwonP-2 strain is administered orally.

[0082] Figure 21 shows the change in enzyme activity when the KwonP-3 strain is administered orally.

[0083] Figure 22 shows the change in enzyme activity when the PicoYP strain is administered orally.

[0084] Figure 23 shows the change in enzyme activity when the PicoYP-01 strain is administered orally.

[0085] Figure 24 shows the change in enzyme activity when the PicoYP-02 strain is administered orally.

[0086] In Figure 19 、 Figure 20 、 Figure 21 、 Figure 22 、 Figure 23 、 Figure 24 , KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01 and PicoYP-02 are orally administered for 90 minutes under conditions similar to the digestion process of the human stomach (1 < pH < 5). The change in ALDH enzyme activity is measured. At pH = 5 (similar to the conditions observed during food intake), the ALDH enzyme activity remains at a minimum of 37.29 units / g and a maximum of 52.24%. It is confirmed that the enzyme activity is maintained when KARC is orally administered.

[0087] Figure 25 shows the growth curve and enzyme activity when the KwonP-1 strain is cultured in a 5 L fermenter.

[0088] Figure 26 shows the growth curve and enzyme activity when the KwonP-2 strain is cultured in a 5 L fermenter.

[0089] Figure 27 ​​​​​​​​​] shows the growth curve and enzyme activity of KwonP-3 strain when it was cultured in a 5 L fermentor.

[0090] [ Figure 28 ] shows the growth curve and enzyme activity of the PicoYP strain when it was cultured in a 5 L fermentor.

[0091] [ Figure 29 ] shows the growth curve and enzyme activity of the PicoYP-01 strain when cultured in a 5 L fermentor.

[0092] [ Figure 30 ] shows the growth curve and enzyme activity of PicoYP-02 strain when cultured in a 5 L fermentor.

[0093] exist[ Figure 25 、 Figure 26 、 Figure 27 、 Figure 28 、 Figure 29 、 Figure 30 ], the new mutant strains KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-01 were cultured in 5 L fermentors using YPD medium under the same conditions. This was carried out at 30°C and 200 rpm for 48 hours. When the growth curve (OD660nm) and ALDH enzyme activity of each strain were compared with the model strain, the ALDH enzyme activity was at least 10.5 times and at most 18.75 times higher. PicoYP-01 had the highest ALDH activity at 52.68 units / g, and KwonP-3 had the lowest value at 29.5 units / g.

[0094] [ Figure 31 ] is a graph showing the decomposition of acetaldehyde in the human body by KARC.

[0095] [ Figure 32 ] is a graph showing the decomposition of malondialdehyde in the human body by KARC.

[0096] [ Figure 33 ] is a graph showing that KARC stabilizes malondialdehyde in the human body.

[0097] In the confirmation of reducing endogenous acetaldehyde in human blood[ Figure 31 ] and reduce blood malondialdehyde [ Figure 32 ] trial, the effects of KARC administration appeared to reduce acetaldehyde and malondialdehyde, biomarkers of hangover, fatigue, and cardiovascular disease. Figure 33 ] showed that in a state where oxidative stress is increased due to taking drugs, KARC administration can reduce oxidative stress and malondialdehyde, a biomarker of reactive oxygen species, thereby reducing oxidative stress. DETAILED DESCRIPTION

[0098] Hereinafter, the method for producing the KARC dry powder of the present invention, i.e., the lysate of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, KCTC14985BP, will be described in more detail.

[0099] These examples are only for the purpose of illustrating compositions that can achieve the purpose of the present invention, and therefore, the scope of the present invention is not limited to the compositions described in the following examples.

[0100] Example

[0101] [Example 1] Screening of wild yeast parent strains for mutation.

[0102] In the present invention, each Makgeolli (Korean traditional liquor) suspension was prepared by mixing various types of Makgeolli with a 0.9% NaCl solution. The Makgeolli suspension was stirred at 200 rpm for 1 hour. The supernatant containing the wild yeast strain was diluted with YPD (yeast extract peptone dextrose broth) medium. The diluted solution was prepared to be 10% of the original solution. -6 times.

[0103] The diluted solution was spread onto YPD agar medium. The agar medium was incubated statically at 30°C under aerobic conditions for one week. Preliminary screening of Saccharomyces cerevisiae was performed based on colony morphology, growth characteristics in YPD agar, and microscopic observation.

[0104] The ALDH activity and glutathione content of the screened Saccharomyces cerevisiae were measured. Parent strains were selected based on ALDH activity and glutathione production.

[0105] 1-1: Measurement of aldehyde dehydrogenase

[0106] Acetaldehyde reacts with dinitrophenylhydrazine (DNPH) to form an acetaldehyde-hydrazone (Ach-DNPH) compound. The Ach-DNPH compound is detected by HPLC equipped with a C18 column at 360 nm. The amount of Ach-DNPH compound detected quantifies the amount of aldehyde reduced by the decomposition reaction by aldehyde dehydrogenase (ALDH).

[0107] The enzyme reaction was performed at 30°C by adding 10 μL of yeast lysate to 990 μL of a reaction mixture [50 mM potassium phosphate buffer (pH 8.0), 1.5 mM acetaldehyde, and 3 mM NADP+]. After the enzyme reaction was complete, 50 μL of 10 mM DNPH was added to induce the formation of Ach-DNPH. Ach-DNPH formation was performed at 22°C for 1 hour.

[0108] The formation of Ach-DNPH was terminated by adding 3M sodium acetate (pH 9). The formed Ach-DNPH compound was isolated by adding two volumes of acetonitrile. The isolated Ach-DNPH compound (in ACN) was analyzed by injection HPLC.

[0109] The concentration of Ach-DNPH compounds was analyzed by HPLC at a wavelength of 360 nm using a mobile phase (acetonitrile, water) developed on a C18 column at a rate of 1 mL / min. The area values of the chromatograms obtained from the HPLC results were converted using a standard curve for aldehyde-DNPH (Sigma-Aldrich) to quantify the concentration of Ach-DNPH compounds. A decrease of 1 mM in Ach-DNPH concentration per minute was calculated as 1 unit of ALDH. ALDH activity was normalized to units per mg of protein.

[0110] 1-2: Glutathione measurement

[0111] Yeast cells were harvested by centrifuging 1 mL of Saccharomyces cerevisiae culture medium. A suspension was prepared by adding 1 mL of water to the harvested yeast cells. Glutathione was extracted by stirring the suspension at 85°C and 1,000 rpm for 2 hours. The suspension was centrifuged to remove the yeast cells, and the supernatant was filtered through a 0.22 μm filter to obtain a glutathione-containing sample.

[0112] The glutathione concentration in the sample was analyzed by HPLC (Shimazu LC-20AD) equipped with a C18 column. Glutathione concentration was analyzed at a wavelength of 210 nm under conditions of developing a mobile phase (2.02 g / L sodium 1-heptanesulfonate monohydrate, 6.8 g / L potassium dihydrogen phosphate, pH 3.0, methanol mixture) at a rate of 1 mL / min. The area value of the chromatogram obtained as the HPLC result was analyzed using a standard curve analysis of glutathione.

[0113] The ALDH activity and glutathione content of 200 different yeasts obtained from Korean Makgeolli were analyzed. The 10 yeasts listed in [Table 1] showed higher ALDH activity or glutathione production capacity than other yeasts.

[0114] Yeast #97 had an ALDH activity of 0.10 units / mg protein, the second highest overall. Yeast #97 also had a glutathione content of 0.42%, the highest of all strains. Yeast #97 was selected as the parent strain and subjected to the mutation induction procedure.

[0115] Table 1

[0116] [Example 2] Identification of parent strains used in the mutagenesis process

[0117] Identification was performed to confirm the exact species of the wild-type parent strain (yeast #97, wild-type yeast). To ensure sufficient yeast cells for DNA extraction, only a single yeast colony was plated on YPD agar medium. DNA was extracted using a genomic DNA preparation kit (HiGene™, BIOFACT Co., Ltd., Daejeon, South Korea) according to the manufacturer's instructions.

[0118] To amplify the rRNA gene in the yeast ITS region, polymerase chain reaction (PCR) was performed on yeast chromosomal DNA using ITS5 (forward) and ITS4 (reverse) primers. PCR results were analyzed by DNA sequencing.

[0119] The DNA sequence of the parent strain was isolated using the Bioedit program. The reverse strand of the PCR result was converted to a paired base sequence using the reverse completion process.

[0120] The forward strand sequence matched its reverse strand counterpart using the Cluster X program. Using the BLAST database provided by the National Center for Biotechnology Information (NCBI), the parent strain was identified as matching the sequence identified in the experimental procedure. The rRNA in the parent strain's ITS was found to be 100% identical to that of Saccharomyces cerevisiae.

[0121] [Example 3] Selection of mutants with improved aldehyde dehydrogenase production

[0122] The mutagenesis procedure for the wild-type S. cerevisiae parent strain was performed according to the methods described in US Patent Application No. 17 / 176,365.

[0123] To induce mutations in the yeast parent strain, wild yeast strains that produce both ALDH and glutathione were treated with ethyl methanesulfonate (EMS) or nitrosoguanidine (NGD). The induced mutant yeast strains were exposed to varying concentrations of methylglyoxal. Mutants with excellent adaptability to methylglyoxal were screened. The selected yeast strains were exposed to varying concentrations of lysine. Mutants with excellent adaptability to lysine were screened. 30 mutants with excellent adaptability to both methylglyoxal and lysine were obtained. Each of the 30 yeast strains was evaluated for five properties: growth curve, ALDH activity, ADH activity, coenzyme content, and glutathione content.

[0124] 3-1: Growth characteristics

[0125] Saccharomyces cerevisiae is a Crabtree-positive microorganism that produces ethanol while growing under aerobic conditions. Cultivating high-yield yeast requires Saccharomyces cerevisiae with high ethanol tolerance.

[0126] YPD medium with different ethanol concentrations (no ethanol, 5%, 7% and 10%) was prepared. Saccharomyces cerevisiae (yeast) medium adjusted to OD=1 at 660nm was prepared. Each mixture of the prepared YPD medium and yeast medium was diluted in a ratio of 99:1. Finally, YPD medium containing yeast with four different alcohol concentrations was prepared. Each YPD medium mixed with yeast was shaken at 30°C and 200rpm. The growth curve of the mutant strain was measured every 3 hours for 48 hours. The growth curve of each mutant strain was evaluated by three characteristics: the time (or period) of the lag phase, the specific growth rate (OD660nm / hr) in the exponential phase and the maximum density (OD660nm).

[0127] The higher the ethanol concentration in YPD medium, the longer the lag phase. The maximum density and specific growth rate decreased. Comparing the maximum density of mutant strains at low (5%) and high (10%) ethanol concentrations revealed that, for nine mutant strains, the high concentration maintained 50% growth compared to the low concentration. The nine mutant strains are distinguished from other strains by their short lag phase and high specific growth rate.

[0128] [Table 2]

[0129] 3-2: Activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH)

[0130] The activity of alcohol dehydrogenase (ADH) was measured by adding 10 μL of yeast lysate to 990 μL of a reaction mixture consisting of 50 mM potassium phosphate buffer (pH 8.0), 2 mM NAD+, and 1% ethanol. The activity of aldehyde dehydrogenase (ALDH) was measured by adding 10 μL of yeast lysate to 990 μL of a reaction mixture consisting of 50 mM potassium phosphate buffer (pH 8.0), 3 mM NAD+, and 1.5 mM acetaldehyde. The enzyme reaction of ADH and ALDH was carried out at 30°C for 5 minutes, and the concentration of NAD(P)H generated by the enzyme reaction was measured by absorbance at 340 nm.

[0131] The enzyme activities of the nine mutant strains (K-1 to K-9) selected in the present invention were measured. The ADH activity of the mutant strains was as low as 382.69 units / g and as high as 975.29 units / g. Compared with the model strain (reference yeast, Saccharomyces cerevisiae KCTC7296), the ADH activity of the mutant strains increased by at least 5.1 times and as much as 13.1 times. The ALDH activity of the mutant strains was as low as 15.23 units / g and as high as 72.16 units / g. Compared with the enzyme activity of the model strain, the ALDH activity of the mutant strains increased by at least 5.3 times and as much as 24.9 times.

[0132] Six mutant strains (K-1, K-4, K-6, K-7, K-8, and K-9) showed similar rates of increase in ADH and ALDH enzyme activities compared to the model strain. The ALDH enzyme activities in three mutant strains (K-2, K-3, and K-5) were 18.3-, 23.2-, and 24.9-fold higher than those in the model strain, respectively. The ADH enzyme activities in three mutant strains (K-2, K-3, and K-5) were 9.7-, 11.6-, and 13.1-fold higher than those in the model strain, respectively. The rate of increase in ALDH enzyme activity in the three mutant strains (K-2, K-3, and K-5) was twice that of ADH.

[0133] The present inventors named three new mutants (K-2, K-3, and K-5) adapted to increase aldehyde dehydrogenase (ALDH) activity PicoYP, PicoYP-01, and PicoYP-02, respectively. These three new mutants were deposited with the Bioresource Center of the Korea Institute of Bioscience and Biotechnology under the accession numbers KCTC14983BP, KCTC14984BP, and KCTC14985BP, respectively.

[0134] 3-3: Content of coenzymes (NAD and NADP)

[0135] NAD total and NADP total were measured in lysates extracted from the mutant strain using the NADH / NAD+ assay kit and the NADPH / NADP+ assay kit, respectively. NAD(P) in the sample was converted to NAD(P)H using an NAD(P) cycling buffer and an NAD(P) cycling enzyme mix. A chromogenic reaction was induced with an NAD(P) chromogen, and the absorbance at 450 nm was measured. The chromogenic reaction was measured by absorbance at 450 nm. The sample absorbance was substituted into the equation corresponding to the standard curve to calculate the NAD(P) total in the yeast lysate.

[0136] The coenzyme content of the nine mutant strains (K-1 to K-9) selected in the present invention was measured. The NADtotal of the mutant strains was as low as 126 nmol / g and as high as 195 nmol / g. Compared with the model strain, the NADtotal of the mutant strain increased by at least 7.3 times and as much as 10.8 times. The NADPtotal content of the mutant strain was as low as 2.4 nmol / g and as high as 5.8 nmol / g. Compared with the model strain, the NADPtotal content of the mutant strain increased by at least 11.4 times and as much as 27.6 times.

[0137] In the six mutants (K-1, K-4, K-6, K-7, K-8, and K-9), the rate of increase in NADPtotal was less than twice the rate of increase in NADtotal. The rates of increase in total NADP content in the three new mutants (PicoYP, PicoYP-01, and PicoYP-02) were 25.7-fold, 22.9-fold, and 27.6-fold, respectively. The rates of increase in total NAD content in the three new mutants were 10.8-fold, 9.9-fold, and 11.3-fold, respectively. The rate of increase in NADPtotal in the three new mutants was greater than twice the rate of increase in NADtotal.

[0138] 3-4: Glutathione (GSH) content

[0139] Glutathione content was measured in nine mutant strains using the same method as in Examples 1-2. Glutathione content ranged from a low of 0.85% to a high of 1.05%. Compared to the model strain, glutathione content increased by at least 2.7 times and as much as 3.3 times in the mutants. Three of the new mutants (PicoYP, PicoYP-01, and PicoYP-02) showed higher increases in ALDH activity and coenzyme content than in the other strains.

[0140] The three new mutant yeasts (PicoYP, PicoYP-01, and PicoYP-02) have similar glutathione production capabilities to existing deposited strains (Kwon P-1, Kwon P-2, and Kwon P-3). Compared to existing deposited strains, the three new mutant yeasts have significantly increased ADH and ALDH enzyme activities and coenzyme content.

[0141] [Table 3]

[0142] [Table 4]

[0143] [Example 4] Comparison of carbon source preferences

[0144] The carbon source preferences of three mutants (KwonP-1, KwonP-2, and KwonP-3) with elevated ALDH and glutathione levels were investigated, for which a domestic patent application was filed on February 18, 2020. The carbon source preferences of a reference yeast strain (KCTC7296) were measured. To determine the maximum capacity for ALDH production, the carbon source preferences of three new mutants (PicoYP, PicoYP-01, and PicoYP-02) were investigated.

[0145] The carbon source preference of the strains was analyzed for specificity and novelty using the API 50CHL kit (API Systems, BIOMERIEUX, SA, France).

[0146] Prepare 15 mL conical tubes containing 8 mL of YPD medium. Inoculate each of the seven mutant strains into the prepared conical tubes.

[0147] After incubating the inoculated conical tubes at 30°C and 200 rpm for 24 hours, each of the seven mutant strains was isolated from the exponential growth phase. To eliminate the effects of residual carbon source in the YPD medium, the yeast was washed three times using a centrifuge. A yeast suspension at a 2 McFarland concentration was prepared using API 50CHL medium. The prepared yeast suspension was placed in a test strip tube. The test strips, upon which the suspension was dispensed, were incubated at 30°C for 24 hours.

[0148] API 50CHL medium, used for API testing, is purple. As acids are produced through energy metabolism, API 50CHL medium turns blue, then green, and finally yellow. The carbon source used by the mutant is recorded based on the color change, such as purple x, blue +, green ++, and yellow +++.

[0149] All seven mutant strains tested used 19 carbon sources for energy production and growth: L-arabinose, ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, N-acetylglucosamine, arbutin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, raffinose, and gentiobiose.

[0150] Only three mutants used rhamnose: KwonP-1, PicoYP-01, and PicoYP-02. Four mutants used sorbitol: KwonP-1, KwonP-3, PicoYP-01, and PicoYP-02. Four mutants used α-methyl-D-mannoside: the model strain, KwonP-1, KwonP-2, and PicoYP-02. Six mutants used amygdalin: KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02. Four mutants used D-turanose: the model strain, KwonP-1, KwonP-3, and PicoYP-02. Three mutants used D-tagatose: the model strain, KwonP-3, and PicoYP-3. Only the model strain used gluconate.

[0151] Mannitol and sorbitol, corresponding to alcoholic carbon sources, had a significant effect on yeast growth. The three new mutants differed from the other four yeast strains in the types of sugars they utilized for growth. The three new mutants (PicoYP, PicoYP-01, and PicoYP-02) showed slight differences in their preferred alcoholic carbon sources [Table 5].

[0152] [Table 5]

[0153] [Example 5] Changes in ALDH activity of mutant strains in gastric juice

[0154] When KARC is administered orally, in order to maintain enzyme activity in the intestine, the enzyme activity must pass safely without being destroyed by gastric acid, which secretes strong proteolytic enzymes such as pepsin.

[0155] NaOH solution was added to artificial gastric fluid at pH 1.17 to create two simulated solutions at pH 3 and pH 5, similar to the human gastric environment during food digestion. 1g of KARC was added to 7mL of artificial gastric fluid and 7mL of each simulated solution and mixed at 36.5°C for 5, 30, 60, and 90 minutes, respectively. NaOH solution was added to each reaction mixture to adjust the acidity to pH 7. 10mL samples were collected from each of the adjusted pH 7 solutions for analysis. ALDH activity was assayed for each sample.

[0156] At pH 1.17, ALDH activity in samples decreased by over 92.88% compared to the control within 5 minutes of reaction. At pH 1.17, ALDH activity in samples decreased by an average of 98.89% over 90 minutes. At pH 3, ALDH activity decreased by an average of 96.66% after 90 minutes, and at pH 5, it decreased by 56.83%. Finally, ALDH activity at pH 3 and 5 remained relatively higher than at pH 1.17 during the 90-minute reaction.

[0157] Specifically, at pH 1.17, the ALDH activity of KwonP-1 (KCTC13925BP) decreased by 90.94% to 5.57 units / g after 5 minutes of reaction compared to the control group. The ALDH activity of KwonP-1 decreased by 98.57% to 0.88 units / g after 90 minutes of reaction. Figure 19 The enzyme activity at pH = 3 and pH = 5 remained relatively higher than that at pH = 1.17. After 90 minutes of reaction, the ALDH activity of KwonP-1 decreased by 96.66% to 5.57 units / g at pH = 3, and decreased by 98.57% to 0.88 units / g at pH = 5.

[0158] At pH = 1.17, the ALDH activity of KwonP-2 (KCTC14122BP) decreased by 91.18% to 5.43 units / g after 5 minutes of reaction. The ALDH activity of KwonP-2 decreased by 98.81% to 0.73 units / g after 90 minutes of reaction. Figure 20 At pH 3 and pH 5, higher enzyme activity was maintained than at pH 1.17. After 90 minutes of reaction, ALDH activity decreased by 97.62% to 1.47 units / g at pH 3, and decreased by 56.11% to 26.99 units / g at pH 5.

[0159] At pH = 1.17, the ALDH activity of KwonP-3 (KCTC14123BP) decreased by 89.99% to 6.16 units / g after 5 minutes of reaction. The ALDH activity of KwonP-3 decreased by 97.85% to 1.32 units / g after 90 minutes of reaction. Figure 21 At pH 3 and pH 5, higher enzyme activity was maintained than at pH 1.17. After 90 minutes of reaction, ALDH activity decreased by 92.61% to 4.55 units / g at pH 3, and decreased by 62.31% to 22.18 units / g at pH 5.

[0160] At pH = 1.17, the ALDH activity of PicoYP (KCTC14983BP) decreased by 92.84% to 4.40 units / g after 5 minutes of reaction. The ALDH activity of PicoYP decreased by 98.33% to 1.03 units / g after 90 minutes of reaction. Figure 22 High enzyme activity was maintained at both pH 3 and pH 5. After 90 minutes of reaction, ALDH activity decreased by 96.66% at pH 3 to 2.05 units / g, and decreased by 53.97% at pH 5 to 28.31 units / g.

[0161] At pH = 1.17, the ALDH activity of PicoYP-01 (KCTC14984BP) decreased by 95.71% to 2.64 units / g after 5 minutes of reaction. The ALDH activity of PicoYP-01 decreased by 99.76% to 0.15 units / g after 90 minutes of reaction. Figure 23 At pH 3 and pH 5, higher enzyme activity was maintained than in gastric juice. After 90 minutes of reaction, ALDH activity decreased by 98.21% at pH 3 to 1.10 units / g, and decreased by 58.74% at pH 5 to 25.38 units / g.

[0162] At pH = 1.17, the ALDH activity of PicoYP-02 (KCTC14985BP) decreased by 96.66% to 2.05 units / g after 5 minutes of reaction. The ALDH activity of PicoYP-02 decreased by 99.76% to 0.15 units / g after 90 minutes of reaction. Figure 24 At pH 3 and pH 5, higher enzyme activity was maintained than in gastric juice. After 90 minutes of reaction, at pH = 3, ALDH activity decreased by 98.21% to 1.10 units / g, and at pH = 5, ALDH activity decreased by 62.08% to 23.32 units / g.

[0163] The pH of secreted gastric juice is 1.17. When food is consumed, the pH of the original gastric juice and food mix in the stomach, raising it from 3 to 5, making it unlikely to reach a pH of 1.17. Despite this, ALDH activity in the mutant strain is maintained even under this extreme pH condition.

[0164] Finally, the ALDH enzyme activity of the new mutants (PicoYP, PicoYP-01, PicoYP-02) remained at 2 to 5 units / g, despite a 92% to 97% decrease under the strongly acidic conditions of pH 1.17. Maintaining 2-5 units of enzyme activity is sufficient to function in the intestine. Compared to pH 1.17, it was even higher at pH 3 and pH 5. This leads to the conclusion that the new mutants (PicoYP, PicoYP-01, PicoYP-02) can be administered orally.

[0165] [Example 6] Growth characteristics of 5L fermenter culture

[0166] Be inoculated in YPD substratum (2% peptone, 1% yeast extract, 2% glucose) respectively, and carry out primary seed culture 18 hours at 30 ℃ and 200rpm.The seed of 20mL culture is inoculated in 1980mL YPD substratum, and in 5L, cultivate again.The cultivation in 5L culture tank was carried out 48 hours at 30 ℃ and 200rpm.Use the 10mL sample analysis OD660nm growth curve and the enzyme activity under collecting from secondary culture.

[0167] The maximum density (OD660nm) of KwonP-1 (KCTC13925BP) was 134.4. The maximum density of KwonP-1 was 4.35% higher than that of the model strain (KCTC7296). The growth curve characteristics and specific growth rate (OD660nm / hr) of KwonP-1 were similar to those of the model strain. The ALDH activity of KwonP-1 was 33.6 units / g. The ALDH activity of KwonP-1 was 11.96 times higher than that of the model strain. Figure 25 ].

[0168] The maximum density (OD660nm) of KwonP-2 (KCTC14122BP) was 133.8. The maximum density of KwonP-2 was 3.88% higher than that of the model strain. The growth of KwonP-2 ended earlier than that of the model strain. The specific growth rate (OD660nm / hr) of KwonP-2 was 14.8% higher than that of the model strain. The ALDH activity of KwonP-2 was 31.5 units / g. The ALDH activity of KwonP-2 was 11.21 times higher than that of the model strain. Figure 26 ].

[0169] The maximum density (OD660nm) of KwonP-3 (KCTC14123BP) was 134.1. The maximum density of KwonP-3 was 4.12% higher than that of the model strain. The growth of KwonP-3 ended earlier than that of the model strain. The specific growth rate (OD660nm / hr) of KwonP-3 was 6.08% higher than that of the model strain. The ALDH activity of KwonP-3 was 29.5 units / g. The ALDH activity of KwonP-3 was 10.5 times higher than that of the model strain. Figure 27 ].

[0170] The maximum density (OD660nm) of PicoYP (KCTC14983BP) was 123.8. The maximum density of PicoYP was 3.88% higher than that of the model strain. The growth curve characteristics of PicoYP were similar to those of the model strain. The specific growth rate (OD660nm / hr) of PicoYP was 6.22% higher than that of the model strain. The ALDH activity of PicoYP was 44.2 units / g. The ALDH activity of PicoYP was 15.73 times higher than that of the model strain. Figure 28 ].

[0171] The maximum density (OD660nm) of PicoYP-01 (KCTC14984BP) was 126.9. The maximum density of PicoYP-01 was 1.47% higher than that of the model strain. The growth curve characteristics of PicoYP-01 were similar to those of the model strain. The specific growth rate (OD660nm / hr) of PicoYP-01 was 2.14% higher than that of the model strain. The ALDH activity of PicoYP-01 was 47.1 units / g. The ALDH activity of PicoYP-01 was 16.76 times higher than that of the model strain. Figure 29 ].

[0172] The maximum density (OD660nm) of PicoYP-02 (KCTC14985BP) was 148.1. The maximum density of PicoYP-02 was 14.99% higher than that of the model strain. Compared with the model strain, the growth curve of PicoYP-02 was at the top. The specific growth rate (OD660nm / hr) of PicoYP-02 was 9.64% lower than that of the model strain. The ALDH activity of PicoYP-02 was 52.68 units / g. The ALDH activity of PicoYP-02 was 18.75 times higher than that of the model strain. Figure 30 ].

[0173] [Example 7] Preparation of mutant strain lysate (KARC)

[0174] To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysate, proteases are removed and inhibited. To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysate, cell debris is removed. The dried products or lysates of the mutant strains are mixed to prepare a KARC composition.

[0175] The mutant strain and the culture medium containing it contain a variety of substances, such as yeast metabolites and proteolytic enzymes secreted by the yeast. To extract and preserve the ALDH, coenzymes, and glutathione present in the yeast, it is necessary to fully remove substances outside the yeast. To this end, a washing procedure was performed. Washing of the mutant strain was performed by dispensing 40 mL of culture medium into a 50 mL conical tube, centrifuging at 13,000 rpm for 15 minutes, and removing the supernatant.

[0176] As a result of centrifugation, the yeast cells aggregated into a pellet containing residual culture medium. Add 30 mL of purified water and vortex to loosen the pellet. Repeat this procedure three times to fully remove the residual culture medium.

[0177] Yeast is known to have an ethanol tolerance of up to 13%, and yeast will die when exposed to high ethanol concentrations. Dissolve the washed pellet thoroughly in 10 mL of 20% ethanol solution to induce yeast death. Stir the pellet in ethanol at 100 rpm for 30 minutes to induce yeast death. At the end of the reaction time, add 30 mL of purified water to bring the ethanol concentration to 5%. Repeat this washing procedure three times to fully remove the ethanol.

[0178] To prevent the degradation of ALDH and ADH by proteases present in yeast cells, 10 mL of 1X PBS was prepared by dissolving two protease inhibitor tablets (Pierce Protease Inhibitor Tablets, EDTA-free, Thermo Scientific). The solution was added to the washed yeast pellet and distributed thoroughly.

[0179] To prepare the lysate of the mutant strain prepared in the present invention, 4 g of glass beads were added and stirred to disrupt the yeast cell walls. To prevent heat generated during yeast disruption from denaturing the enzyme, vortexing for 30 seconds and incubating on ice for 30 seconds were repeated 6 times.

[0180] After the yeast cell wall was broken, 10 mL of 100 mM potassium phosphate buffer was added and mixed by vortexing for 3-5 seconds. Centrifugation was performed 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 The KARC composition was prepared by filtration through a syringe filter (Sartorius, Goettingen, Germany).

[0181] To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysates, remove and inhibit intracellular proteases, and remove cellular debris such as cell walls, KARC compositions were prepared using lysates of six mutant strains (KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, PicoYP-02) or mixtures thereof in a free ratio [Table 6].

[0182] KARC 1 is made from Kwon P-1. KARC 1's ADH and ALDH enzyme activities are 461.4 units / g and 28.6 units / g, respectively. The NAD and NADP total coenzyme contents in KARC 1 are 176.2 nanomoles / g and 5.1 nanomoles / g, respectively. KARC 1 also contains 0.98 wt% GSH.

[0183] KARC 2 is made from Kwon P-2. KARC 2 has ADH and ALDH enzyme activities of 482.1 units / g and 29.8 units / g, respectively. The NAD total and NADP total coenzyme contents in KARC 2 are 175.4 nanomoles / g and 5.2 nanomoles / g, respectively. KARC 2 also has a GSH content of 0.96 wt%.

[0184] KARC 3 is made from Kwon P-3. KARC 2 has ADH and ALDH enzyme activities of 477.5 units / g and 28.1 units / g, respectively. KARC 3 has NAD total and NADP total coenzyme contents of 177.2 nanomoles / g and 5.1 nanomoles / g, respectively. KARC 3 also has a GSH content of 1.00 wt%.

[0185] KARC 4 is made from PicoYP. KARC 2 has ADH and ALDH enzyme activities of 586.8 units / g and 33.8 units / g, respectively. KARC 4 has NAD total and NADP total coenzyme contents of 184.3 nanomoles / g and 5.7 nanomoles / g, respectively. KARC 4 also has a GSH content of 0.84 wt%.

[0186] KARC 5 is produced from PicoYP-01. KARC 5 exhibits ADH and ALDH enzyme activities of 621.6 units / g and 38.2 units / g, respectively. The NAD and NADP total coenzyme contents in KARC 5 are 186.9 nanomoles / g and 5.6 nanomoles / g, respectively. KARC 5 also contains 0.84 wt% GSH.

[0187] KARC 6 is made from PicoYP-02. KARC 5 has ADH and ALDH enzyme activities of 664.1 units / g and 41.6 units / g, respectively. KARC 6 has NAD total and NADP total coenzyme contents of 195.0 nanomoles / g and 5.8 nanomoles / g, respectively. KARC 6 also has a GSH content of 0.88 wt%.

[0188] KARC was prepared by freely mixing dry powders and lysates prepared from six deposited strains. The average enzyme activities of ADH and ALDH in the KARC composition were 547.6 units / g and 33.1 units / g, respectively. The average contents of the coenzyme NAD total and coenzyme NADP total in the KARC composition were 180.4 nanomoles / g and 5.4 nanomoles / g, respectively. The average glutathione content in the KARC composition was 0.84 wt%.

[0189] The aldehyde decomposition ability of KARC was maintained during the lysate production process. KARC exhibited the ability to remove endogenous aldehydes such as HNE, MDA, and 3,4-dihydroxyphenylacetaldehyde (DOPAL).

[0190] [Table 6]

[0191] [Example 8] Sequence analysis of ALDH contained in mutant strains.

[0192] The differences in ALD (yeast aldehyde dehydrogenase) between the mutants and the parent strain were investigated. Whole-genome sequencing was performed on the parent strain and mutants KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02. Mutant cells were isolated by culturing pure strains on solid culture medium. The resulting mutant genome sequences were then analyzed.

[0193] In the ALD (yeast aldehyde dehydrogenase) of the new mutant strain, ALD2 (SEQ ID NO: 3) and ALD3 (SEQ ID NO: 4) were found to be densely distributed on chromosome 13. A 689-nucleotide noncoding region was located between the ALD2 and ALD3 encoding genes.

[0194] ALD2 and ALD3 coexist in the same genome. ALD2 and ALD3 encode separate aldehyde dehydrogenases. The ALD2 gene is nearly identical to ALD3, consisting of 1,521 nucleotides and 506 amino acids, but differs by 8.2% in sequence. ALD2 and ALD3 were identified as separate aldehyde dehydrogenases, differing by 125 bases (8.2%).

[0195] In six mutant strains (KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02), the ALD2 sequence lacks a stop codon, allowing protein synthesis to proceed. Consequently, a new, larger ALDH enzyme was created by linking a portion of ALD2 to ALD3 [SEQ ID NO: 1].

[0196] ALD2 [SEQ ID NO. 3] of the model strain (KCTC7296) consists of a 30-nucleotide sequence (5'-GTTCACATAAATCTCTCTTTGGACAACTAA-3') encoding 9 amino acids (N-VHINLSLDN-C) at the terminal, excluding the stop codon.

[0197] ALD2 of the six mutants consists of a specific 42-nucleotide sequence (5'-AGATATAGATTATACACATTTAGAAAATTAGCCAAAAGAAAA-3') encoding 14 amino acids (N-RYRLYTFRKLAKRK-C) between the 5' end of ALD2 and ALD3 [SEQ ID NO. 2].

[0198] The ALD2 gene encoding ALD2 is deleted from nucleotide 1492 to nucleotide 647 in the noncoding region, eliminating the termination codon. Ultimately, the six deposited mutants contain a novel mutant gene consisting of 3,054 bases encoding a novel ALD2 gene [SEQ ID NO: 1].

[0199] [Example 9] Effectiveness of KARC in reducing endoplasmic reticulum (ER stress)-induced liver fat

[0200] Administration of tunicamycin (Tm) to animals inhibits N-glycosylation of proteins, leading to the formation of unfolded proteins (UPs). Tunicamycin administration to animals causes ER stress. Tunicamycin inhibits N-acetylglucosamine transferase (NAT) in eukaryotic cells. Inhibition of NAT inhibits the formation of N-acetylglucosamine lipid metabolites and the glycosylation of newly synthesized proteins.

[0201] In the endoplasmic reticulum (ER), proteins synthesized on ribosomes undergo higher-order structural and post-translational modifications, leading to the folding and assembly of glycoproteins. Inhibiting glycoprotein production with tunicamycin interferes with protein maturation. Disrupted protein maturation leads to the production of misfolded or unfolded proteins. These misfolded or unfolded proteins cannot assemble properly and accumulate in the ER, causing ER stress. ER stress can lead to cell death.

[0202] To investigate the efficacy of KARC in suppressing endoplasmic reticulum (ER) stress-mediated acute fatty liver, the present inventors analyzed the effects of KARC and fat-related liver tissue morphology in model animals induced by ER stress.

[0203] Specifically, C57BL / 6J male mice (Jungang Laboratories, South Korea), a model animal for ER stress induction, were housed in a 12-hour light / dark cycle (6:00 AM to 6:00 PM, 6:00 PM to 6:00 AM) at 23°C and 60-70% humidity. Mice had free access to water in a pathogen-free facility.

[0204] Liver sample size of experimental animals was evaluated based on the analysis.ER stress was induced by intraperitoneal administration of 2 mg / kg tunicamycin to mice, and the effect of KARC was tested at 24 and 48 hour intervals.

[0205] KARC is prepared from dried powder or lysate of Saccharomyces cerevisiae KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, or PicoYP-02. The prepared KARC contains glutathione, aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), and coenzymes NAD and NADP.

[0206] The mice were anesthetized with isoflurane (Gyeonggi Hana Pharmaceutical) and sacrificed at 9:00 am. To analyze the morphology of liver tissue, H&E staining was used to confirm its structure. H&E tissue staining was performed.

[0207] Lipids in 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.

[0208] Triglyceride levels in liver tissue were quantified using a triglyceride assay kit (AM 157S-K, Asan Pharmaceuticals, Seoul, South Korea). Quantified triglyceride levels in liver tissue were measured by measuring the absorbance of samples and standards at 550 nm using an Infinite 200PRO (Tecan Trading AG, Switzerland).

[0209] Tunicamycin induces the appearance of fatty liver in liver tissue[ Figure 1 ].

[0210] In the tunicamycin-injected group, fatty liver persisted and 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 recovery rate of fatty liver.

[0211] Histomorphometric analysis by H&E staining also showed that the amount of fat in the liver tissue of the KARC-administered group was less than that of the vehicle group [ Figure 2 ].

[0212] The triglyceride content in liver tissue after defoamer stress was determined. Figure 3 ] and total cholesterol[ Figure 4 ]. In the livers of mice subjected to ER stress, triglycerides and total cholesterol increased. In the livers of mice administered KARC, both triglycerides and total cholesterol decreased. KARC, a lysate of Saccharomyces cerevisiae, is effective in treating fatty liver disease.

[0213] [Example 10] Changes in ER stress-related factors after KARC administration

[0214] Tunicamycin blocks the transfer of N-acetylglucosamine 1-phosphate to the dolichol phosphate site of proteins. Thus, tunicamycin inhibits the binding of sugars to the NH3 of the protein building block asparagine.

[0215] Tunicamycin inhibits glycosylated protein synthesis by inhibiting entry into the S phase of the cell cycle and DNA synthesis by prolonging the G1 phase. An ER stress model was created by injecting tunicamycin into normal mice. The effects of KARC were investigated by administering 20 units / kg of mutant yeast lysate KARC to the ER stress model for 24 or 48 hours.

[0216] Acute fatty liver-related factors were analyzed in the liver tissues of mice immediately after tunicamycin administration or KARC administration.

[0217] To investigate the effects of inhibiting acute fatty liver formation, we analyzed in detail the factors related to ER stress, inflammation, lipid synthesis, and fatty acid oxidation in the liver tissues of mice injected with tunicamycin or KARC.

[0218] The expression of ER stress-related gene Chop[ Figure 5 ]、Grp78[ Figure 6 ]、Ire-1α[ Figure 7 ]、Gadd34[ Figure 8 ] and Atf4[ Figure 9 The mRNA and protein expression levels of the inflammatory gene F4 / 80[ Figure 10]、Mcp1[ Figure 11 ]、Tnf-α[ Figure 12 ] and Il-6[ Figure 13 The expression levels of Ppar-α[ Figure 14 ]、Pgc-1α[ Figure 15 ]、Cpt-1α[ Figure 16 ] and Fgf21[ Figure 17 ] gene expression levels.

[0219] Total RNA was prepared from liver tissue of mice with induced ER stress by the TriZol procedure (Invitrogen). Using total RNA as a template, cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA).

[0220] To measure the mRNA expression levels of various genes, qPCR was performed using the CFX96™ Real-Time PCR System (Bio-Rad Laboratories).

[0221] 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 liver tissue of ER stress-induced mice.

[0222] Liver tissue lysates (extracts or digests) were extracted using T-PER (Tissue Protein Extraction Reagent; Thermo Scientific, Rockford, IL, USA) supplemented with protease and phosphatase inhibitors (Thermo Scientific). Proteins in liver tissue lysates were diluted with 5X sample buffer (EBA-1052, ELPIS BIOTECH, Seoul, South Korea) and heated at 95°C for 5 min. Proteins were separated by 5-15% Tris-HCl SDS / PAGE gel electrophoresis and transferred to nitrocellulose membranes (GE Healthcare, Uppsala, Sweden).

[0223] All immunoblots were probed with HRP-conjugated secondary antibodies (Clarity Western ECL Substrate, Bio-Rad Laboratories) and protein bands were detected using a chemiluminescence imaging system (Fusion Fx, VilberLourmat, Eberhardzell, Germany).

[0224] The expression of ER stress-related genes in the liver tissues of KARC-administered mice after ER stress induction was analyzed by qPCR. Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 and Figure 9 ] As shown in the results of a study published in the journal Cell Reports, all KARC-administered groups showed improved expression of ER stress-related genes, whose expression is increased by ER stress induction. It is known that the expression of inflammatory genes is increased in the liver tissue of mice with induced ER stress.

[0225] The expression of marker genes related to inflammatory genes was analyzed. Figure 10 、 Figure 11 、 Figure 12 and Figure 13 ] As shown in Figure 5, the expression of all genetic markers was reduced by KARC.

[0226] The expression of genes related to fatty acid oxidation was analyzed. Figure 14 、 Figure 15 、 Figure 16 and Figure 17 ] As shown in FIG, in the group administered with KARC, fatty acid oxidation was improved by reducing ER stress.

[0227] When the expression of proteins associated with ER stress was analyzed, 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 analyzed. In all cases, KARC administration improved ER stress and reduced the expression of proteins related to lipid metabolism and fatty acid oxidation. Figure 18 ].

[0228] [Table 7]

[0229] [Example 11] Effect of reducing oxidative stress.

[0230] When drinking alcohol, excessive acetaldehyde (ACh) is produced by alcohol dehydrogenase (ADH), leading to increased reactive oxygen species and oxidative stress. Aldehyde dehydrogenase (ALDH) converts ACh into acetic acid and excretes it from the body. Excessive aldehyde production due to mutations in the ALDH gene or from excessive alcohol consumption can lead to lipid peroxidation.

[0231] The resulting acetaldehyde and malondialdehyde exacerbate oxidative stress and interfere with mitochondrial energy metabolism. Endoplasmic reticulum stress is caused by the accumulation of denatured proteins in cells, leading to cell death.

[0232] Blood acetaldehyde concentration was measured over time after drinking. Figure 31When drinking alone, the area under the curve (AUC) of blood acetaldehyde (Ach) was 13.02±1.18 mg·h / dL. When administered at a dose of 10 units / kg of KARC, the area under the curve (AUC) of blood acetaldehyde (Ach) decreased significantly by 26.13% compared to drinking alone, measuring 9.39±1.07 mg·h / dL (P=0.005).

[0233] When administered at a dose of 20 units / kg, the AUC of blood acetaldehyde (ACh) significantly decreased by 55.71% compared to alcohol consumption alone, reaching 5.22 ± 0.99 mg·h / dL (P < 0.001). Compared to the group administered 10 units / kg of KARC, the 20 units / kg group showed a significant decrease in blood acetaldehyde (ACh) (P = 0.034). KARC demonstrated a dose-dependent reduction in total blood acetaldehyde (ACh) over time.

[0234] The reduction in blood acetaldehyde (Ach) concentration due to KARC administration has a positive impact on reducing oxidative stress and promoting health.

[0235] Measurement of blood malondialdehyde (MDA) concentration during chemotherapy Figure 32 The blood MDA concentration in the control group was 0.607±0.161 μM. The group receiving KARC treatment showed a significant 63.3% decrease in blood MDA concentration compared to the control group, measuring 0.223±0.033 μM (P<0.001).

[0236] In the control group, blood MDA concentration ranged from 0.427 μM to 0.885 μM, showing a wide variation. In the KARC-administered group, the range was significantly narrowed, with values ranging from 0.158 μM to 0.269 μM. This confirms not only the effect of lowering blood MDA concentration but also stabilizing it, as shown in [ Figure 33 ] confirmed.

[0237] Various factors, including drug ingestion, stress, and intense physical exercise, lead to an increase in intracellular reactive oxygen species (ROS). This triggers lipid peroxidation and the oxidation of endogenous amines (such as dopamine, norepinephrine, serotonin, and histamine). Reactive aldehyde compounds, including 4-hydroxynonenal (HNE), malondialdehyde (MDA), acetaldehyde (ACh), and dopamine-induced aldehydes, accumulate within cells, exacerbating oxidative stress.

[0238] 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 (ALPEs). The accumulation of these products has toxic effects on various cells, further exacerbating oxidative stress.

[0239] This cumulative oxidative stress disrupts mitochondrial energy metabolism within the cell and leads to the accumulation of aldehyde intermediates in aldose sugar metabolism, including methylglyoxal (MG) and glyceraldehyde-3-phosphate (GA3P). Chain reactions involving aldehydes lead to the accumulation of stable final sugar oxidation products, known as advanced glycation end products (AGEs), which weaken intracellular antioxidant defense systems such as glutathione (GSH). These processes increase endoplasmic reticulum (ER) stress, leading to increased apoptosis in neuronal cells.

[0240] Increased reactive oxygen species and oxidative stress are associated with elevated levels of reactive aldehydes, such as HNE and MDA, and modified proteins, such as advanced glycation end products (AGEs) and advanced lipid peroxidation end products (ALEs). This series of events is known to reinforce and amplify each other, leading to increased endoplasmic reticulum stress (ER stress).

[0241] KARC administration effectively regulated malondialdehyde, a marker of reactive oxygen species and oxidative stress, showing the potential to reduce oxidative stress and improve endoplasmic reticulum (ER) stress stability. KARC significantly reduced malondialdehyde concentrations in the bloodstream, indicating its ability to reduce reactive oxygen species and oxidative stress.

[0242] By reducing acetaldehyde and malondialdehyde levels in human blood, KARC exhibits the potential to prevent and correct ER stress by reducing reactive oxygen species and oxidative stress.

[0243] [Example 12] Acute oral administration test

[0244] 12-1. Preparation of experimental animals

[0245] The experimental animals were female and male ICR mice (7 weeks old). The received ICR mice were adapted for 7 days. The general symptoms of the adopted mice during the adaptation period were observed, and only healthy animals were used for short-term toxicity testing. Feed and water were available freely. Based on an average body weight of about 20g the day before oral administration, the group was divided into 10 groups of 5 mice each.

[0246] 12-2. Administration of test substance

[0247] The test substances were prepared by dissolving in physiological saline so that the doses for experimental animals were 0, 750, 3,000, and 5,000 mg / kg, respectively, based on the content of the mutant yeast lysate KARC of the present invention.

[0248] The dosage standards were in accordance with the Korea National Toxicology Program (KNTP) Toxicity Test Manual of the Ministry of Food and Drug Safety. The maximum dose of 5,000 mg / kg, as specified in the KNTP Manual, was used as the maximum concentration for this experiment. Samples prepared for each group were administered orally once to each test animal. The normal group (G1) received normal saline.

[0249] 12-3. Observation and autopsy

[0250] For all test groups, mice were observed for symptoms at least once daily from the date of acquisition until the date of necropsy. Symptoms were observed for 7 days after oral administration. Necropsy was performed after observing the symptoms of the rats. During the rat necropsy, changes in various organs were observed visually.

[0251] Single-dose toxicity testing of the ALDH-containing KARC composition of the present invention was conducted in mice. Results showed that no mouse mortality was observed within 7 days at concentrations of up to 5,000 mg / kg of mutant yeast KARC. No abnormalities, such as weight gain or changes in feed intake, were observed in the mice. Post-observation autopsies revealed no abnormal findings.

[0252] [Example 13] Preparation of food and pharmaceutical compositions for preventing and improving fatty liver disease and liver dysfunction by detoxifying endogenous acetaldehyde.

[0253] [1] Food and pharmaceutical compositions containing KARC as an active ingredient for preventing and restoring fatty liver disease and liver dysfunction were prepared. Food or pharmaceutical compositions containing KARC powder in various composition ratios can be prepared. 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 compositions containing the powder composition are shown in [Table 8].

[0254] [Table 8] Industrial Applicability

[0255] KARC dry powder, excipients, and natural sweeteners such as oligofructose, enzyme-treated stevia, anhydrous citric acid, isomaltodextrin, and xylitol, as well as citrus juice powder and citrus flavoring powder, are added to food and pharmaceutical compositions. The processing and testing of the raw materials and final products of the food or pharmaceutical compositions are conducted in accordance with general testing methods and the Health Functional Food Act as outlined in the Korean Food Code.

[0256] A food or pharmaceutical composition comprising KARC can prevent or improve deterioration of liver function.

[0257] The above examples describe in detail the mutant yeast composition KARC containing aldehyde dehydrogenase, including therapeutically effective dosages in disease models, methods of manufacture, pharmacological effects, methods of administration, acute oral toxicity, and representative examples of food or pharmaceutical compositions. While the above examples describe the efficacy of KARC in detail, they are merely exemplary of the present invention.

[0258] 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.

[0259] Even foods or therapeutic agents containing modified forms of aldehyde dehydrogenase that embody the technical gist of the present invention as described in the patent claims fall within the scope of legal protection of the present invention.

[0260]

Claims

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

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

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

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

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