Microorganism with high tripeptide production capacity and use thereof

Abstract

This invention relates to a strain of Candida utilis with high tripeptide production capacity and / or alcohol dehydrogenase activity and its uses.

Description

Technical Field

[0001] This invention relates to a strain of Candida utilis with high tripeptide production capacity and / or alcohol dehydrogenase activity and its uses. Background Technology

[0002] Unlike other foods, alcohol is a xenobiotic that cannot accumulate in the body. Its breakdown products, such as acetaldehyde, are known to be a major cause of oxidative stress-induced cell damage and hangover symptoms. The effects of alcohol on liver metabolism depend on intake, but it is nicotinamide adenine dinucleotide phosphate (NADP) and acetaldehyde, produced during oxidation rather than alcohol itself, that become the primary mediators of liver damage through condensation reactions with reactive amines in the body. Acetaldehyde transported to the brain is known to be converted into many harmful compounds that cause hangover symptoms such as hot flashes, rapid pulse, nausea, and vomiting.

[0003] 80-90% of the alcohol that enters the body is first broken down into acetaldehyde by alcohol dehydrogenase (ADH) in liver cells. Then, it is metabolized by aldehyde dehydrogenase (ALDH) to form acetic acid. Acetic acid is then hydrolyzed into carbon dioxide and water, thus undergoing a complete breakdown process. A crucial step in the breakdown of ethanol in the liver is the oxidation reaction of ADH, which converts ethanol into acetaldehyde.

[0004] Glutathione (L-γ-glutamyl-L-cysteine ​​glycine, GSH) is a bioactive substance found in cells, existing as a tripeptide composed of three amino acids: glutamic acid, cysteine, and glycine. In vivo, it exists in two forms: reduced glutathione (GSH) and oxidized glutathione (GSSG). Glutathione is present in the cells of animals, plants, and microorganisms at concentrations of 0.1–10 mM, accounting for over 90% of total non-protein cellular activity. The various functions of glutathione are important not only in agriculture but also in many areas of medicine, including enzymology, transport, pharmacology, therapy, toxicology, endocrinology, and microbiology.

[0005] Glutathione can be produced primarily through microbial fermentation or enzymatic synthesis. Due to current high production costs, enzymatic synthesis methods are not yet commercially viable, while methods involving culturing microorganisms and extracting them from microbial cells are widely used in industry. Yeast is a widely used microbial strain for glutathione production due to its high intracellular glutathione content and its reputation as a safe microorganism for food production. Strains of the genera *Saccharomyces* and *Candida* are particularly representative. The already relatively high glutathione concentrations (0.1-1% dry weight) in wild-type strains of these yeast species, along with the advantages of high-density cell culture and rapid growth in low-cost media, make yeast fermentation production more competitive. Summary of the Invention

[0006] The technical problem to be solved by the present invention

[0007] One embodiment of the present invention is to provide a Candida utilis strain with high ADH activity.

[0008] One embodiment of the present invention is to provide a Candida utilis strain with tripeptide production capacity and high ADH activity.

[0009] Another embodiment of the present invention provides a composition for the decomposition of alcohol, said composition comprising at least one selected from the group consisting of: cells of a Candida utilis strain, a culture of the strain, lysates of the cells, fragments of the cells, and extracts thereof.

[0010] Another embodiment of the present invention provides a composition for preventing, improving or relieving hangovers, said composition comprising at least one selected from the following: cells of a Candida utilis strain, a culture of the strain, lysates of the cells, fragments of the cells, and extracts thereof.

[0011] Another embodiment of the present invention provides a composition or antioxidant composition for reducing oxidative stress, comprising at least one selected from the following: cells of a Candida utilis strain, a culture of the strain, lysates of the cells, fragments of the cells, and extracts thereof.

[0012] A further embodiment of the present invention provides a composition for preventing, improving or treating oxidative stress-related diseases, the composition comprising at least one selected from the following: cells of Candida utilis strain, cultures of the strain, cell lysates, cell fragments and extracts thereof.

[0013] A further embodiment of the present invention provides a food composition comprising at least one selected from the following: cells of a Candida utilis strain, a culture of the strain, lysates of the cells, fragments of the cells, and extracts thereof.

[0014] Technical means to solve technical problems

[0015] The *Candida utilis* strain according to the present invention possesses glutathione-producing activity and / or alcohol dehydrogenase (ADH) activity; more specifically, it possesses both alcohol dehydrogenase activity and high glutathione-producing activity, thereby having the advantage of more effectively detoxifying and excreting alcohol in vivo. As a non-GMO strain isolated from nature rather than a recombinant strain, the yeast strain according to the present invention has the advantage of high availability.

[0016] The Candida utilis strain of the present invention may have a glutathione yield of more than 0.8% by weight and / or an ADH activity of more than 0.18 mU / ml per cell dry weight (g), preferably at least about 1.5% by weight of glutathione and / or at least 0.2 mU / ml per cell dry weight (g).

[0017] The *Candida utilis* strain of the present invention possesses alcohol dehydrogenase (ADH) activity and alcohol tolerance, particularly the ability to grow at ethanol concentrations of 2-15% (v / v). More specifically, when cultured at ethanol concentrations of 6-15% (v / v), the optical density (OD) value of the *Candida utilis* strain of the present invention can be 120-200% of that of the *Candida utilis* strain preserved with KCCM 11355, based on 100% of the OD value.

[0018] The *Candida utilis* strain of the present invention may have at least one culture characteristic selected from the following: (i) increased glutathione production during shaking culture at 30°C; (ii) based on 100% of the cell concentration (OD at 600 nm) during shaking culture in a glucose-containing medium, the lower limit of the cell concentration during shaking culture in a sucrose-containing medium is 105% or more, 110% or more, or 115% or more, and the upper limit of the cell concentration is 120% or less, or a combination of the lower and upper limits, for example, a cell concentration (OD at 600 nm) of 105-120%. OD), or based on 100% of the glutathione content (mg / L) when cultured with shaking in a glucose-containing medium, where the glutathione content (mg / L) when cultured with shaking in a sucrose-containing medium can be 105% or more, 110% or more, 115% or more, less than 150%, less than 140%, less than 130%, or less than 120%, for example, 105-150% or 110-140% of the glutathione content, (iii) when cultured with the addition of cysteine, the glutathione yield increases, and (iv) at a concentration of 6 g / L·h -1 The feeding rate provides the ability to maintain glutathione production under fed-batch culture conditions, for example, with a glutathione content of 50-120% based on the glutathione content at the end of the batch, or with a glutathione content of 80-120% based on the maximum glutathione amount in the batch when cultured at a concentration of ethanol above 2 g / L.

[0019] This invention relates to health functional foods and food additives with alcohol decomposition activity, which can be widely used in the manufacture of foods for the prevention of alcoholic liver disease, because the Candida utilis strain of this invention has alcohol tolerance and alcohol decomposition ability, thus having a hangover relief effect before and after drinking and helping the liver detoxify.

[0020] Because the strain of the present invention has excellent alcohol (ethanol) degrading enzyme activity, it can effectively inhibit the absorption of ingested alcohol (ethanol), thereby preventing liver dysfunction, alcoholic liver disease and alcoholic intestinal disease caused by excessive alcohol metabolism.

[0021] Specifically, alcohol cannot be stored in the body and must be metabolized. This metabolic process mainly takes place in the liver through alcohol dehydrogenases. These enzymes break down alcohol into acetaldehyde. Acetaldehyde is toxic and damages liver cells. The large amounts of fatty acids produced during alcohol metabolism lead to fat accumulation in the liver, causing alcoholic liver disease. These alcoholic liver diseases are mainly classified as alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, and alcoholic cirrhosis. The mechanisms causing liver damage include alcohol itself, metabolites such as acetaldehyde, and immune responses. In particular, acetaldehyde is a major cause of hepatotoxicity, such as lipid peroxidation, binding to the cytoplasm, disrupting the electron transport chain in mitochondria, interfering with microtubule function, producing substances that bind to proteins, and increasing collagen synthesis.

[0022] Furthermore, since the strain of the present invention has excellent glutathione production activity, it promotes the excretion of toxic substances produced by the metabolism of ingested alcohol (ethanol) and protects hepatocytes from damage caused by harmful reactive oxygen species, thereby preventing the occurrence of liver dysfunction, alcoholic liver disease and intestinal diseases.

[0023] Specifically, glutathione is an antioxidant that promotes the excretion of toxic substances through urine or bile, particularly by acting on the detoxification process of alcohol metabolites in the liver. Furthermore, glutathione protects hepatocytes from damage by harmful reactive oxygen species. Therefore, the strain of the present invention possesses alcohol tolerance and the ability to break down alcohol, thus not only preventing alcohol absorption and breakdown but also promoting the excretion of toxic substances produced by alcohol breakdown due to the high glutathione content in the strain, thereby achieving the purpose of preventing liver damage and liver disease.

[0024] This invention relates to a food, food additive, beverage, or health supplement containing ingredients that effectively lower blood alcohol levels and enhance antioxidant activity in the body.

[0025] The strains of this invention can be cultured aerobically in a culture medium containing a carbon source, a nitrogen source, and inorganic salts.

[0026] As a component of the culture medium for this strain, the carbon source may be at least one or two selected from the following: dextrin, glucose, sucrose, acetic acid, ethanol, molasses, and sulfite pulping waste liquid, etc. Sucrose is preferred, taking into account the production of glutathione. More specifically, the *Candida utilis* strain of the present invention may have at least one culture characteristic selected from the group consisting of: (ii) a cell concentration (OD value at 600 nm) of 100% when cultured with shaking in a glucose-containing medium and 105-120% when cultured with shaking in a sucrose-containing medium; or a glutathione content (mg / L) of 105-150% or 110-140% when cultured with shaking in a glucose-containing medium and shaking in a sucrose-containing medium; (iii) an increase in glutathione production when cultured with the addition of cysteine; and (iv) a glutathione production at a concentration of 6 g / L·h. -1 Under fed-batch culture conditions with a feed rate that maintains glutathione production.

[0027] The nitrogen source can be at least one or two selected from the following: urea, ammonia, ammonium sulfate, inorganic salts such as ammonium chloride or ammonium phosphate, and nitrogen-containing organic substances such as corn steep liquor (CSL), casein, yeast extract, or peptone. In addition, phosphate, potassium, or magnesium components can be added to the culture medium; examples of these can be common industrial raw materials such as superphosphate, ammonium phosphate, potassium chloride, potassium hydroxide, magnesium sulfate, and magnesium hydrochloride. Furthermore, inorganic salts such as zinc, copper, manganese, and iron ions can be used. In addition, vitamins and nucleic acid-related substances can also be added.

[0028] The culture temperature suitable for this invention can be the culture conditions of yeast, such as 20-40°C, preferably 25-35°C, and pH 3.5-8.0, especially 4.0-6.0.

[0029] As a cultivation method applicable to the present invention, any one of batch culture, fed-batch culture, or continuous culture can be used, but fed-batch culture or continuous culture is used in industry.

[0030] This invention provides a method for increasing glutathione production by culturing Candida utilis strains.

[0031] The strain can be cultured using sugar Alternatively, glucose can be used as a carbon source. The culture strain can be cultured by measuring the ethanol content in the fermentation broth and adjusting the carbon source supply rate. For example, when the ethanol content in the fermentation broth is below 5 g / L, the carbon source supply rate can be 5-10 g / L·h. -1 5.5-8 g / L·h -1 5.5-7 g / L·h -1 Or 5.5-6.5 g / L·h-1 The carbon source supply rate is a continuous supply of carbon source.

[0032] More specifically, considering glutathione production, the *Candida utilis* strain of the present invention is preferably cultured using fed-batch culture. More preferably, at a concentration of 6 g / L·h... -1 Under conditions of fed-batch culture with a specific feeding rate, glutathione production can be maintained.

[0033] Preferably, the method for increasing glutathione yield can be performed according to at least one method selected from the group consisting of: controlling the type of carbon source, the carbon source supply rate, cysteine ​​addition, and other culture conditions. For example, the method can be at least one method selected from the group consisting of: (i) cysteine-added culture, (ii) using sucrose or glucose as a carbon source, (iii) controlling the carbon source supply rate, (iv) culture temperature, and (v) stirring conditions, etc. Specifically, at least one method selected from the group consisting of: shaking culture at 30°C, using sucrose as a carbon source under shaking culture conditions, cysteine-added culture, and fed-batch culture can be used. Fed-batch culture can be performed by measuring the ethanol content in the fermentation broth and adjusting the carbon source supply rate; for example, fed-batch culture can be performed at a rate of 6 g / L·h. -1 The feeding rate is used for this purpose.

[0034] According to the method of the present invention, a culture containing yeast cells contains a high concentration of glutathione, and can be processed to obtain glutathione-containing fractions. As a method for separating glutathione-containing fractions from the culture medium, any commonly used method can be used, examples include hot water extraction, extraction by cell disruption, etc. Furthermore, the glutathione-containing fractions can be concentrated to a high concentration by supporting the obtained extract on a carrier. In addition, yeast extracts can be produced from the cultures obtained by the above-described culture methods. As a method for preparing yeast extracts, any conventional method can be used, and self-digestion, enzymatic hydrolysis, or alkaline extraction methods are all industrially applicable.

[0035] Furthermore, dried cells can be obtained from the culture medium cultured using the methods described above. Any method can be used to prepare dried yeast cells, as long as it is a commonly used method; however, freeze-drying, spray drying, and drum drying are commonly employed industrially.

[0036] One embodiment of the present invention provides a composition for reducing oxidative stress, an antioxidant composition, or a composition for preventing, improving, alleviating, or treating oxidative stress-related diseases, comprising at least one selected from the group consisting of: cells of a Candida utilis strain, a culture of the strain, lysates of the cells, fragments of the cells, and extracts thereof. The composition may be a pharmaceutical composition, a food composition, or a cosmetic composition.

[0037] This invention relates to the use of Candida utilis strains as antioxidants, which are now applicable to the treatment of disorders, symptoms, pathologies and diseases caused by or related to the adverse effects of oxidative stress and / or free radicals in the body's cells, tissues and organs.

[0038] Oxidative stress damages proteins, DNA, and lipids, and plays a crucial role in the progression of neurodegenerative and age-related diseases. Low-molecular-weight hydrophobic antioxidant compounds are beneficial for conditions such as acute respiratory distress syndrome, amyotrophic lateral sclerosis (ALS), atherosclerotic cardiovascular disease, multiple organ dysfunction syndrome, and peripheral systemic symptoms of central nervous system neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, and Kreutzfeldt-Jacob disease. Oxidative stress is known to have a causal relationship with the pathogenesis of Parkinson's disease, Alzheimer's disease, Kreutzfeldt-Jacob disease, and other types of diseases.

[0039] A lack of antioxidants in cells produces excess free radicals, leading to macrocell damage, lipid peroxidation, and toxin accumulation, ultimately resulting in cell death. Due to the importance of antioxidant compounds in preventing cellular oxidation, tissues receive a continuous supply of natural antioxidants, such as glutathione (GSH) ([γ]-glutamylcysteine). GSH is synthesized in almost all cells and is one of the essential cellular antioxidants responsible for maintaining proper oxidative states in the body. When oxidized, glutathione forms a dimer called GSSG, which is recycled in organs that produce glutathione reductase. In adults, reduced GSH is primarily produced by GSSG in the liver and synthesized in relatively small amounts in skeletal muscle, erythrocytes, and leukocytes, and is also transported to other tissues of the body via the bloodstream.

[0040] However, under certain conditions, the normal physiological supply of GSH is insufficient, its distribution is inadequate, or excessive local oxidative demand prevents it from preventing cellular oxidation. Under other conditions, the production and demand of cellular antioxidants such as GSH are mismatched, resulting in insufficient concentrations of these molecules in the body. In other cases, antioxidants are depleted by certain tissues or biological pathways, thereby inhibiting the concentration of intracellular antioxidants. In any of the above situations, increased serum concentrations of antioxidants such as glutathione will increase the amount of antioxidants entering the cells. This leads to an enhanced concentration gradient for induced uptake in transport systems used for cellular uptake.

[0041] This invention is intended to prevent, alleviate, or treat symptoms, diseases, disorders, or pathological conditions associated with excessive antioxidant production, wherein said symptoms, diseases, disorders, or pathological conditions are AIDS, diabetes, macular degeneration, congestive heart failure, cardiovascular disease, coronary restenosis, lung disease, inflammatory disease, asthma, RNA virus infection, DNA virus infection, sepsis, osteoporosis, bone disease, microbial infection, toxin exposure, radiation exposure, burns, prions, nervous system diseases, blood diseases, blood cell diseases, arterial diseases, and muscle diseases.

[0042] Glutathione-containing yeast extract refers to a cell extract that is rich in glutathione, a tripeptide composed of the amino acids glutamic acid, cysteine, and glycine. In the composition according to the invention for the prevention or treatment of hangovers, the glutathione-containing yeast extract preferably contains more than 5% glutathione.

[0043] The food composition according to the present invention can be any food or beverage to which dry yeast or yeast extract can be added, but examples include alcoholic beverages, soft drinks, fermented food seasonings, soups, general foods, candies, etc. Therefore, the present invention can effectively produce foods and beverages containing high concentrations of glutathione.

[0044] One embodiment of the present invention provides a composition for breaking down alcohol; a composition for preventing, improving, alleviating, or reducing hangovers; or a composition for preventing, improving, alleviating, or treating alcoholic liver disease, comprising at least one selected from the group consisting of: cells of a Candida utilis strain, a culture of the strain, lysates of the cells, fragments of the cells, and extracts thereof. Alcoholic liver disease includes, but is not limited to, alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, and alcoholic cirrhosis. The composition may be a pharmaceutical composition or a food composition.

[0045] The *Candida utilis* strain of the present invention has alcohol tolerance and alcohol decomposition activity, thus contributing to hangover relief and liver detoxification due to alcohol decomposition before or after drinking. The present invention also relates to pharmaceutical compositions, food compositions, health functional foods, and food additives using microbial cells of the strain that can prevent, alleviate, improve, or treat alcoholic liver disease.

[0046] In addition to the main components, the hangover-related compositions of the present invention may also contain vitamins such as B vitamins, vitamin C, vitamin E, or beta-carotene; minerals such as Ca, Mg, or Zn; phospholipids such as lecithin; amino acids such as alanine or taurine; fructose; oligosaccharides; Ganoderma lucidum; or mixtures thereof as auxiliary components. Components such as amino acids contained in yeast extracts have the function of promoting alcohol metabolism, thereby reducing, improving, or preventing hangovers.

[0047] The *Candida utilis* strain of this invention exhibits alcohol tolerance and alcohol-degrading ability, thus it can prevent alcoholic liver disease by helping to alleviate hangovers before or after drinking and by aiding liver detoxification. Because the strain of this invention possesses excellent alcohol (ethanol)-degrading enzyme activity, it can effectively inhibit the absorption of ingested alcohol (ethanol), thereby preventing liver dysfunction, alcoholic liver disease, and alcoholic intestinal disorders caused by excessive alcohol metabolism. Furthermore, because the strain of this invention produces glutathione, it can prevent, improve, or reduce hepatotoxicity caused by toxic substances produced during alcohol metabolism, and promote the excretion of alcohol metabolites.

[0048] The pharmaceutical compositions according to the invention can be prepared into conventional dosage forms by selecting and adding pharmaceutically acceptable carriers and additives in addition to the active ingredient. The pharmaceutical compositions can be formulated into solutions, suspensions, powders, granules, tablets, capsules, pills, or extract formulations for oral administration, but are not limited thereto. Pharmaceutically acceptable carriers may be selected from one or more of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and pharmaceutically acceptable additives include at least one selected from flavoring agents, coloring agents, friction reducers, and acidifiers.

[0049] In addition, as additives to enhance the taste, natural flavorings such as plum, lemon, pineapple and vanilla, natural colorings such as natural fruit juice, chlorophyll and flavonoids, sweeteners such as fructose, honey, sugar alcohol and sugar, and acidifiers such as citric acid and sodium citrate can be used.

[0050] To achieve the effect of preventing or treating hangovers, the pharmaceutical composition can be administered orally multiple times, such that for an adult weighing 60 kg, the total daily dose based on the active ingredient is 0.3-10 g, preferably 0.7-4.2 g, but not limited thereto. Appropriate amounts can be used to achieve the desired effect, but are not limited thereto.

[0051] Furthermore, the compositions of the present invention can be used as health supplements. These health supplements can be formulated into teas, jellies, liquid extracts, beverages, etc., using extracts as active ingredients.

[0052] Invention Effects

[0053] This invention relates to Candida utilis strains with tripeptide production capacity and / or high ADH activity, compositions for breaking down alcohol, compositions for relieving, improving or preventing hangovers, or compositions for relieving oxidative stress or antioxidant compositions. Attached Figure Description

[0054] Figure 1 A graph illustrating the glutathione production capacity of the candidate strains initially selected according to the present invention.

[0055] Figure 2 A graph illustrating the NADH concentration produced per unit time by the candidate strains initially selected according to the present invention.

[0056] Figure 3 A graph illustrating the rate of change of glutathione content depending on the sucrose supply rate according to the present invention.

[0057] Figure 4 A graph showing the ethanol content in the culture medium depending on the sucrose supply rate according to the present invention.

[0058] Figure 5 A graph illustrating the change in cell concentration over time depending on the sucrose injection rate according to the present invention. Detailed Implementation

[0059] The present invention will be described in more detail with reference to the following embodiments, but the present invention is not limited to the following embodiments.

[0060] Example 1: Isolation of glutathione-producing microorganisms

[0061] 1-1: Strain Culture

[0062] To screen for glutathione-producing microorganisms, makgeolli (rice wine), koji (fermented rice wine starter), and traditional soybean paste were purchased from traditional markets throughout South Korea and used as samples. 1 g of sample was suspended in 10 mL of 0.85% NaCl, and 100 μL of the suspension was spread onto YPD (yeast extract 10 g / L, peptone 20 g / L, dextrose 20 g / L) agar plates and incubated on solid-state at 30°C for 2 days. 150 colonies were isolated from the colonies grown on solid-state media by selecting colonies of different shapes and sizes. These colonies were then cultured in test tubes containing YPD broth (yeast extract 10 g / L, peptone 20 g / L, dextrose 20 g / L) at 30°C with stirring for 2 days to obtain cell cultures.

[0063] 1-2: Measurement of cell growth extent (absorbance)

[0064] Cell concentration was determined by measuring the absorbance of cell cultures at 600 nm, and the results were expressed as cell optical density (OD) values.

[0065] 1-3: Determination of glutathione content in culture medium

[0066] The bacterial culture was centrifuged to remove the supernatant, and the cells were collected after washing once with distilled water. 40–70% ethanol was added to the collected cells, and intracellular glutathione was extracted using a fine mixer for 10–30 minutes. After centrifugation of the extract, the supernatant was reacted with 10 mM DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)) dissolved in 0.5 M pH 8.0 potassium phosphate buffer at 40°C for 20 minutes. The glutathione content (GSH mg / L) was determined by measuring the absorbance at 412 nm. DTNB, commonly used for glutathione content analysis, is known as Ellman's reagent and is used to detect thiols. GSH reacts with DTNB to produce yellow 2-nitro-5-benzoic acid and GSSG; the concentration of GSH can be calculated by measuring the OD value at 412 nm. GSSG is reduced to GSH by glutathione reductase and reacts again with DTNB, forming a cycle.

[0067] 1-4: Measurement of glutathione content per cell dry weight (g)

[0068] The bacterial culture was centrifuged to remove the supernatant and washed once with distilled water to collect the cells. The absorbance of the collected cells was measured, and the weight of the dried cells was calculated based on the absorbance. Specifically, to measure the dry weight of the cells (g), the cell culture was centrifuged to remove the supernatant, and only the cells were collected. The collected cells were washed with 0.9% NaCl and diluted with distilled water to prepare samples with an absorbance of 0.1–1. For the diluted bacterial samples, the absorbance was measured at 600 nm, and the dry weight of the cells was calculated based on the absorbance. After measuring the absorbance, the cells were filtered through 0.2 μm filter paper under reduced pressure. The filter paper containing the cells was dried at 60°C for more than 12 hours, placed in a desiccator containing silica gel for more than 6 hours, and then weighed. The amount of dried cells was determined by calculating the weight difference between the empty filter paper and the filter paper containing the filtered cells. Therefore, the concentration of dried cells (g / L) can be determined based on the absorbance value.

[0069] After measuring absorbance, glutathione was extracted from the cells in essentially the same manner as in Examples 1-3. The extract was centrifuged to obtain the supernatant, and the amount of glutathione produced (g / L) was measured. The measured amount of glutathione was divided by the calculated stem cell weight (g / L) and then multiplied by 100 to calculate the GSH% per stem cell weight (g). The glutathione content per cell dry weight (g) was measured and is shown in Table 1 as GSH (%) / g-cells.

[0070] The results of measuring the glutathione production of 150 strains using this method showed that the glutathione yield per cell dry weight (g) (GSH(%) / g-cell) ranged from approximately 0.3 to 2% by weight. Based on the glutathione content per cell dry weight (g), the top seven (70) candidate strains were isolated. As a result of the analysis of the selected seven strains, the cell OD of Examples 1-2, the glutathione content of Examples 1-3, and the glutathione content per cell dry weight (g) are shown in Table 1. Figure 1 middle.

[0071] As a control, the same experiment was performed on the *Candida utilis* KCCM 11355 standard strain, and the results are shown in Table 1 and... Figure 1 middle.

[0072] [Table 1]

[0073]

[0074] As shown in Table 1, as a result of the analysis of cell growth and glutathione production in ethanol-free medium, the glutathione content of the seven selected strains was higher than that of the standard strain *Candida utilis* KCCM 11355, specifically by approximately 0.7–2% by weight. The six strains other than SYC-7D showed the same or higher levels of cell growth as the standard strain, thus exhibiting more desirable characteristics.

[0075] Therefore, considering glutathione yield (mg / L) and glutathione yield per cell dry weight (GSH(%) / g-cell) as selection criteria, SYC-7D, SYC-JH, SYC-P1, SYC-P3, SYC-PR9, SYC-PR19, and SYC-PR20 can be selected. Preferably, SYC-7D, SYC-JH, SYC-P1, SYC-P3, SYC-PR9, SYC-PR19, and SYC-PR20 have a glutathione yield per cell dry weight (g) (GSH(%) / g-cell) of 0.8% by weight or more, or more preferably, SYC-PR9, SYC-PR19, and SYC-PR20 have a glutathione yield per cell dry weight (g) (GSH(%) / g-cell) of 1.5% by weight or more.

[0076] In particular, when considering large-scale industrial production, it is more preferable to consider both cell OD value and glutathione yield per cell dry weight (GSH(%) / g-cell). From this perspective, strains SYC-P1, SYC-PR9, SYC-PR19, and SYC-PR20, which have higher cell OD values ​​and higher glutathione yields than standard strains, are confirmed to be more preferred.

[0077] Example 2: Assay of alcohol dehydrogenase (ADH) activity

[0078] ADH activity was measured by culturing seven (7) strains selected in Example 1 that had high glutathione content.

[0079] Specifically, ADH activity was analyzed using an ADH activity assay kit (Abcam). Cells were cultured in YPD medium at 30°C for 24–48 hours, and then collected to a volume of 1 × 10⁻⁶ cells. 6 CFU / ml. Collected cells were washed with distilled water, then ADH analysis buffer was added, and cell walls were disrupted using a bead beater. As components of the reaction solution, 50 μl of sample or NADH standard was mixed with 82 μl of ADH analysis buffer, 8 μl of chromogenic solution, and 10 μl of isopropanol for each concentration. After reacting at 37°C for 3 minutes, the absorbance of the experimental group (A0) and the control group was measured at 450 nm. After reacting for another 30 minutes at 37°C, the change in absorbance at 450 nm was measured, and the amount of NADH produced per unit time (minute) was calculated to compare the ADH activity of each strain. Figure 2 The NADH production of each strain is shown. It was confirmed that the NADH production of these seven strains ranged from 24 to 160 nmol. As a control, the same experiment was performed on the *Candida utilis* KCCM 11355 standard strain, and the results are shown in Table 2.

[0080] Table 2 shows the results of comparing the ADH activity of seven bacterial strains with the supernatant obtained after yeast cell lysis. The values ​​listed in Table 2 refer to the titer (mUnit, mU) of alcohol dehydrogenase (ADH) per reaction volume (ml), expressed in mU / ml. This represents the ADH activity calculated based on the concentration of NADH produced per unit time (minute). Additionally, the concentration of NADH produced per unit time (minute) is as follows: Figure 2 As shown.

[0081] [Table 2]

[0082]

[0083]

[0084] As shown in Table 2, the ADH activities of the seven selected strains ranged from 0.03 to 0.26 mU / ml. The strains with higher ADH activity than the standard strain *Candida utilis* KCCM 11355 (control) were SYC-P1, SYC-PR9, SYC-PR19, and SYC-PR20, with SYC-PR20 exhibiting the highest activity. Therefore, in terms of ADH activity, SYC-P1, SYC-PR9, SYC-PR19, and SYC-PR20 can be considered as secondary selections. Preferably, SYC-P1, SYC-PR9, and SYC-PR20 with an ADH activity of 0.20 mU / ml or higher can be selected.

[0085] Considering the combination of the glutathione content per cell dry weight (g) shown in Table 1 of Example 1 and the ADH activity analysis results in Table 2, strains with high glutathione yield and ADH activity are preferred. Therefore, SYC-PR9, SYC-PR19, and SYC-PR20 can be selected as secondary candidate strains.

[0086] Example 3: Evaluation of cell growth and GSH production based on culture time

[0087] For SYC-PR20 selected in Example 1, cell growth and GSH production were evaluated based on culture time.

[0088] YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L dextrose) was prepared as the culture medium for culturing strain SYC-PR20. 3 ml of the medium was dispensed into test tubes and cultured with shaking at 30°C for 24–60 hours. Culture medium was collected at 24, 36, 48, and 60 hours, and cell OD values, glutathione content, and glutathione content per cell dry weight (g) were measured. The results are shown in Table 3 below.

[0089] [Table 3]

[0090]

[0091] As a result of measuring cell OD values ​​and GSH production over bacterial strain culture times from 24 to 60 hours, cell OD increased from 12 at 24 hours, 15 at 36 hours, 18 at 48 hours, and 19 at 60 hours. GSH production was confirmed to be 69 mg / L at 24 hours, 82 mg / L at 36 hours, 111 mg / L at 48 hours, and 120 mg / L at 60 hours. Therefore, the GSH% per gram of cell dry weight was confirmed to increase from 1.4% at 24 hours to 1.6% at 60 hours.

[0092] Example 4: Evaluation of microbial tolerance to ethanol

[0093] For the SYC-PR20 selected in Example 1, cell growth was evaluated in an ethanol-containing culture medium.

[0094] Specifically, 5×YPD (yeast extract 50 g / L, peptone 100 g / L, dextrose 100 g / L) medium was prepared and mixed with 100% ethanol to achieve a final ethanol concentration of 0, 2, 4, 6, 8, 10, or 15% (v / v). 3 ml of each concentration of ethanol-containing medium was dispensed into test tubes and incubated at 30°C with shaking for 60 hours to obtain the culture medium. Cell concentration was analyzed by measuring absorbance at 600 nm in essentially the same manner as in Examples 1-2, and the results are shown in Table 4 below.

[0095] As a control, the same experiment was performed on the standard strain of Candida utilis KCCM 11355, and the results are shown in Table 4.

[0096] [Table 4]

[0097]

[0098] Saccharomyces cerevisiase is considered a strain with high alcohol tolerance, capable of growing at 7-11% (v / v) ethanol concentrations. The SYC-PR20 strain, selected twice according to Examples 2 and 3, exhibits high alcohol tolerance, particularly showing high cell growth in alcohol-containing media compared to the Candida utilis KCCM 11355 standard strain (control group), especially exceeding 110% cell growth at 0.5-15% (v / v) ethanol concentrations.

[0099] In Table 4, the SYC-PR20 strain, exhibiting relatively high ADH activity, showed higher cell concentrations than the control group at the same ethanol concentration. Therefore, this confirms that the SYC-PR20 strain possesses excellent glutathione production capacity, ADH activity, and alcohol tolerance.

[0100] Example 5: Strain Identification

[0101] In Example 4, the SYC-PR20 strain exhibited the most preferred characteristics among the seven selected strains, with a glutathione yield of 1.6% and an ADH activity of 0.26 mU / ml.

[0102] The 18S rRNA sequence of strain SYC-PR20 was analyzed using universal primers ITS1 (SEQ ID NO: 2: 5'-TCCGTAGGTGAACCTGC GG-3') and ITS4 (SEQ ID NO: 3: 5'-TCCGTAGGTGAACCTGCGG-3'). The 18S rDNA sequence of strain SYC-PR20 is shown in SEQ ID NO: 1. As a result of strain identification based on the 18S rDNA sequence, it was identified as *Candida utilis* (Pichia jadinii).

[0103] The Candida utilis strain SYC-PR20 was deposited on August 7, 2020, at the Korea Microbial Collection Center located at 45 Hongjenae 2-ga, Seodaemun-gu, Seoul, with the accession number KCCM 12777P.

[0104] Example 6: Evaluation of glutathione yield in fermenter culture

[0105] Under flask culture conditions used for microbial isolation and identification, such as in Examples 1-4, mechanical limitations make it difficult to increase stirring speeds beyond a certain level and to control a constant culture environment (air volume, pH, etc.). On the other hand, in industrial-scale cultivation, by supplying air to the culture medium and stirring, the medium components and oxygen are evenly distributed throughout the space, and temperature, pH, etc., can be adjusted, allowing cell culture to proceed under optimized conditions. Therefore, fermenter culture exhibits a higher cell growth rate than flask culture, resulting in rapid consumption of added sugar and increased productivity. Thus, fermenter culture experiments for industrial applications of microorganisms are needed.

[0106] Specifically, colonies formed on YPD agar plates inoculated with Candida utilis strain SYC-PR20 according to Example 5 were inoculated into 3 mL of YPD broth (10 g / L yeast extract, 20 g / L peptone, 20 g / L dextrose) and cultured at 30 °C and 240 rpm for 24 hours to obtain seed cultures. 3 mL of the previously obtained seed culture was then inoculated into 100 mL of the same YPD broth and cultured under the same conditions to prepare seed cultures for use in a 5 L fermenter.

[0107] To evaluate cell growth and glutathione production based on carbon source, 5L fermenter cultures were performed. Specifically, glucose-based and sucrose-based media were prepared in the 5L fermenter with the media compositions shown in Table 5, with a final culture volume of 2L. The prepared seed cultures for the 5L fermenter culture were inoculated into the 5L fermenter and cultured with stirring under the conditions shown in Table 6.

[0108] [Table 5]

[0109]

[0110] [Table 6]

[0111] Cultivation conditions Primary Seed Culture Seed culture Main training volume 3mL 100mL 2L temperature 30℃ 30℃ 28℃ Stirring speed 240rpm 240rpm 500rpm Culture pH No control No control 5.0 Training time 24 hours 24 hours 29 hours

[0112] The cell concentration and glutathione yield of the obtained culture medium were measured in essentially the same manner as in Example 1, and the results are shown in Table 7 below.

[0113] [Table 7]

[0114]

[0115] As shown in Table 7, both glucose-based and sucrose-based cultures essentially consumed the initial sugar input by the end of 29 hours of culture. The OD value at 600 nm was used as the cell concentration at this point, which was 28.4 for glucose-based culture and 31.9 for sucrose-based culture, indicating a 112% increase in cell yield compared to the sucrose-based culture. The glutathione content based on sucrose was 223.3 mg / L, showing approximately 122% of the result for the same culture time as the glucose-based culture.

[0116] Example 7: Evaluation of glutathione yield based on culture conditions

[0117] Colonies formed on YPD agar plates inoculated with Candida utilis strain SYC-PR20 according to Example 5 were inoculated into 3 mL of YPD broth (10 g / L yeast extract, 20 g / L peptone, 20 g / L dextrose) and cultured at 30 °C and 240 rpm for 24 hours to obtain seed cultures. 3 mL of the previously obtained seed culture was then inoculated into 100 mL of the same YPD broth and cultured under the same conditions to prepare seed cultures for use in a 5 L fermenter.

[0118] To evaluate the high concentration of cells and the yield of glutathione, seed cultures and 5L fermenter cultures were prepared according to the method in Example 5. In batch culture, when the cumulative ethanol concentration after initial sugar consumption was below 5 g / L, fed-batch culture was carried out by continuously supplying a sugar solution containing 600 g / L sucrose to maintain high cell concentration and glutathione production.

[0119] As a result, the change in glutathione content in the culture medium over time was determined according to the sucrose feeding rate and shown in the figure. Figure 3 In this study, the change in ethanol content in the culture medium over time was determined and presented. Figure 4 In addition, the change in cell concentration with sucrose feeding rate was measured and shown in [the figure]. Figure 5 In. Figure 3 , Figure 4 and Figure 5 In the diagram, the white spheres indicate a sucrose feeding rate of 4.5 g / L·h. -1 The black spheres indicate a sucrose feeding rate of 6 g / L·h. -1 .

[0120] When at 6g / L·h -1 When sucrose is fed at a high rate, the ethanol content in the culture medium remains above 3 g / L, therefore the rate of change in glutathione content is almost constant. On the other hand, at a low sucrose feeding rate (4.5 g / L·h), the ethanol content in the culture medium remains above 3 g / L, thus the rate of change in glutathione content is almost constant. -1 Under these conditions, ethanol content remained at a low level, and intracellular glutathione content decreased. This fact indicates that when the sucrose feeding rate is high, the energy required for glutathione biosynthesis is adequately provided by the sufficient sugar supply in the culture medium, but when the sucrose feeding rate is low, the energy supply is insufficient, leading to restricted cell growth and reduced glutathione content.

[0121] Therefore, when the *Candida utilis* strain SYC-PR20 according to the present invention is used at 6 g / L·h -1 When the sugar feeding rate is set, ethanol accumulates in the culture medium as a cell metabolite due to the ample sugar supply, and the glutathione content can be maintained at the level of batch culture in the high-concentration culture portion, which is therefore preferred.

[0122] [Registration Number]

[0123] Name of depository: Korea Center for Microbial Preservation (overseas)

[0124] Registration No.: KCCM12777P

[0125] Registration Date: 20200807

[0126] (Translation)

[0127] Registration certificate for microbial preservation used in patent procedures

[0128] To: Sanyo Co., Ltd.

[0129] No. 295, Banqiao Road

[0130] Bundang-gu, Seongnam City, Gyeonggi Province

[0131] Postal code: 13488

[0132]

[0133] The above translation is identical to the original text. <110> Sanyo Co., Ltd. <120> Microorganisms with high tripeptide production capacity and their uses <130> KHP232110962.6 <150> KR 10-2020-0177834 <151> 2020-12-17 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 473 <212> DNA <213> Artificial Sequence <220> <223> 18S rDNA of Candida utilis <400> 1 acctgggcct gcgcttctag cgcggctcca accaatacac agtgtatttt gcttcttttg 60 ctttggctct gccaaaggtt ttaaacacag aaatttattt tctctagaaa ctagtcaatt 120 tgaattttaa tcttcaaaac tttcaacaac ggatctcttg gttctcgcaa cgatgaagaa 180 cgcagcgaaa tgcgatacgt aatgtgaatt gcaggttttc gtgaatcatc gaatctttga 240 acgcatattg cgctctctgg cattccagag agcatgcctg tttgagcgtc atttctctct 300 caagatcctc taggggactt ggtattgagt gatactctgt gttaacttga aatactctag 360 gcagagctcc ccctagaaat cctctgggcc gaaataatgt attaggttct accaactcgt 420 tattttccag acagacttcc aggcagagct cggctgaaca acctttctaa gct 473 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer ITS1 <400> 2 tccgtaggtg aacctgcgg 19 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer ITS4 <400> 3 tccgtaggtg aacctgcgg 19

Claims

1. A strain of Candida utilis possessing alcohol dehydrogenase (ADH) activity, alcohol tolerance, and glutathione production capacity. The registration number of the *Candida utilis* strain mentioned therein is KCCM 12777P.

2. The strain according to claim 1, wherein the ADH activity per gram of dry cell weight is ≥0.18 mU / ml and the glutathione yield per gram of dry cell weight is ≥0.8% by weight.

3. The strain according to claim 1, (i) wherein the strain has alcohol tolerance capable of growing at ethanol concentrations of 2-15% (v / v), and (ii) wherein, when cultured at an ethanol concentration of 6-15% (volume / volume), the strain has a cell optical density (OD) value of 120-200% based on 100% of the cell optical density (OD) value of Candida utilis with accession number KCCM11355.

4. An antioxidant composition comprising at least one selected from the group consisting of: cells of a Candida utilis strain as described in any one of claims 1 to 3, a culture of the strain, lysates of the cells, fragments of the cells, and extracts thereof.

5. A composition for the decomposition of alcohol, the composition comprising at least one selected from the group consisting of: cells of a Candida utilis strain as described in any one of claims 1 to 3, a culture of said strain, lysate of said cells, fragments of said cells, and extracts thereof.

6. The composition for decomposing alcohol according to claim 5, wherein the alcohol dehydrogenase (ADH) activity of the strain is greater than 0.18 mU / ml per gram of cell dry weight.

7. A composition for reducing, improving, alleviating or preventing hangovers, said composition comprising at least one selected from the group consisting of: cells of a Candida utilis strain as described in any one of claims 1 to 3, cultures of said strains, cell lysates, cell fragments, and extracts thereof.

8. A method for increasing glutathione production by culturing *Candida utilis* strains according to any one of claims 1 to 3.

9. The method of claim 8, wherein the strain is cultured by using sucrose or glucose as a carbon source.

10. The method of claim 8, wherein the strain is cultured by measuring the ethanol content in the fermentation broth and adjusting the carbon source supply rate.

11. The method of claim 8, wherein the strain is cultured by adding cysteine.

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