Microorganisms with high tripeptide production ability and uses thereof

Abstract

The present invention relates to a Candida utilis strain having high tripeptide-producing ability and / or alcohol decomposition enzyme activity, and uses thereof.

Description

[Technical field]

[0001] The present invention relates to a Candida utilis strain having high tripeptide-producing ability and / or alcohol decomposition enzyme activity, and uses thereof. [Background technology]

[0002] Unlike other foods, alcohol is a type of xenobiotic that does not accumulate in the body, and its decomposition products such as acetaldehyde are known to be the main cause of cell damage and hangover symptoms due to oxidative stress. Alcohol has various effects on liver metabolism depending on the amount consumed, and nicotinamide adenine dinucleotide phosphate (NADP) and acetaldehyde produced during the oxidative decomposition process, rather than alcohol itself, act as the main mediator of liver damage through condensation reactions with bioactive amines. At the same time, acetaldehyde delivered to the brain is transformed into many harmful compounds, causing hangover symptoms such as facial flushing, increased pulse, nausea, and vomiting.

[0003] 80-90% of alcohol that enters the body is first broken down into acetaldehyde by alcohol dehydrogenase (ADH) present in liver cells, and the acetaldehyde is then metabolized again by aldehyde dehydrogenase (ALDH) enzyme to form acetic acid, after which it is hydrolyzed into carbon dioxide and water and completely broken down. The key step in ethanol breakdown in the liver is the conversion to acetaldehyde via an oxidation reaction by ADH.

[0004] Glutathione (L-γ-glutamyl-L-cysteinylglycine, GSH) is a biologically active substance present in cells, and is a tripeptide composed of three amino acids, glutamate, cysteine, and glycine. It exists in the body in two forms: reduced glutathione (GSH) and oxidized glutathione (GSSG). Glutathione is present in animal, plant, and microbial cells at concentrations of 0.1-10 mM, and accounts for more than 90% of the total non-protein active matter in cells. The diverse functions of glutathione are important in many medical fields, including enzymology, transport, pharmacology, therapy, toxicology, endocrinology, and microbiology, as well as in the agricultural field.

[0005] Glutathione can be produced mainly by fermentation or enzyme synthesis using microorganisms, but currently, the enzyme synthesis process has not yet been commercialized due to the high production cost. On the other hand, the method of culturing microorganisms and extracting it from the cells is widely used industrially. As a microbial strain for glutathione production, yeast, which contains a high content of glutathione in its cells and is recognized as a safe microorganism for food production, is widely used, and in particular, Saccharomyces and Candida strains are representative. In the case of wild-type strains of this yeast species, the glutathione concentration is already very high at about 0.1-1% of the cell dry weight, and the advantages of high-density cell culture and fast growth in a low-cost medium make the fermentation production of glutathione using yeast competitive. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides a Candida utilis strain having tripeptide-producing ability and / or high ADH activity, a composition for decomposing alcohol, a composition for relieving, improving, or preventing a hangover, a composition for reducing oxidative stress, or an antioxidant composition using the same. [Means for solving the problem]

[0007] One example of the present invention provides a Candida utilis strain having high ADH activity.

[0008] One example of the present invention provides a Candida utilis strain having tripeptide producing ability and high ADH activity.

[0009] Another example of the present invention provides a composition for alcohol decomposition, comprising at least one selected from the group consisting of a Candida utilis strain, a culture of the strain, a lysate of the strain, a homogenate of the strain, and extracts thereof.

[0010] Another example of the present invention provides a composition for relieving, improving, or preventing a hangover, comprising one or more selected from the group consisting of cells of the Candida utilis strain, a culture of the strain, a disrupted product of the cells, a lysate of the cells, and extracts thereof.

[0011] Another example of the present invention provides a composition for reducing oxidative stress or an antioxidant composition, comprising at least one selected from the group consisting of a Candida utilis strain, a culture of the strain, a disrupted product of the strain, a lysate of the strain, and extracts thereof.

[0012] Another example of the present invention provides a composition for preventing, ameliorating, or treating an oxidative stress-related disease, comprising at least one selected from the group consisting of cells of the Candida utilis strain, a culture of the strain, a disrupted cell, a lysate of the cell, and extracts thereof.

[0013] A further embodiment of the present invention provides a food composition comprising at least one selected from the group consisting of cells of the Candida utilis strain, a culture of the strain, a disrupted product of the cells, a lysate of the cells, and extracts thereof. [Brief description of the drawings]

[0014] [Figure 1] 1 is a graph showing glutathione productivity of candidate strains primarily screened according to the present invention. [Diagram 2] 1 is a graph showing the NADH concentration produced per unit time of candidate strains primarily screened according to the present invention. [Diagram 3] 1 is a graph showing the rate of change in glutathione content depending on the sucrose feeding rate according to the present invention. [Figure 4] 1 is a graph showing the ethanol content in a culture medium according to the sucrose injection rate according to the present invention. [Diagram 5] 1 is a graph showing the change in bacterial cell concentration over time depending on the sucrose injection rate according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The Candida utilis strain according to the present invention has glutathione production ability and / or alcohol dehydrogenase (ADH) activity, and more specifically, has high glutathione production ability as well as alcohol dehydrogenase activity, which is advantageous in more smoothly detoxifying and excreting alcohol from the body. The yeast strain according to the present invention is a non-GMO strain isolated from nature, not produced by recombinant technology, and has the advantage of being highly usable.

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

[0017] The Candida utilis strain of the present invention has an alcohol decomposition ability (alcohol dehydrogenase, ADH) and alcohol tolerance, specifically, has alcohol tolerance that allows it to grow under conditions where the ethanol concentration is 2 to 15% (v / v). More specifically, when cultured under conditions where the alcohol concentration is 6 to 15% (v / v), the Candida utilis strain of the present invention may have a cell optical density (OD) value of 120 to 200% based on the cell optical density (OD) value of Candida utilis having deposit number KCCM 11355 being 100%.

[0018] The Candida utilis strain according to the present invention (i) exhibits increased glutathione production during shaking culture at 30°C, and (ii) exhibits a significantly increased glutathione production rate when cultured in a sucrose-containing medium compared to a bacterial cell concentration (OD value at 600nm) of 100% when cultured in a glucose-containing medium under shaking culture conditions. (iii) the OD value of the culture medium may have a lower limit of 105%, 110%, or 115% or more, and the upper limit of the bacterial cell concentration may be 120% or less, or may have a range that combines the lower limit and the upper limit, for example, 105 to 120%, or, based on the glutathione content (mg / L) of 100% when cultured in a glucose-containing medium under shaking culture conditions, the glutathione content when cultured in a sucrose-containing medium may be more specifically 105% or more, 110% or more, or 115% or more, but may be 150% or less, 140% or less, 130% or less, or 120% or less, for example, 105 to 150%, or 110 to 140%, (iii) the amount of glutathione produced is increased when cultured with the addition of cysteine, and (iv) the feeding rate is 6 g / L h -1 The culture characteristics may be one or more selected from the group consisting of: maintaining the amount of glutathione produced under fed-batch culture conditions with the amount of glutathione supplied in the culture medium at the end of the batch, for example, having a glutathione content of 50% to 120% based on the glutathione content at the end of the batch, or having a glutathione content of 80% to 120% of the maximum glutathione content per batch at an ethanol concentration of 2 g / L or more.

[0019] The Candida utilis strain according to the present invention has alcohol tolerance and alcohol decomposition ability, and relates to a health functional food and food additive having alcohol decomposition ability that can be widely used in the manufacture of foods that can relieve hangovers by decomposing alcohol before or after drinking and can help the liver detoxify, thereby preventing alcoholic liver disease.

[0020] The bacterial strain of the present invention has excellent alcohol (ethanol) decomposition enzyme activity, and therefore can effectively suppress the absorption of ingested alcohol (ethanol), thereby preventing liver dysfunction and the occurrence of alcoholic liver disease and intestinal disease caused by excessive alcohol metabolism.

[0021] Specifically, alcohol cannot be stored in the body and must be metabolized, and this process is mostly carried out in the liver, where this metabolism is carried out by alcohol dehydrogenase present in the liver. Alcohol is broken down by this enzyme into a substance called acetaldehyde, and the acetaldehyde produced is toxic and damages liver cells, and as a result of alcohol metabolism, a large amount of fatty acids are produced, and fat accumulates in the liver, causing alcoholic liver disease. Such alcoholic liver disease is largely classified into alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, and alcoholic cirrhosis, and the mechanism of liver damage can be alcohol itself, metabolic products such as acetaldehyde, or immune reactions. In particular, acetaldehyde acts as the main culprit of liver toxicity, such as peroxidation of fat, binding to cytoplasm, disruption of the mitochondrial electron transport chain, interference with the function of microtubules, formation of binding substances with proteins, and increased collagen synthesis.

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

[0023] Specifically, glutathione is an antioxidant that promotes the excretion of toxic substances into urine and bile, and in particular, it acts on the process of alcohol metabolic products being detoxified in the liver to promote the excretion of toxic substances. In addition, glutathione protects liver cells from damage caused by harmful active oxygen, etc. Therefore, the strain according to the present invention has alcohol tolerance and alcohol decomposition ability, and not only has the function of preventing and decomposing alcohol absorption, but also promotes the excretion of toxic substances through the decomposition of alcohol due to its high glutathione content, thereby preventing liver damage and liver disease.

[0024] The present invention relates to a food, food additive, beverage or dietary supplement containing an ingredient effective in reducing blood alcohol levels and enhancing antioxidant capacity in the body.

[0025] The strain of the present invention can be aerobically cultured in a medium containing a carbon source, a nitrogen source, inorganic salts, and the like.

[0026] The medium composition for these strains can use one or more carbon sources selected from the group consisting of dextrin, glucose, sucrose, acetic acid, ethanol, molasses, and sulfite pulp waste liquor, which are commonly used in the cultivation of microorganisms, and sucrose is preferred in terms of glutathione production. More specifically, the Candida utilis strain of the present invention has (ii) a bacterial cell concentration in a sucrose-containing medium of 105-120% based on a bacterial cell concentration (OD value at 600 nm) of 100% when cultured in a glucose-containing medium under shaking culture conditions, or a glutathione content in a sucrose-containing medium of 105-150% or 110-140% based on a glutathione content (mg / L) of 100% when cultured in a glucose-containing medium under shaking culture conditions, (iii) an increase in glutathione production when cultured with the addition of cysteine, and (iv) a feeding rate of 6 g / L h -1and maintaining the amount of glutathione produced under fed-batch culture conditions with the amount of glutathione supplied by the strain.

[0027] As the nitrogen source, one or more selected from the group consisting of inorganic salts such as urea, ammonia, ammonium sulfate, ammonium chloride, or ammonium phosphate, and nitrogen-containing organic substances such as corn steep liquor (CSL), casein, yeast extract, or peptone, are used. Phosphate components, potassium components, and magnesium components may also be added to the medium, and these may be ordinary industrial raw materials such as calcium superphosphate, ammonium phosphate, potassium chloride, potassium hydroxide, magnesium sulfate, and magnesium hydrochloride. Inorganic salts such as zinc, copper, manganese, and iron ions may also be used. Vitamins, nucleic acid-related substances, and the like may also be added.

[0028] The culture temperature applicable to the present invention may be the culture conditions for yeast, for example, 20 to 40° C., preferably 25 to 35° C., and the pH is preferably 3.5 to 8.0, particularly preferably 4.0 to 6.0.

[0029] The culture format applicable to the present invention may be any of batch culture, fed-batch culture, and continuous culture, but in industrial applications, fed-batch culture or continuous culture is used.

[0030] The present invention provides a method for increasing the amount of glutathione produced by culturing the Candida utilis strain.

[0031] The strain may be cultured using sugar or glucose as a carbon source. The strain may 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 5 g / L or less, the carbon source may be continuously supplied, and the carbon source supply rate may be 5 to 10 g / L·h. -1 , 5.5-8g / L·h-1 , 5.5-7g / L·h -1 , or 5.5 to 6.5 g / h -1 may be also possible.

[0032] More specifically, in consideration of glutathione production, the Candida utilis strain according to the present invention is preferably cultured in a fed-batch manner, more preferably at a feeding rate of 6 g / L h -1 The amount of glutathione produced can be maintained under fed-batch culture conditions with the culture medium supplied with 0.1% glutathione.

[0033] Preferably, the method for increasing the amount of glutathione produced can be one or more methods selected from the group consisting of the type of carbon source, adjustment of the supply rate of the carbon source, cysteine ​​addition conditions, and other culture condition adjustments. For example, the method can be one or more methods selected from the group consisting of (i) cysteine-added culture, (ii) use of sucrose or glucose as a carbon source, (iii) adjustment of the carbon source supply rate, (iv) culture temperature, and (v) stirring conditions, specifically, one or more methods selected from the group consisting of shaking culture at 30°C, use of sucrose as a carbon source under shaking culture conditions, cysteine-added culture, and fed-batch culture. The fed-batch culture can be performed by adjusting the carbon source supply rate by measuring the ethanol content in the fermentation liquid, for example, a feeding rate of 6 g / L h. -1 This can be carried out in a fed-batch culture.

[0034] According to the method of the present invention, a culture containing high concentrations of glutathione in yeast cells can be obtained, and a fraction containing glutathione can be obtained from the culture. The method for fractionating a glutathione-containing fraction from a culture can be any method that is commonly used, and includes hot water extraction and extraction by cell disruption. In addition, it is possible to concentrate a fraction containing glutathione at a high concentration by supporting the obtained extract on a carrier. In addition, a yeast extract can be produced from the culture cultured by the above method. The method for producing a yeast extract can be any method that is commonly used, and autolysis, enzymatic decomposition, alkaline extraction, and the like are industrially adopted.

[0035] In addition, dried yeast cells can be produced from the culture obtained by the above-mentioned method. Any method that is commonly used may be used to produce dried yeast cells, but freeze-drying, spray-drying, drum-drying, etc. are used industrially.

[0036] One example of the present invention relates to a composition for reducing oxidative stress, an antioxidant composition, or a composition for preventing, improving, alleviating or treating an oxidative stress-related disease, comprising at least one selected from the group consisting of a Candida utilis strain, a culture of the strain, a disrupted product of the strain, a lysate of the strain, and extracts thereof. The composition may be a pharmaceutical composition, a food composition or a cosmetic composition.

[0037] The present invention provides the use of Candida utilis strains as antioxidants in novel applications for treating diseases, symptoms, pathologies and disorders resulting from or associated with the adverse effects of oxidative stress and / or the generation of free radicals in cells, tissues and organs of the body.

[0038] Oxidative stress plays a critical role in the progression of neurodegenerative and aging-related diseases, causing damage to proteins, DNA, and lipids. Low molecular weight hydrophobic antioxidant compounds are useful in treating peripheral tissue conditions such as acute respiratory distress syndrome, amyotrophic lateral sclerosis, atherosclerotic cardiovascular disease, multiple organ dysfunctions, and central nervous system degenerative neurological diseases such as Parkinson's disease, Alzheimer's disease, and Creutzfeldt-Jakob's disease. Oxidative stress is known to be causally linked to the pathogenesis of Parkinson's disease, Alzheimer's disease, and Creutzfeldt-Jakob's disease, as well as other types of diseases.

[0039] Lack of antioxidants in cells can lead to the generation of excessive free radicals, which can destroy large cells, peroxidize lipids, accumulate toxins, and ultimately cause cell death. Due to the importance of antioxidant compounds in preventing cellular oxidation, natural antioxidants such as glutathione (GSH) ([γ]-glutamylcysteinylglycine) are continuously provided to tissues. GSH is synthesized in almost all cells and is one of the fundamental cellular antioxidants responsible for maintaining an appropriate oxidative state in the body. When oxidized, GSH forms a dimer called GSSG, which is recycled in organs that produce glutathione reductase. In human adults, reduced GSH is primarily produced from GSSG in the liver, and in smaller amounts in skeletal muscles and red and white blood cells, where it can be transported via the bloodstream to other tissues in the body.

[0040] However, under some conditions, the normal physiological supply of GSH is insufficient, its distribution is inadequate, or local oxidative demand is too high to prevent cellular oxidation. Under other conditions, the production and demand of cellular antioxidants, e.g., GSH, are mismatched, resulting in insufficient concentrations of the molecule in the body. In other cases, antioxidants are depleted by certain tissues or biological pathways, suppressing intercellular antioxidant concentrations. In such cases, increasing serum concentrations of antioxidants, e.g., glutathione, increases the amount of antioxidant available to enter cells. In facilitated transport systems for cellular absorption, the concentration gradient that induces absorption is increased.

[0041] The present invention relates to a method for preventing, ameliorating, or treating a condition, disease, disorder, or pathological condition associated with overproduction of oxidants, wherein the condition, disease, disorder, or pathological condition can be selected from the group consisting of AIDS, diabetes, macular degeneration, congestive heart failure, cardiovascular disease, coronary artery restenosis, lung disease, inflammatory disease, asthma, RNA virus infection, DNA virus infection, sepsis, osteoporosis, bone disease, microbial infection, toxin exposure, radiation exposure, burn trauma, prion disease, neurological disease, blood disease, blood cell disease, arterial disease, and muscle disease.

[0042] The glutathione-containing yeast extract means an extract of fungal cells, and is rich in glutathione, a tripeptide composed of three amino acids, glutamic acid, cysteine, and glycine. In the composition for preventing or treating hangover according to the present invention, it is preferable to use a glutathione-containing yeast extract containing 5% or more of glutathione.

[0043] The food composition according to the present invention may be any food or drink to which dry yeast or yeast extract can be added, such as alcoholic beverages, soft drinks, fermented food seasonings, soups, general foods, confectioneries, etc. Thus, according to the present invention, food or drink containing high concentrations of glutathione can be efficiently produced.

[0044] One example of the present invention relates to a composition for alcohol decomposition comprising a Candida utilis strain, a culture of the strain, a disrupted product of the strain, a lysate of the strain, and one or more selected from the group consisting of the strain, the culture of the strain, the disrupted product of the strain, the lysate of the strain, and extracts thereof; a composition for hangover relief, improvement, reduction, or prevention; or a composition for preventing, improving, reducing, or treating alcoholic liver disease. The alcoholic liver disease includes, but is not limited to, alcoholic fatty liver, alcoholic hepatitis, alcoholic hepatic fibrosis, and alcoholic cirrhosis. The composition may be a pharmaceutical composition or a food composition.

[0045] The Candida utilis strain according to the present invention has alcohol tolerance and alcohol decomposition ability, and has a hangover relieving effect by decomposing alcohol before or after drinking and helps the liver detoxify, and relates to a pharmaceutical composition, food composition, health functional food, and food additive that can prevent, reduce, improve, or treat alcoholic liver disease using the bacterial cells of the strain.

[0046] In addition to the above main ingredients, the hangover-related composition of the present invention may further contain, as auxiliary ingredients to further enhance the hangover relieving effect, vitamins such as B vitamins, vitamin C, vitamin E, or beta-carotene, mineral ingredients such as Ca, Mg, or Zn, phospholipids such as lecithin, amino acids such as alanine or taurine, fructose, oligosaccharides, porcelain mushroom, or mixtures thereof. The amino acids and other ingredients contained in the yeast extract have the function of promoting alcohol metabolism and have the function of relieving, improving, or preventing a hangover.

[0047] The Candida utilis strain according to the present invention has alcohol tolerance and alcohol decomposition ability, and can prevent alcoholic liver disease by relieving hangovers and assisting the liver detoxification function by decomposing alcohol before or after drinking. The strain according to the present invention has excellent alcohol (ethanol) decomposition enzyme activity, and can effectively inhibit the absorption of ingested alcohol (ethanol), thereby preventing liver dysfunction, alcoholic liver disease, and intestinal disease caused by excessive alcohol metabolism. Furthermore, the strain according to the present invention produces glutathione, which can prevent, improve, or reduce liver toxicity caused by toxic substances generated during alcohol metabolism, as well as promote the excretion of alcohol metabolic products.

[0048] The pharmaceutical composition according to the present invention can be prepared in a conventional dosage form by adding pharma- ceutical acceptable carriers and additives in addition to the active ingredient. The pharmaceutical composition can be orally administered in the form of, but not limited to, a liquid, suspension, powder, granules, tablets, capsules, pills, or extract. The pharma-ceutical acceptable carrier can be one or more selected from diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and the pharma-ceutical acceptable additive can be one or more selected from flavors, dyes, antifriction agents, and acidulants.

[0049] In addition, additives for stimulating the taste can be used by mixing natural flavors such as plum flavor, lemon flavor, pineapple flavor, and herb flavor, natural fruit juice, natural colorings such as chlorophyllin and flavonoids, sweeteners such as fructose, honey, sugar alcohol, and sugar, or acidulants such as citric acid and sodium citrate.

[0050] The pharmaceutical composition may be orally administered several times a day in a total amount of 0.3 to 10 g, preferably 0.7 to 4.2 g, of an active ingredient based on an adult weighing 60 kg in order to obtain a hangover prevention or treatment effect, but is not limited thereto and may be used in an appropriate amount for the desired effect.

[0051] The composition of the present invention can also be used as a dietary supplement, which can be prepared as tea, jelly, extract, beverage, etc., containing the extract of the medicinal plant as an active ingredient. EXAMPLES

[0052] The present invention will be described in more detail with reference to the following examples, but it is not intended that the present invention be limited to the following examples.

[0053] Example 1: Isolation of glutathione-producing strains 1-1: Strain culture To search for glutathione-producing microorganisms, makgeolli, koji, traditional sauces, etc. were purchased from traditional markets nationwide and used as samples. 1g of sample was suspended in 10mL of 0.85% NaCl, and 100μl of the suspension was spread on a YPD (yeast extract 10g / L, peptone 20g / L, dextrose 20g / L) agar plate and cultured on a solid medium at 30℃ for 2 days. 150 colonies were isolated by selecting those with different shapes and sizes from the colonies grown on the solid medium, and then cultured in a test tube with YPD broth (yeast extract 10g / L, peptone 20g / L, dextrose 20g / L) at 30℃ for 2 days with shaking to obtain a bacterial strain culture.

[0054] 1-2: Measurement of bacterial growth level (absorbance) The bacterial cell concentration of the culture was determined by measuring absorbance at 600 nm, and the result was expressed as cell OD value.

[0055] 1-3: Measurement of glutathione content in cultures The culture solution of the strain was centrifuged to remove the supernatant, and the cells were washed once with distilled water to recover the cells. The recovered cells were added with 40-70% ethanol, and intracellular glutathione was extracted for 10-30 minutes using a fine mixer. The extract solution was centrifuged, and the supernatant was taken and reacted with 10 mM DTNB (5,5'-Dithiobis-(2-Nitrobenzoic Acid)) dissolved in 0.5 M potassium phosphate pH 8.0 buffer at 40°C for 20 minutes, and the glutathione content was confirmed by measuring the absorbance at 412 nm, and the glutathione content (GSH mg / L) was measured. DTNB, which is mainly used for glutathione analysis, is known as Ellman's reagent and is a reagent designed to detect thiol compounds. The reaction of DTNB and GSH produces yellow 2-nitro-5-benzoic acid and GSSG, and the concentration of GSH can be calculated by measuring the OD value at 412 nm. GSSG is reduced to GSH by glutathione reductase and then reacts with DTNB again to form a recycling system.

[0056] 1-4: Measurement of glutathione content per dry weight (g) of bacteria The strain culture solution was centrifuged to remove the supernatant, and the cells were washed once with distilled water to recover the cells. The absorbance of the recovered cells was measured, and the dry cell concentration was calculated based on the absorbance. Specifically, the dry cell weight (g) was determined by centrifuging the culture solution, removing the supernatant, recovering only the cells, washing the recovered cells with 0.9% NaCl, and diluting them with distilled water to prepare a sample with an absorbance between 0.1 and 1. The absorbance of the diluted cell sample was measured at 600 nm, and the dry cell weight was calculated based on the absorbance. After measuring the absorbance, the cells were filtered under reduced pressure using a 0.2 μm filter paper. The filter paper on which the cells were filtered was dried at 60° C. for 12 hours or more, and left in a desiccator containing silica gel for 6 hours or more, and then the weight was measured. The weight difference between the empty filter paper and the filter paper on which the cells were filtered was calculated to confirm the dry cell amount. Therefore, the dry cell concentration (g / L) based on the absorbance value was confirmed.

[0057] After measuring the absorbance, glutathione was extracted from the cells in a manner substantially similar to that of Examples 1-3, and the extract was centrifuged to obtain the supernatant, and the amount of glutathione produced (g / L) was measured. The measured amount of glutathione produced was divided by the calculated dry cell concentration (g / L) and multiplied by 100 to calculate GSH% per dry cell weight (g). The glutathione content per dry cell weight (g) was measured, and the GSH content (%) per dry cell weight (g) is shown in Table 1 below as GSH (%) / g-cell.

[0058] As a result of confirming glutathione productivity of 150 strains by the above method, the glutathione content per dry weight (g) of cells (GSH (%) / g-cell) was distributed in the range of about 0.3 to 2% by weight, and the top 7 candidate strains were isolated based on the glutathione content per dry weight (g) of cells. As the analysis results of the 7 selected strains, the cell OD of Example 1-2, the glutathione content of Example 1-3, and the glutathione content per dry weight (g) of cells are shown in Table 1, and the glutathione content per dry weight (g) of cells is shown in Figure 1.

[0059] As a control, the same experiment was carried out on the standard strain C. utilis KCCM 11355, and the results are shown in Table 1 and FIG.

[0060] [Table 1]

[0061] As shown in Table 1, the results of analyzing the bacterial cell growth and glutathione production in an ethanol-free medium showed that the glutathione content of the seven selected strains was higher than that of the standard strain C. utilis KCCM 11355, specifically, about 0.7 to 2% by weight of glutathione content and production. It was also confirmed that the six strains except for the SYC-7D strain had the same or higher bacterial cell growth rate as the standard strain and had more preferable characteristics.

[0062] Therefore, when considering the glutathione production amount (mg / L) and the glutathione production amount per dry cell weight (GSH (%) / g-cell) as the criteria for selecting a strain, 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 with a production amount per dry cell weight (GSH (%) / g-cell) of 0.8 wt% or more can be selected, and more preferably SYC-PR9, SYC-PR19, and SYC-PR20 with a production amount per dry cell weight (GSH (%) / g-cell) of 1.5 wt% or more can be selected.

[0063] In particular, when considering industrial production, it was found that it is more preferable to consider both the bacterial cell OD value and the production amount per dry cell weight (GSH (%) / g-cell). From this perspective, it was confirmed that SYC-P1, SYC-PR9, SYC-PR19, and SYC-PR20 are more preferable strains that have higher bacterial cell OD values ​​and higher glutathione production than the standard strain.

[0064] Example 2: Alcohol decomposition enzyme (ADH) activity assay The seven strains with high glutathione content selected in Example 1 were cultured and the ADH activity was confirmed.

[0065] Specifically, ADH activity was measured using the ADH Activity Assay Kit (Abcam). After culturing in YPD medium at 30°C for 24 to 48 hours, the cells were 1*10 6 The cells were collected to a concentration of CFU / ml. After washing the collected cells with distilled water, ADH assay buffer was added and the cell walls were broken using a bead beater. The reaction mixture consisted of 82μl of ADH assay buffer, 8μl of developer, 10μl of isopropanol, and 50μl of sample or NADH standard substance at each concentration. After 3 minutes of reaction at 37℃, the absorbance of the experimental group (A0) and the control group was measured at 450nm. After an additional 30 minutes of reaction at 37℃, the change in absorbance was measured at 450nm, and the NADH concentration produced per unit time was calculated to compare the ADH activity of each strain. The measured NADH production amount of each strain is shown in Figure 2. It was confirmed that the 7 strains had NADH production amounts with a distribution in the range of 24 to 160 nm. The same experiment was performed on the standard strain C.utilis KCCM 11355 as a control group. The results are shown in Table 2.

[0066] The results of comparing the ADH activity of seven strains in the supernatant obtained after disrupting yeast cells are shown in Table 2 below. The values ​​in Table 2 indicate the titer (mUnit, mU) of alcohol dehydrogenase (ADH) per ml of reaction solution, in mU / ml, and are the results calculated from the ADH activity based on the concentration of NADH produced per unit time (min). The concentration of NADH produced per unit time (min) is also shown in Figure 2.

[0067] [Table 2]

[0068] As shown in Table 2, the ADH activity of the seven selected strains ranged from 0.03 to 0.26 mu / ml. The strains with higher ADH activity than the standard strain C. utilis KCCM 11355 (control group) were SYC-P1, SYC-PR9, SYC-PR19, and SYC-PR20, and the strain with the highest activity was confirmed to be SYC-PR20. Therefore, SYC-P1, SYC-PR9, SYC-PR19, and SYC-PR20 were selected as the second selection in terms of ADH activity. Preferably, SYC-P1, SYC-PR9, and SYC-PR20 with ADH activity of 0.20 (mU / ml) or more were selected.

[0069] Considering the glutathione content per cell weight and cell dry weight (g) shown in Table 1 in Example 1 and the ADH activity analysis results of the strains in Table 2, strains with high glutathione production and ADH activity are preferred. Therefore, SYC-PR9, SYC-PR19, and SYC-PR20 can be selected as the second candidate strains.

[0070] Example 3: Evaluation of bacterial growth and GSH production depending on the culture time The SYC-PR20 selected in Example 1 was evaluated for its cell growth and GSH production depending on the culture time.

[0071] YPD (yeast extract 10 g / L, peptone 20 g / L, dextrose 20 g / L) was prepared as a medium for culturing the SYC-PR20 strain. 3 ml of the medium was dispensed into a test tube, and cultured with shaking at 30°C for 24 to 60 hours. The culture was taken at 24, 36, 48, and 60 hours, and the cell OD, glutathione content, and glutathione content per cell dry weight (g) were measured in a manner substantially similar to that of Examples 1-2 to 1-4. The results are shown in Table 3 below.

[0072] [Table 3]

[0073] The OD value of the bacterial cell and the amount of GSH produced were measured from 24 to 60 hours of culture time. The OD value of the bacterial cell increased from 12 at 24 hours to 15 at 36 hours, 18 at 48 hours, and 19 at 60 hours. The amount of GSH produced 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, it was confirmed that the GSH% per g of dry bacterial cell increased from 1.4% at 24 hours to 1.6% at 60 hours.

[0074] Example 4: Evaluation of ethanol tolerance of microorganisms The bacterial growth of SYC-PR20 selected in Example 1 was evaluated in an ethanol-containing medium.

[0075] Specifically, 5X YPD (yeast extract 50g / L, peptone 100g / L, dextrose 100g / L) medium was prepared and mixed with 100% ethanol to adjust the final ethanol concentration in the medium to 0, 2, 4, 6, 8, 10, or 15v / v%. 3ml of medium containing ethanol at different concentrations was dispensed into test tubes and cultured at 30°C for 60 hours with shaking to obtain cultures. The cultures were measured for bacterial cell concentration by measuring absorbance at 600nm in a manner substantially similar to that in Example 1-2, and the results are shown in Table 4 below.

[0076] As a control, the same experiment was carried out on the standard strain C. utilis KCCM 11355 (control group), and the results are shown in Table 4.

[0077] [Table 4]

[0078] Saccharomyces cerevisiae, known to have high alcohol tolerance, can grow at ethanol concentrations of 7-11% (v / v). The SYC-PR20 strain, which was selected in the second round in Examples 2 and 3, has high alcohol tolerance. In particular, compared to the standard strain C. utilis KCCM 11355 (control group), the SYC-PR20 strain showed high bacterial cell growth in an alcohol-containing medium, and in particular, it showed high bacterial cell growth of over 110% at ethanol concentrations of 0.5-15v / v%.

[0079] As shown in Table 4, the SYC-PR20 strain, which has relatively high ADH activity, had a higher bacterial cell concentration at the same ethanol concentration compared to the control group. Therefore, it was confirmed that the SYC-PR20 strain has excellent glutathione production ability, ADH activity, and alcohol tolerance.

[0080] Example 5: Strain Identification In Example 4, the SYC-PR20 strain showed the most excellent characteristics among the seven strains selected, with a glutathione yield of 1.6% and an ADH activity of 0.26 mU / ml.

[0081] The SYC-PR20 strain was subjected to 18S rRNA sequence analysis using universal primers ITS1 (SEQ ID NO: 2: 5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (SEQ ID NO: 3: 5'-TCCGTAGGTGAACCTGCGG-3'). The 18S rDNA sequence of the SYC-PR20 strain is shown in SEQ ID NO: 1. The strain was identified based on the 18S rDNA sequence information and was identified as Candida utilis (Pichia jadinii).

[0082] The Candida utilis SYC-PR20 strain was deposited at the Korea Microorganism Collection, located at 45, Hongjenae 2-ga-gil, Seodaemun-gu, Seoul, on August 7, 2020, and was assigned the accession number KCCM 12777P.

[0083] Example 6: Evaluation of glutathione production by fermentation tank culture In flask culture conditions such as those in Examples 1-4 for the isolation and characteristic investigation of microorganisms, it is difficult to increase the stirring speed beyond a certain level due to mechanical limitations, and it is difficult to adjust the culture environment (air volume, pH, etc.) to a certain level. In contrast, in industrial culture, air is supplied to the culture solution and stirring is performed, so that medium components and oxygen are uniformly distributed throughout the entire space, and temperature, pH, etc. can be adjusted, making it possible to culture under optimal conditions. Therefore, fermenter culture shows a higher bacterial growth rate than flasks, which leads to rapid consumption of the input sugar and increased productivity. Therefore, fermenter culture experiments are necessary for the application of industrial strains.

[0084] Specifically, a colony formed on a YPD agar plate inoculated with the Candida utilis SYC-PR20 strain of Example 5 was inoculated into 3 mL of YPD (yeast extract 10 g / L, peptone 20 g / L, dextrose 20 g / L) broth and cultured for 24 hours under conditions of a temperature of 30° C. and 240 rpm. The seed culture was then inoculated with 3 mL of the pre-cultured seed culture into 100 mL of the same YPD broth and cultured under the same conditions to prepare a seed culture for a 5 L fermentation tank.

[0085] To evaluate the bacterial growth and glutathione production depending on the carbon source, a 5L fermenter was cultured, and a glucose-based culture medium and a sugar-based culture medium were prepared in a 5L fermenter with a final culture volume of 2L and the medium composition shown in Table 5. The prepared seed bacteria were inoculated into the 5L fermenter and cultured with stirring under the conditions shown in Table 6.

[0086] [Table 5]

[0087] [Table 6]

[0088] The bacterial cell concentration and the amount of glutathione produced in the obtained culture solution were measured in substantially the same manner as in Example 1, and the measurement results are shown in Table 7 below.

[0089] [Table 7]

[0090] As shown in the results in Table 7, the initial sugar input was almost completely consumed when both glucose- and sugar-based cultures were completed at 29 hours, at which point the bacterial cell concentration was 28.4 at 600 nm for the glucose-based culture and 31.9 at 600 nm for the sugar-based culture, confirming that the bacterial cell yield per sugar increased by 112%. The glutathione content was 223.3 mg / L in the sugar-based culture, which was about 122% higher than that in the glucose-based culture for the same time of culture.

[0091] Example 7: Evaluation of glutathione production depending on culture conditions The colonies formed on the YPD agar plate inoculated with the selected Candida utilis SYC-PR20 strain were inoculated into 3 mL of YPD (yeast extract 10 g / L, peptone 20 g / L, dextrose 20 g / L) broth and cultured for 24 hours under conditions of 30°C and 240 rpm. The same YPD broth (100 mL) was again inoculated with 3 mL of the pre-cultured seed culture and cultured under the same conditions to prepare seed culture for a 5 L fermentation tank.

[0092] In order to evaluate high-concentration bacterial cells and glutathione production, a seed culture and a 5L fermenter culture were prepared as in Example 5. When the ethanol concentration accumulated after the initial sugar consumption in the batch culture was 5g / L or less, a fed-batch culture was performed by continuously supplying 600g / L sucrose solution to perform an experiment on a method of maintaining high bacterial cell concentration culture and glutathione productivity.

[0093] As a result, the change in glutathione content in the culture medium due to the sucrose feeding rate was measured over the culture time and is shown in Figure 3, and the ethanol content in the culture medium is shown in Figure 4. The change in bacterial cell concentration over time due to the sucrose feeding rate is shown in Figure 5. In Figures 3, 4, and 5, the white cells were -1 The black ball represents the sucrose feeding rate of 6g / L h -1 means.

[0094] Sucrose feeding rate: 6g / L·h -1 During feeding, the ethanol content in the culture medium was maintained at 3 g / L or more, and the rate of change in glutathione content was almost constant. On the other hand, at a low feeding rate (4.5 g / L h -1 ), the ethanol content was maintained low and the intracellular glutathione content was decreased. This was because when the sucrose feeding rate was high, the energy required for glutathione biosynthesis was sufficiently supplied due to the sufficient supply of sugar in the culture medium, but when the sucrose feeding rate was low, the energy supply was insufficient, causing the bacteria to grow at a limited rate, resulting in a decrease in glutathione content.

[0095] Therefore, the Candida utilis SYC-PR20 strain according to the present invention can be cultured at a feeding rate of 6 g / L h -1 When the sugar is supplied at a sufficient rate, ethanol accumulates in the culture medium as a bacterial metabolic product due to the sufficient sugar supply, and the glutathione content can be maintained at the batch level in the high-concentration culture section, which is more preferable.

Claims

1. It has alcohol dehydrogenase (ADH) activity and alcohol tolerance, It has glutathione producing ability, The accession number is KCCM 12777P. Candida utilis strains.

2. 2. The strain according to claim 1, wherein the strain has an ADH activity of 0.18 mU / ml or more per dry weight (g) of the bacterial cells and produces glutathione in an amount of 0.8% by weight or more per dry weight (g) of the bacterial cells.

3. The strain according to claim 1, which has alcohol tolerance and can grow under conditions in which the ethanol concentration is 2 to 15% (v / v).

4. The strain according to claim 3, wherein the strain has a cell optical density (OD) value of 120 to 200% based on OD value of 100% of Candida utilis having deposit number KCCM 11355 when cultured under conditions of an alcohol concentration of 6 to 15% (v / v).

5. A composition for alcohol decomposition comprising at least one selected from the group consisting of a Candida utilis strain according to any one of claims 1 to 4, a culture of said strain, a disrupted product of said strain, a lysate of said strain, and extracts thereof.

6. 6. The composition for alcohol decomposition according to claim 5, wherein the bacterial strain has an alcohol dehydrogenase (ADH) activity of 0.18 mU / ml or more per dry weight (g) of bacterial cells.

7. A composition for relieving, improving, alleviating, or preventing a hangover, comprising at least one selected from the group consisting of a Candida utilis strain according to any one of claims 1 to 4, a culture of the strain, a disrupted product of the strain, a lysate of the strain, and extracts thereof.

8. A method for increasing glutathione production by culturing the Candida utilis strain according to any one of claims 1 to 4.

9. The method of claim 8, wherein the culture of the strain uses sugar or glucose as a carbon source.

10. The method according to claim 8, wherein the culture of the strain is carried out by controlling the carbon source supply rate based on the ethanol content in the fermentation broth.

11. 9. The method of claim 8, comprising culturing the strain with the addition of cysteine.

Images