Specification
Title of the invention: Yeast strain producing glutathione and glutathione production method using the same
technical field
[One]
The present application relates to a novel glutathione-producing yeast strain and a glutathione production method using the same.
[2]
background
[3]
Glutathione (GSH) is an organic sulfur compound most commonly present in cells, and is in the form of a tripeptide in which three amino acids, glycine, glutamate, and cysteine, are combined.
[4]
Glutathione exists in the body in two forms: reduced glutathione (GSH) and oxidized glutathione (GSSG). Reduced glutathione (GSH), which is present in a relatively high proportion under normal circumstances, is mainly distributed in the liver and skin cells of the human body. It plays an important role such as a whitening action that inhibits the production.
[5]
Since the amount of glutathione produced gradually decreases as aging progresses, the decrease in the production of glutathione, which plays an important role in antioxidant and detoxification, promotes the accumulation of active oxygen, the main culprit of aging, so the supply of glutathione is required from the outside (Sipes IG et al. al, The role of glutathione in the toxicity of xenobiotic compounds: metabolic activation of 1,2-dibromoethane by glutathione, Adv Exp Med Biol. 1986;197:457-67.).
[6]
As such, glutathione with various functions is spotlighted as a material in various fields such as pharmaceuticals, health functional foods, and cosmetics, and is also used in the manufacture of flavor materials, food and feed additives. It is known that glutathione has a great effect of increasing the taste of the raw material and maintaining rich taste, and can be used alone or in combination with other substances as a kokumi flavor enhancer. In general, Kokumi material has a richer feeling than umami material such as nucleic acid and MSG, and is known to be produced by decomposition and aging of proteins.
[7]
However, despite the increasing demand for glutathione that can be used in various fields as described above, the industrial production of glutathione requires a considerable cost, so the market is not greatly activated.
[8]
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[9]
The present inventors completed the present application by developing a new strain excellent in glutathione-producing ability as a result of earnest efforts to solve the above problems.
[10]
means of solving the problem
[11]
An object of the present application is to provide a novel Saccharomyces cerevisiae strain that produces glutathione.
[12]
Another object of the present application is to provide a method for producing glutathione comprising the step of culturing the strain.
[13]
Another object of the present application is the step of culturing the strain and mixing the cultured strain, its dried product, extract, culture, lysate, and at least one material selected from glutathione recovered therefrom and additives; It provides a method for preparing a glutathione-containing composition comprising a.
[14]
Another object of the present application is the strain; dried products, extracts, cultures, and lysates of the strain; And glutathione recovered from any one or more of the strain, dried product, extract, culture, and lysate; It provides a composition for antioxidant function, detoxification, immune enhancement, cosmetics, food, and feed containing one or more substances selected from among.
[15]
Another object of the present application, the strain; dried products, extracts, cultures, and lysates of the strain; And glutathione recovered from any one or more of the strain, dried product, extract, culture, and lysate; To provide a pharmaceutical composition or pharmaceutical composition for use in the prevention or treatment of diseases caused by glutathione deficiency, comprising one or more substances selected from among.
[16]
Effects of the Invention
[17]
The strain of the present application can produce a remarkably large amount of glutathione compared to the existing glutathione-producing strain. Therefore, the strain of the present application can be usefully used for glutathione production.
[18]
Also the strain of the present application; dried products, extracts, cultures, and lysates of the strain; And glutathione recovered from any one or more of the strain, dried product, extract, culture, and lysate; Since the composition comprising one or more substances selected from among them has excellent active oxygen scavenging ability, it can be usefully used as a composition for antioxidant function, detoxification, and immunity enhancement.
[19]
Accordingly, the composition may be usefully used in cosmetic compositions, pharmaceutical compositions, and manufacturing thereof.
[20]
In addition, when the composition is used for food, it can be usefully used in the preparation of food compositions and feed compositions because it improves the cleanliness of food as well as the above-described functions.
[21]
Brief description of the drawing
[22]
1 is a sensory evaluation result of a food composition comprising an extract of Saccharomyces cerevisiae CEN.PK2-1D, CJ-5, and CJ-37 strains.
[23]
Figure 2 is the result of confirming the radical removal ability of the extract of Saccharomyces cerevisiae CEN.PK2-1D, CJ-5, CJ-37 strain.
[24]
3 is a result confirming the active oxygen absorption capacity of Saccharomyces cerevisiae CEN.PK2-1D, CJ-5, CJ-37 strains.
[25]
Best mode for carrying out the invention
[26]
This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.
[27]
In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present application described herein. Also, such equivalents are intended to be covered by this application.
[28]
[29]
One aspect of the present application may provide a novel Saccharomyces cerevisiae strain producing glutathione.
[30]
The glutathione-producing strain of the present application may be a Saccharomyces cerevisiae CJ-5 strain with accession number KCCM12568P.
[31]
[32]
The glutathione-producing strain of the present application is 18S (ITS, 5.8S) rRNA sequence of Saccharomyces cerevisiae, specifically Saccharomyces cerevisiae or Saccharomyces cerevisiae YJM1592 of other homologous species ( SEQ ID NO: 3) and 90% or more, specifically 91%, 92%, 93%, 93.2%, 93.5% or 93.7% or more homology or identity with 18S (ITS, 5.8S) rRNA sequence, but may contain , but not limited thereto. More specifically, the glutathione-producing strain of the present application is at least 90%, specifically 91%, 92%, 93%, 93.2%, 93.5% or 93.7% of the 18S (ITS, 5.8S) rRNA sequence of YJM1592. 18S (ITS, 5.8S) rRNA sequence having homology or identity and having less than 96%, 95.5% or 95.2% identity. The 18S (ITS, 5.8S) rRNA sequence is an internal transcribed spacer (ITS) sequence that exists between 18S rRNA and 5.8S and is used for species classification.
[33]
The glutathione-producing strain of the present application may have an 18S (ITS, 5.8S) rRNA sequence of SEQ ID NO: 1. According to one embodiment, the 18S (ITS, 5.8S) rRNA sequence of the strain is 90% or more with SEQ ID NO: 1, specifically 95%, 96%. It may have 97%, 98%, or 99% or more homology or identity, but is not limited thereto.
[34]
In the present application, the term 'homology' or 'identity' refers to a degree related to two given amino acid sequences or base sequences, and may be expressed as a percentage. The terms homology and identity can often be used interchangeably.
[35]
Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially homologous or identical sequences generally have moderate or high stringency conditions along at least about 50%, 60%, 70%, 80% or 90% of the entire or full-length sequence. It can hybridize under stringent conditions. Hybridization is also contemplated for polynucleotides containing degenerate codons instead of codons in the polynucleotides.
[36]
Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, using a known computer algorithm such as the "FASTA" program. or, as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine. (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA(Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073). For example, BLAST from the National Center for Biotechnology Information Database, or ClustalW, can be used to determine homology, similarity or identity.
[37]
Homology, similarity or identity of polynucleotides or polypeptides is described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. 48: 443 by comparing the sequence information using a GAP computer program. In summary, the GAP program is defined as the total number of symbols in the shorter of two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids). Default parameters for the GAP program are: (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.
[38]
In addition, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be confirmed by comparing the sequences by Southern hybridization experiments under defined stringent conditions, and the defined appropriate hybridization conditions are within the scope of the art. , can be determined by methods well known to those skilled in the art.
[39]
[40]
In the present application, "glutathione" is used interchangeably with "glutathione" and "GSH", and refers to a tripeptide composed of three amino acids: glutamate, cysteine, and glycine. Glutathione may be used as a raw material for pharmaceuticals, health functional foods, flavoring materials, food, feed additives, cosmetics, etc., but is not limited thereto.
[41]
The novel Saccharomyces cerevisiae strain of the present application is characterized by high glutathione-producing ability.
[42]
The strain may be a strain with enhanced glutathione-producing ability, a strain with high glutathione-producing ability, or a strain with increased glutathione-producing ability.
[43]
"Strain producing glutathione" of the present application may be used interchangeably with terms such as "strain having glutathione-producing ability" and "glutathione-producing strain".
[44]
The Saccharomyces cerevisiae strain according to the present invention contains 0.6% by weight or more, specifically 0.7% by weight or more, 0.8% by weight or more, 0.9% by weight or more, 1.0% by weight or more, 1.1% by weight or more of glutathione relative to the dry cell weight. It may include, but is not limited to.
[45]
[46]
Another aspect of the present application may provide a composition for producing glutathione comprising the strain.
[47]
In the present application, "glutathione production" to "glutathione production" may include accumulating glutathione in cells.
[48]
The composition for producing glutathione is a composition capable of producing glutathione by the strain of the present application, for example, the composition includes the strain, and further includes a configuration capable of producing glutathione using the strain without limitation. can
[49]
The composition for glutathione production of the present application may further include any suitable additive commonly used in the composition for glutathione production. The additive may be naturally occurring or non-naturally occurring, but is not limited thereto.
[50]
The additives may include excipients and/or emulsifiers. The excipient may be appropriately selected and used according to the intended use or form, for example, starch, glucose, cellulose, lactose, glycogen, D-mannitol, sorbitol, lactitol, maltodextrin, calcium carbonate, synthetic aluminum silicate, Calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium hydrogen carbonate, purified lanolin, dextrin, sodium alginate, methylcellulose, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose, propylene glycol, casein, calcium lactate , Primogel, and gum arabic. Specifically, it may be one or more components selected from starch, glucose, cellulose, lactose, dextrin, glycogen, D-mannitol, and maltodextrin, but is not limited thereto. Emulsifiers include glycerol ester, susorbitan ester, monoglyceride, diglyceride, triglyceride, sucrose ester, sorbitan ester, propellin glycol ester, glycerin fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, sucrose fatty acid ester , lecithin or a mixture thereof may be used, but not limited thereto, and those known in the art may be appropriately used.
[51]
The excipient may include, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizing agent, or an isotonic agent, but is not limited thereto.
[52]
[53]
Another aspect of the present application may provide a glutathione production method comprising the step of culturing the strain. Through the culture of the strain, glutathione may be accumulated in the cells.
[54]
The medium and other culture conditions used for culturing the strain of the present application may be used without any particular limitation as long as it is a medium used for culturing the microorganisms of the genus Saccharomyces. It can be cultured under aerobic or anaerobic conditions in a normal medium containing a nitrogen source, phosphorus, inorganic compounds, amino acids and/or vitamins while controlling temperature, pH, etc.
[55]
As the carbon source in the present application, carbohydrates such as glucose, fructose, sucrose, maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; Amino acids such as glutamic acid, methionine, lysine, and the like may be included, but are not limited thereto. In addition, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice winter, cassava, sugarcane offal and corn steep liquor can be used, including glucose and sterilized pretreated molasses (i.e. molasses converted to reducing sugar). Carbohydrates such as, etc. may be used, and other appropriate amounts of carbon sources may be variously used without limitation. These carbon sources may be used alone or in combination of two or more thereof.
[56]
Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Organic nitrogen sources such as amino acids, peptones, NZ-amines, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysates, fish or decomposition products thereof, defatted soybean cakes or decomposition products thereof and the like can be used. These nitrogen sources may be used alone or in combination of two or more, but is not limited thereto.
[57]
The phosphorus may include potassium first potassium phosphate, second potassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like may be used.
[58]
In addition, the medium may contain amino acids, vitamins and/or appropriate precursors. Specifically, L-amino acid and the like may be added to the culture medium of the strain. Specifically, glycine, glutamate, and/or cysteine ​​may be added, and if necessary, L-amino acids such as lysine may be further added, but not necessarily limited thereto. does not
[59]
[60]
The medium or precursor may be added to the culture in a batch or continuous manner, but is not limited thereto.
[61]
In the present application, by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the culture in an appropriate manner during the culture of the strain, the pH of the culture can be adjusted. In addition, during culturing, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen or carbon dioxide gas may be injected with or without gas to maintain anaerobic and microaerobic conditions.
[62]
The temperature of the culture may be 25°C to 40°C, and more specifically, 28°C to 37°C, but is not limited thereto. The incubation period may be continued until a desired production amount of a useful substance is obtained, and specifically, it may be 1 hour to 100 hours, but is not limited thereto.
[63]
[64]
The glutathione production method may further include an additional process after the culturing step. The additional process may be appropriately selected depending on the use of glutathione.
[65]
Specifically, the glutathione production method may include recovering glutathione from at least one material selected from the strain, its dried product, extract, culture, and lysate after the culturing step.
[66]
In the present application, "culture" refers to the strain of the present application.It may be a result obtained by culturing. For example, it may be a medium containing by-products generated through metabolism and ingestion of nutrients by culturing the strain in a medium, a dilution or a concentrate of the medium, a dried product obtained by drying the medium, a prepared product or purification of the medium It includes cultures of all formulations that can be formed using the above medium, such as water or a mixture thereof. The culture of the microorganism of the present application may or may not include the microorganism. The culture is the same as described above.
[67]
In the present application, "lysate" may be a product obtained by crushing or lysing the strain or a culture thereof, or a supernatant obtained by centrifuging the lysate.
[68]
The culture, lysate, supernatant thereof, and fractions thereof are also included in the scope of the present application. The method may further include a step of lysing the strain before or at the same time as the recovery step. The lysis of the strain may be carried out by a method commonly used in the art to which the present application belongs, for example, a lysis buffer solution, a sonicator, heat treatment, and a French presser. In addition, the lysis step may include an enzymatic reaction such as a cell wall degrading enzyme, a nucleic acid degrading enzyme, a nucleic acid transfer enzyme, a proteolytic enzyme, but is not limited thereto.
[69]
For the purpose of the present application, dry yeast containing a high content of glutathione through the glutathione manufacturing method, yeast extract, yeast extract mix powder, pure purified glutathione (pure) glutathione), but is not limited thereto, and may be appropriately manufactured according to the desired product.
[70]
In the present application, dry yeast may be used interchangeably with terms such as "dried strain". The dry yeast may be prepared by drying yeast cells accumulating glutathione, and specifically, may be included in a composition for feed, a composition for food, and the like, but is not limited thereto.
[71]
In the present application, yeast extract may be used interchangeably with terms such as "strain extract". The extract of the strain may refer to a material remaining after separating the cell wall from the cells of the strain. Specifically, it may mean the remaining components excluding the cell wall from the components obtained by lysing the cells. The extract of the strain includes glutathione, and components other than glutathione may include one or more components of protein, carbohydrate, nucleic acid, and fiber, but is not limited thereto.
[72]
In the recovery step, glutathione, a target material, may be recovered using a suitable method known in the art.
[73]
The recovery step may include a purification process. The purification process may be pure purification by separating only glutathione from the strain. Through the purification process, pure purified glutathione may be prepared.
[74]
If necessary, the glutathione production method may further include mixing a material selected from among the strains obtained after the culturing step, their dried products, extracts, cultures, and lysates, and glutathione recovered therefrom and additives. Through the mixing step, yeast extract mix powder may be prepared.
[75]
The additives may include excipients and/or emulsifiers. Excipients and emulsifiers may be appropriately selected and used by those skilled in the art, and examples thereof are as described above.
[76]
[77]
Another aspect of the present application, the step of culturing the strain; and mixing a material selected from among cultured strains, dried products thereof, extracts, cultures, lysates, and glutathione recovered therefrom and additives; It can provide a method for preparing a composition comprising glutathione, including.
[78]
The composition of the present application may further include a naturally occurring material or a non-naturally occurring material. The material may include, but is not limited to, a pharmaceutically acceptable carrier, a food pharmaceutically acceptable carrier, a cosmetically acceptable carrier, and the like, depending on the intended use of the composition. The carrier can be appropriately selected by those skilled in the art based on known contents.
[79]
In the composition, the content of the material selected from the strains of the present application, their dried products, extracts, cultures, lysates, and glutathione recovered therefrom relative to the total composition may be 3 to 30% by weight based on the total weight of the composition.
[80]
The additives may include excipients and/or emulsifiers. Excipients and emulsifiers may be appropriately selected and used by those skilled in the art, and examples thereof are as described above.
[81]
[82]
Another aspect of the present application is the strain; dried products, extracts, cultures, and lysates of the strain; And glutathione recovered from any one or more of the strain, dried product, extract, culture, and lysate; It is possible to provide a composition for antioxidant function, detoxification, immune enhancement, cosmetics, food or feed containing one or more substances selected from among.
[83]
Another aspect of the present application is the strain; dried products, extracts, cultures, and lysates of the strain; And glutathione recovered from any one or more of the strain, dried product, extract, culture, and lysate; It is possible to provide a method for preparing a composition for antioxidant function, detoxification, immune enhancement, cosmetics, food, or feed containing one or more substances selected from among.
[84]
For the purpose of the present application, the composition may include the strain itself, and may include the culture of the strain, the dried product of the strain, the extract of the strain, the lysate of the strain, the culture of the strain, the extract, It may include, but is not limited to, recovering glutathione from dry matter and crushed matter. That is, since the strain, its dried product, extract, culture, and lysate contain glutathione, the form containing glutathione may vary slightly depending on the specific use of the composition, but as long as the content of the desired glutathione can be increased. , may be included in any form without limitation.
[85]
In the composition, the content of the material selected from the strains of the present application, their dried products, extracts, cultures, lysates, and glutathione recovered therefrom relative to the total composition may be 3 to 30% by weight based on the total weight of the composition. In addition, the composition may further comprise any suitable additives commonly used in compositions.
[86]
The composition is a strain producing glutathione of the present application; dried products, extracts, cultures, and lysates of the strain; And glutathione recovered from any one or more of the strain, dried product, extract, culture, and lysate; It may include one or more of the substances to have an increased glutathione content.
[87]
[88]
One aspect of the present application is the strain; dried products, extracts, cultures, and lysates of the strain; And glutathione recovered from any one or more of the strain, dried product, extract, culture, and lysate; It is possible to provide a composition for antioxidant function comprising one or more substances selected from among.

Claims

[Claim 1]
Saccharomyces cerevisiae strain with accession number KCCM12568P to produce glutathione.
[Claim 2]
The strain according to claim 1, wherein the strain comprises the nucleotide sequence of SEQ ID NO: 1 as an 18S rRNA sequence.
[Claim 3]
A method for producing glutathione, comprising the step of culturing the strain of claim 1.
[Claim 4]
The method of claim 3, further comprising recovering glutathione from at least one material selected from the cultured strain, the dried product of the strain, the extract of the strain, the culture of the strain, and the lysate of the strain. manufacturing method.
[Claim 5]
Culturing the strain of claim 1; And mixing the cultured strain, its dried product, extract, culture, lysate, and at least one material selected from glutathione recovered therefrom and an additive;
[Claim 6]
The method of claim 5, wherein the additive comprises an excipient or an emulsifier.
[Claim 7]
The method according to claim 5, wherein the weight of one or more substances selected from the cultured strain, its dried material, extract, culture, and lysate and glutathione recovered therefrom is 3 to 30% by weight relative to the total weight of the composition. Preparation of a composition containing glutathione Way.
[Claim 8]
The strain of claim 1; dried products, extracts, cultures, and lysates of the strain; And glutathione recovered from any one or more of the strain, dried product, extract, culture, and lysate; A composition for antioxidant function comprising one or more substances selected from among.
[Claim 9]
The strain of claim 1; dried products, extracts, cultures, and lysates of the strain; And glutathione recovered from any one or more of the strain, dried product, extract, culture, and lysate; A composition for detoxification comprising one or more substances selected from among.
[Claim 10]
The strain of claim 1; dried products, extracts, cultures, and lysates of the strain; And glutathione recovered from any one or more of the strain, dried product, extract, culture, and lysate; A composition for enhancing immunity, comprising one or more substances selected from among.
[Claim 11]
The strain of claim 1; dried products, extracts, cultures, and lysates of the strain; And glutathione recovered from any one or more of the strain, dried product, extract, culture, and lysate; A cosmetic composition comprising one or more substances selected from among.
[Claim 12]
The strain of claim 1; dried products, extracts, cultures, and lysates of the strain; And glutathione recovered from any one or more of the strain, dried product, extract, culture, and lysate; A composition for food comprising one or more substances selected from among.
[Claim 13]
The strain of claim 1; dried products, extracts, cultures, and lysates of the strain; And glutathione recovered from any one or more of the strain, dried product, extract, culture, and lysate; A composition for feed comprising one or more substances selected from among.
[Claim 14]
The strain of claim 1; dried products, extracts, cultures, and lysates of the strain; And glutathione recovered from any one or more of the strain, dried product, extract, culture, and lysate; A pharmaceutical composition for use in the prevention or treatment of diseases caused by glutathione deficiency, comprising one or more substances selected from among.
[Claim 15]
The strain of claim 1; dried products, extracts, cultures, and lysates of the strain; And glutathione recovered from any one or more of the strain, dried product, extract, culture, and lysate; A pharmaceutical composition for preventing or treating diseases caused by glutathione deficiency, comprising one or more substances selected from among.