Yeast mutant strain HSY-NGM-029 having improved crude protein content and method for producing same

Abstract

The present invention relates to yeast mutant strain HSY-NGM-029 having an improved crude protein content and a method for producing same. More specifically, the present invention provides: a Saccharomyces yeast mutant strain prepared by UV irradiation of wild-type Saccharomyces cerevisiae under specific conditions, and having an improved crude protein content compared to the wild type; a method for producing the yeast mutant strain; a method for producing a crude protein using the yeast mutant strain; and a food and a feed composition including a crude protein produced from the yeast mutant strain.

Description

Yeast mutant strain HSY-NGM-029 with improved crude protein content and its production method

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2024-0086183, filed July 1, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a yeast mutant strain HSY-NGM-029 having an improved crude protein content and a method for producing the same, and more particularly, to a Saccharomyces yeast mutant strain having an improved crude protein content compared to the wild type, which is produced by irradiating wild-type Saccahromyces cerevisiae with ultraviolet rays under specific conditions, a method for producing the yeast mutant strain, a method for producing crude protein using the yeast mutant strain, and a food and feed composition comprising crude protein produced from the yeast mutant strain.

[0003] The demand for protein for human and animal nutrition is increasing alongside the global population. However, social issues such as food shortages and animal welfare are also emerging, and alternative protein sources are gaining attention as a solution. Consequently, the development and launch of alternative protein products is actively underway, and the alternative protein market is experiencing continuous growth.

[0004] Alternative protein foods are processed foods that resemble real meat and seafood in texture and flavor by utilizing various alternative protein sources, such as plant-based, cell-cultured, and fermented, instead of traditional agricultural and livestock production methods. Fermented alternative proteins are high-quality single-cell proteins extracted and propagated from natural sources such as yeast, bacteria, fungi, and algae, and are rich in amino acids, vitamin B, nucleic acids, physiologically active peptides, and immunosaccharides. As disclosed in Patent Document 1, these single-cell proteins are being used as raw materials for artificial meat products as a substitute for animal proteins. However, the protein content of yeast is generally lower than that of bacteria, and therefore, research is needed to improve the protein content of yeast for the commercialization of yeast-derived single-cell proteins.

[0005] Among yeasts, Saccahromyces cerevisiae is a unicellular eukaryote and a GRAS (Generally Recognized as Safe) strain that has been used for alcohol and bakery purposes in food processing for a long time. In the present invention, we attempted to develop a technology that can improve the crude protein content of yeast in a simpler way using wild-type yeast. Accordingly, among various yeast strains already used in the food industry, Saccahromyces cerevisiae KCCM12638 was selected as a parent strain, and the parent strain was irradiated with ultraviolet rays under specific conditions to produce a mutant strain with an improved crude protein content compared to the wild type, thereby completing the present invention.

[0006] [Prior Art Literature]

[0007] [Patent Document]

[0008] (Patent Document 1) Republic of Korea Publication No. 10-2022-0123019

[0009] The present invention aims to provide a Saccharomyces yeast mutant strain having an improved crude protein content compared to the wild type, which is produced by irradiating wild type Saccahromyces cerevisiae with ultraviolet rays under specific conditions.

[0010] Another object of the present invention is to provide a method for producing a Saccharomyces yeast mutant strain having an improved crude protein content compared to the wild type, and a method for producing crude protein using the yeast mutant strain.

[0011] Another object of the present invention is to provide a feed or food composition comprising crude protein produced from the yeast mutant strain.

[0012] Another object of the present invention is to provide a method for producing feed, feed additive, food or food additive using the yeast mutant strain.

[0013] Another object of the present invention is to provide a use of the yeast mutant strain for improving crude protein production.

[0014] Another object of the present invention is to provide a use of the yeast mutant strain for the production of feed, feed additive, food or food additive having improved crude protein content.

[0015] To solve the above-described problem, the present invention provides a Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) having an improved crude protein content compared to the wild type.

[0016] At this time, the mutant strain HSY-NGM-029 may be derived from Saccharomyces cerevisiae KCCM12638, a yeast strain for producing American whiskey.

[0017] At this time, the mutant strain HSY-NGM-029 was exposed to ultraviolet rays of 250 to 260 nm wavelength at 3000 to 9000 mW / cm at a position 5 to 50 cm away in the vertical direction from Saccharomyces cerevisiae KCCM12638. 2 It can be manufactured by irradiating for 8 to 10 hours with a dose of .

[0018] In the present invention, the mutant strain HSY-NGM-029 can have a crude protein content increased by 29 to 60% compared to the wild type.

[0019] In the present invention, the mutant strain HSY-NGM-029, compared to Saccharomyces cerevisiae KCCM12638, has 26171G>C, 194499A>C mutations in chromosome 1, 813110T>G mutations in chromosome 2, 4090A>C, 316502T>G mutations in chromosome 3, 224264T>C, 364680T>A mutations in chromosome 8, 248948T>C mutations in chromosome 9, 502139A>C mutations in chromosome 10, 432073C>A mutations in chromosome 11, and 1064798T>G, 1064817T>G, 1064832T>A in chromosome 12. It may have single nucleotide polymorphisms (SNPs) of 1064858A>C, 1064858A>T mutations, 354374G>T, 908306G>A, 908390C>T mutations on chromosome 13, 6714C>A, 783656G>A, 783659T>G, 783791C>T, 783810G>T mutations on chromosome 14, 30149T>A mutation on chromosome 15, and 19717C>A mutation on mitochondrial chromosomes.

[0020] The present invention also provides a method for producing a Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) having an improved crude protein content compared to the wild type, comprising the following steps:

[0021] a) a step of pre-cultivating Saccharomyces cerevisiae KCCM12638; and

[0022] b) A step of irradiating the above-mentioned pre-cultured Saccharomyces cerevisiae KCCM12638 with ultraviolet rays.

[0023] In the present invention, the manufacturing method may additionally include, between steps a) and b), a step a') of diluting the pre-cultured strain so that the optical density at 600 nm becomes 0.5 to 1.5.

[0024] In the present invention, step b) is performed by applying ultraviolet rays of 250 to 260 nm wavelength to Saccharomyces cerevisiae KCCM12638 at a vertical distance of 5 to 50 cm, irradiating 3000 to 9000 mW / cm 2 It can be performed by investigating for 8 to 10 hours with an investigation amount of .

[0025] In the present invention, after step b), compared to Saccharomyces cerevisiae KCCM12638, 26171G>C, 194499A>C mutations in chromosome 1, 813110T>G mutations in chromosome 2, 4090A>C, 316502T>G mutations in chromosome 3, 224264T>C, 364680T>A mutations in chromosome 8, 248948T>C mutations in chromosome 9, 502139A>C mutations in chromosome 10, 432073C>A mutations in chromosome 11, 1064798T>G, 1064817T>G, 1064832T>A in chromosome 12, The method may further include a step of selecting a mutant rice strain having the SNPs of 1064858A>C, 1064858A>T mutations, 354374G>T, 908306G>A, 908390C>T mutations in chromosome 13, 6714C>A, 783656G>A, 783659T>G, 783791C>T, 783810G>T mutations in chromosome 14, 30149T>A mutation in chromosome 15, and 19717C>A mutation in mitochondrial chromosome.

[0026] In addition, the present invention provides a method for producing crude protein using the aforementioned Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP).

[0027] In the present invention, the crude protein production method may include the following steps:

[0028] a) a step of culturing Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP); and

[0029] b) A step of recovering crude protein from the above strain.

[0030] Furthermore, the present invention provides a food or food additive composition comprising a Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP), a culture of the strain, or a crude protein produced from the strain.

[0031] Additionally, the present invention provides a feed or feed additive composition comprising a Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP), a culture of the strain, or crude protein produced from the strain.

[0032] In addition, the present invention provides a method for producing feed, feed additive, food or food additive using Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) or a culture of the strain.

[0033] The present invention also provides the use of the yeast mutant strain for improving crude protein production.

[0034] Furthermore, the present invention provides a use of the yeast mutant strain or the strain culture for use in the production of feed, feed additive, food or food additive having improved crude protein content.

[0035] The Saccharomyces cerevisiae mutant strain HSY-NGM-029 (Accession No. KCTC 15779BP) according to the present invention is produced by irradiating the wild-type Saccharomyces cerevisiae KCCM12638, which is already used in the food industry, with ultraviolet rays under specific conditions, thereby enabling the securing of a yeast strain exhibiting a high crude protein content in a much simpler and simpler manner than the existing GMO yeast. The Saccharomyces cerevisiae mutant HSY-NGM-029 (Accession No. KCTC 15779BP) produced in this manner exhibits a crude protein content that is improved by up to 60% or more compared to the wild-type, enabling more efficient crude protein production.

[0036] Figure 1 is a graph showing the survival rate of Saccharomyces cerevisiae KCCM12638 strain according to ultraviolet irradiation time.

[0037] Figure 2 shows the results of measuring the relative content (%) of total protein using the Bradford protein assay after culturing the Saccharomyces cerevisiae KCCM12638 strain irradiated with UV rays, and a mutant strain with a 20% increase in protein content compared to the wild-type Saccharomyces cerevisiae KCCM12638 strain was first selected.

[0038] Figure 3 shows the results of measuring the relative content (%) of total protein using the Bradford protein assay after culturing the first-selected strain. A mutant strain with a 20% increase in protein content compared to the first-selected mutant strain HS-Y001 was selected for the second time.

[0039] Figure 4 shows the results of measuring the concentration of total protein (mg / L) using the Bradford protein assay after culturing the second-selected strain. The mutant strain with the highest increase in protein concentration compared to the first-selected mutant strain HS-Y001 was selected for the third time.

[0040] Figure 5 shows the results of BLAST comparison of the ITS (internal transcribed spacer) portion (ITS1, 5.8S rRNA, and ITS2 base sequences) of S. cerevisiae S288C and S. cerevisiae KCCM12638, the parent strain of the mutant yeast of the present invention. Here, the query is the ITS portion (ITS1, 5.8S rRNA, ITS2) of S. cerevisiae S288C, and the subject is the ITS portion (ITS1, 5.8S rRNA, ITS2) of S. cerevisiae KCCM12638. The query sequence is shown in SEQ ID NO: 1, and the subject sequence is shown in SEQ ID NO: 2.

[0041] Figure 6 shows the results of BLAST comparison of the ITS portion (ITS1, 5.8S rRNA, and ITS2 base sequences) of the parent strain Saccharomyces cerevisiae KCCM12638 and the Saccharomyces cerevisiae mutant strain derived therefrom (accession number KCTC 15779BP). Here, the query is the ITS portion (ITS1, 5.8S rRNA, ITS2) of Saccharomyces cerevisiae KCCM12638, and the subject is the ITS portion (ITS1, 5.8S rRNA, ITS2) of Saccharomyces cerevisiae KCTC 15779BP. The query sequence is shown in sequence number 2, and the subject sequence is shown in sequence number 3.

[0042] Hereinafter, the present invention will be described in more detail.

[0043] All technical terms used in this invention, unless otherwise defined, have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of this invention.

[0044] All numbers expressing sizes, amounts, and physical properties of features used in this specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that may vary depending on the desired properties sought to be obtained by those skilled in the art utilizing the teachings disclosed herein.

[0045] The present inventors, in response to the need for developing a technology capable of improving the crude protein content of yeast in a simpler manner using wild-type yeast, selected a yeast strain already used in the food industry as a parent strain and treated it with ultraviolet rays under specific conditions to produce a mutant strain with improved crude protein content compared to the wild-type strain.

[0046] Accordingly, the first aspect of the present invention relates to a Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) having an improved crude protein content compared to the wild type, and a method for producing the same.

[0047] As used herein, the term "crude protein" refers to the protein obtained by measuring total nitrogen in general analysis of food and feed and multiplying this by 6.25 (the reciprocal of the average nitrogen content of the protein, 1 / 0.16).

[0048] In a specific embodiment of the present invention, in order to produce a high-protein content yeast strain that can be directly applied to the food industry, Saccharomyces cerevisiae KCCM12638, a yeast strain for producing American whiskey, was selected as a parent strain among various yeast strains already used in the food industry. Accordingly, the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) of the present invention may be derived from Saccharomyces cerevisiae KCCM12638.

[0049] In another specific embodiment of the present invention, a mutant yeast strain having an improved crude protein content compared to the wild type was produced by irradiating the parent strain, Saccharomyces cerevisiae KCCM12638, with ultraviolet light. To produce the mutant strain, the parent strain was placed at a distance of 30 cm under a 254 nm UV lamp and irradiated with 3000 to 9000 mW / cm 2 The strain was treated with UV light at 2-hour intervals for 0 to 24 hours at a dose of 100 μg / mL. As a result, as shown in Fig. 1, it was confirmed that the survival rate was 5% compared to the parent strain at 10 hours after UV light exposure, and this time period was selected as the optimal UV light exposure time.

[0050] After UV irradiation, a mutant strain with a crude protein content improved by more than 60% compared to the wild type Saccharomyces cerevisiae KCCM12638 was discovered through an additional selection process, and named Saccharomyces cerevisiae HSY-NGM-029, and deposited in the Korea Center for Bioscience and Biotechnology (KCTC) Gene Bank on January 18, 2024, and assigned the accession number KCTC 15779BP.

[0051] Therefore, the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) having an improved crude protein content compared to the wild type was irradiated with ultraviolet rays of 250 to 260 nm wavelength at a distance of 5 to 50 cm, preferably 5 to 30 cm, in a vertical direction at a wavelength of 3000 to 9000 mW / cm2 to Saccharomyces cerevisiae KCCM12638. 2 It can be manufactured by irradiating for 8 to 10 hours with a dose of .

[0052] More specifically, the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) according to the present invention can be produced by a production method comprising the following steps a) and b):

[0053] a) a step of pre-cultivating Saccharomyces cerevisiae KCCM12638; and

[0054] b) A step of irradiating the above-mentioned pre-cultured Saccharomyces cerevisiae KCCM12638 with ultraviolet rays.

[0055] In the present invention, the pre-culture of step a) may be performed to enrich the strain before irradiating Saccharomyces cerevisiae KCCM12638 with ultraviolet rays.

[0056] The culture conditions for the above-mentioned enrichment may be applied without limitation as long as they are known in the art as a method for culturing yeast. More preferably, the method may be a method known for culturing Saccharomyces cerevisiae. For example, step a) may be a liquid culture of Saccharomyces cerevisiae KCCM12638 in a test tube, but is not limited thereto.

[0057] In the present invention, the manufacturing method may additionally include, between steps a) and b), a step a') of diluting the pre-cultured strain so that the optical density at 600 nm becomes 0.5 to 1.5.

[0058] In the present invention, step b) is to irradiate Saccharomyces cerevisiae KCCM12638 with ultraviolet rays having a wavelength of 250 to 260 nm at a distance of 5 to 50 cm in the vertical direction, preferably 5 to 30 cm away, at a wavelength of 3000 to 9000 mW / cm. 2 It can be performed by investigating for 8 to 10 hours with an investigation amount of .

[0059] At this time, if the distance to which ultraviolet rays are irradiated is less than 5 cm in the vertical direction, a problem may arise in which the mortality rate significantly increases even with short-term ultraviolet irradiation, and if the distance to which ultraviolet rays are irradiated is more than 50 cm in the vertical direction, a problem may arise in which mutations are not induced even with long-term ultraviolet irradiation.

[0060] At this time, if the UV irradiation time is less than 8 hours, the mortality rate is low, making it difficult to select mutant strains. After 10 hours of UV irradiation, the mortality rate reaches 100%, making it impossible to select viable mutant strains.

[0061] In the present invention, the manufacturing method may include a step of additionally culturing the colonies obtained after step b) to select mutant strains with improved crude protein content compared to the wild type. At this time, the additional culturing may be performed one or more times, for example, 1 to 5 times, preferably 1 to 3 times. At this time, it is preferable that the culture scale also increases as the number of culturing times increases.

[0062] In the present invention, the term “cultivation” means growing the strain under appropriately artificially controlled environmental conditions.

[0063] In an exemplary embodiment, the colonies obtained after step b) were first cultured at the well plate level, and then only mutant strains confirmed to have increased total protein concentration compared to the wild-type strain through the Bradford assay were first selected.

[0064] In another exemplary embodiment, the first-selected mutant strains were sub-cultured at the test tube level, and only mutant strains that were confirmed to have increased total protein concentration compared to the first-selected mutant strains through the Bradford assay were secondarily selected.

[0065] In another exemplary embodiment, the secondary selected mutant strains were tertiarily cultured at the flask level, and only mutant strains that were confirmed to have increased total protein concentration compared to the secondary selected mutant strains through the Bradford assay were tertiarily selected. The final selected mutant strain showed a crude protein content that was more than 60% higher than the initial wild type, confirming that the crude protein content was significantly improved.

[0066] In another exemplary embodiment, the crude protein content of the third-selected mutant strain was compared to the first-selected mutant strain through crude protein analysis of the 5L fermenter-scale culture. It was confirmed that the third-selected mutant strain exhibited a crude protein content that was increased by at least about 16% to 29% compared to the first-selected mutant strain.

[0067] Accordingly, the Saccharomyces cerevisiae mutant strain produced by the production method of the present invention can exhibit a crude protein content that is increased by at least about 16%, preferably at least 29%, compared to the wild type, and can be increased by up to about 60%, but is not limited thereto.

[0068] In the present invention, the medium used for the pre-culture of step (a) and the culture performed after step (b) may include carbon sources, nitrogen sources, amino acids, vitamins, etc. commonly used in yeast culture, and a person skilled in the art can appropriately select the types and concentrations of these components included in the medium according to the culture conditions. An appropriate culture temperature may be 20 to 30°C, but is not limited thereto.

[0069] The present inventors attempted to identify the distribution and pattern of mutations at the genome level in the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) with improved crude protein content compared to the wild type through whole genome re-sequencing. To this end, first, using Saccharomyces cerevisiae S288C as the whole genome, single nucleotide polymorphism (SNP) information was obtained between the parent strain KCCM12638 and the UV mutant strain HSY-NGM-029 (accession number KCTC 15779BP), and the ITS1, 5.8S rRNA, and ITS2 sequences (hereinafter referred to as “ITS portions”) were extracted from the base sequences obtained through whole genome resequencing, and BLAST was performed to compare the ITS portions of HSY-NGM-029 (accession number KCTC 15779BP) and the parent strain KCCM12638. As a result, as shown in Fig. 5, 841 base sequences out of 848 KCCM12638 ITS portions matched S288C, and as shown in Fig. 6, 848 out of 848 base sequences matched between the ITS portions of KCCM12638 and UV mutant strain HSY-NGM-029 (accession number KCTC 15779BP), confirming that UV mutant strain HSY-NGM-029 (accession number KCTC 15779BP) is also a Saccharomyces cerevisiae strain derived from KCCM12638.

[0070] Next, to confirm whether the UV mutant strain HSY-NGM-029 (accession number KCTC 15779BP) is an independently produced strain modified from KCCM12638, the SNPs of KCCM12638 and HSY-NGM-029 (accession number KCTC 15779BP) were compared. As a result, as shown in Table 2, HSY-NGM-029 (accession number KCTC 15779BP) had at least 25 SNPs compared to the KCCM12638 strain, namely, 26171G>C, 194499A>C mutations on chromosome 1, 813110T>G mutations on chromosome 2, 4090A>C, 316502T>G mutations on chromosome 3, 224264T>C, 364680T>A mutations on chromosome 8, 248948T>C mutations on chromosome 9, 502139A>C mutations on chromosome 10, 432073C>A mutations on chromosome 11, 1064798T>G, 1064817T>G on chromosome 12, It was confirmed to have the following SNPs: 1064832T>A, 1064858A>C, 1064858A>T mutations on chromosome 13, 354374G>T, 908306G>A, 908390C>T mutations on chromosome 14, 6714C>A, 783656G>A, 783659T>G, 783791C>T, 783810G>T mutations on chromosome 14, 30149T>A mutation on chromosome 15, and 19717C>A mutation on mitochondrial chromosome.

[0071] Therefore, in order to select a mutant strain having an improved crude protein content compared to the wild type, the manufacturing method of the present invention, after step b), compares Saccharomyces cerevisiae KCCM12638 with 26171G>C mutations in chromosome 1, 813110T>G mutations in chromosome 2, 4090A>C mutations in chromosome 3, 224264T>C mutations in chromosome 8, 502139A>C mutations in chromosome 9, 432073C>A mutations in chromosome 11, 1064798T>G mutations in chromosome 12, The method may further include a step of selecting a mutant strain having the SNPs of 1064832T>A, 1064858A>C, 1064858A>T mutations, 354374G>T, 908306G>A, 908390C>T mutations in chromosome 13, 6714C>A, 783656G>A, 783659T>G, 783791C>T, 783810G>T mutations in chromosome 14, 30149T>A mutation in chromosome 15, and 19717C>A mutation in mitochondrial chromosome.

[0072] The Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) manufactured in this manner exhibits improved crude protein content compared to the parent strain, wild-type Saccharomyces cerevisiae KCCM12638, and can be used in a method for mass-producing crude protein industrially.

[0073] Accordingly, the second aspect of the present invention relates to a method for producing crude protein using the aforementioned Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP).

[0074] In relation to the second aspect, the present invention also provides the use of a Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) for improving crude protein production.

[0075] Specifically, the protein production method may include a) a step of culturing a Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP); and b) a step of recovering a protein from the strain.

[0076] In the present invention, the culture in step a) may be performed using the pre-culture solution in which the strain is cultured, using an isolated strain, or using a strain or culture thereof stored frozen.

[0077] In the present invention, the culture in step a) includes batch culture, continuous culture, and fed-batch culture, and specifically, continuous culture can be performed in a batch process or a fed batch or repeated fed batch process, but is not limited thereto.

[0078] The cultivation according to the present invention is not particularly limited, but can be conducted using, for example, a liquid culture tank, a rotary drum fermentor, or a tray fermentor. In addition to the rotary drum or tray fermentor, any device useful for fermenting strains can be used in the method of the present invention without limitation in its form, and an appropriate device can be selected and used depending on the production scale.

[0079] In the present invention, the pH of the culture can be adjusted by appropriately adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture during cultivation. During cultivation, foaming can be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, oxygen or an oxygen-containing gas can be injected into the culture to maintain an aerobic state.

[0080] According to a specific embodiment of the present invention, the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) of step a) can be cultured in the same medium as the medium for cultivating its parent strain, Saccharomyces cerevisiae KCCM12638.

[0081] For example, the strain of step a) above can be cultured in a medium containing at least one selected from the group consisting of glucose, fructose or galactose as a carbon source, and yeast extract, peptone, tryptone, malt extract, beef extract, casein, soytone, sodium nitrate (NaNO3), ammonium chloride (NH4Cl) and ammonium sulphate ((NH4)2SO4) as a nitrogen source. Specifically, the strain of step a) can be cultured in a medium containing 0.5 to 5% (w / v) of glucose, fructose or galactose as a carbon source, 0.5 to 6% (w / v) of yeast extract as a nitrogen source and 0.1 to 3% (w / v) of peptone, but is not limited thereto, and a person skilled in the art can appropriately select the types and concentrations of carbon sources and nitrogen sources included in the medium according to culture conditions for the purpose of increasing crude protein productivity.

[0082] In addition, the culture conditions of the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) of step a) above can be cultured under the same conditions as the culture conditions of its parent strain, Saccharomyces cerevisiae KCCM12638.

[0083] For example, the strain of step a) above can be fed-batch cultured at a pH of 3 to 6, more preferably at a pH of 3 or more and less than 5.5, at a stirring speed of 100 to 1500 rpm, and with air injected at a speed of 0.5 to 10 vvm, but is not limited thereto, and a person skilled in the art can appropriately adjust the pH, stirring speed, air injection speed, etc. for the purpose of increasing crude protein productivity.

[0084] At this time, an additional supply solution may be injected at the point where all of the initial glucose has been consumed in step a). The supply solution may contain various carbon sources and / or various nitrogen sources, similar to the initial medium, and may contain the same or different carbon sources and / or nitrogen sources as the initial medium. A person skilled in the art can appropriately select and adjust the components and concentrations included in the supply solution, taking into account the components and culture conditions of the initial medium.

[0085] The injection rate of the above-mentioned supply solution can be determined within a range where the glucose concentration of the culture medium is close to 0 g / L and the ethanol concentration does not exceed 10 g / L. The injection rate of the supply solution including a carbon source and a nitrogen source within the above range may be, for example, 1 to 50 mL / hr, preferably 2 to 35 mL / hr, but is not limited thereto.

[0086] In the crude protein production method of the present invention, the culture medium used in step a) may be optimized to increase the crude protein content of the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP).

[0087] In the crude protein production method of the present invention, the culture temperature in step a) may be 20 to 30°C, but is not limited thereto.

[0088] In the crude protein production method of the present invention, step b) can be performed using a method known in the art for recovering crude protein by crushing a yeast strain. Known methods for recovering crude protein include, but are not limited to, centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC. For example, after recovering the cells from the culture solution by centrifugation, etc., and then drying them by a conventional method if necessary, the strain can be crushed to recover the crude protein. The cell wall of the strain can be disrupted by a method for dissolving the cell wall by enzyme treatment or a method for physically disrupting the cell wall. In addition, after centrifuging the culture solution, the cells can be washed with distilled water to obtain a yeast suspension, and then the yeast suspension can be disrupted by a known method to disrupt the cell wall, thereby recovering the crude protein.

[0089] The crude protein produced as described above can be used in various foods or feeds.

[0090] Accordingly, the third aspect of the present invention relates to a composition for food or food additives and a composition for feed or feed additives comprising a Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP), a culture of the strain or crude protein produced from the strain.

[0091] In relation to the third aspect, the present invention also provides a method for producing a food or food additive and a feed or feed additive using a Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP), a culture of the strain, or a crude protein produced from the strain.

[0092] Furthermore, the present invention provides a use of a Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) or a culture of the strain for use in the production of food or food additives and feed or feed additives having improved crude protein content.

[0093] The Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) according to the present invention, a culture of the strain, and a food or food additive composition comprising crude protein produced from the strain exhibit an improved crude protein content compared to the parent strain Saccharomyces cerevisiae KCCM12638, and thus has high nutritional value as an alternative protein source.

[0094] In the present invention, the strain may be at least one selected from the group consisting of the strain itself, a strain lysate, a strain concentrate, a strain extract, and a strain dried product.

[0095] In the present invention, the term "crusted material" may be obtained by physically and chemically treating the strain itself, by high-pressure sterilization or ultrasonic treatment, or by crushing the supernatant, fermented product or culture solution obtained by centrifuging the culture of the strain, but is not limited thereto.

[0096] In the present invention, the term "fermented product" may include a medium containing a strain, its metabolites, and extra nutrients obtained by culturing a microorganism in a medium for a certain period of time, a culture solution obtained by removing the strain from the medium, a dilution or concentrate of the medium or culture solution, or a dried product thereof. The term "fermentation" refers to a process in which a microorganism uses its own enzymes to decompose organic matter, other than a putrefaction reaction.

[0097] In the present invention, the method for obtaining a fermented product from the strain is not particularly limited, and the fermented product can be obtained according to a method commonly used in the relevant technical field or a similar field.

[0098] In the present invention, the fermentation product obtained from the strain may include all kinds of materials including the fermentation product generated from the strain, such as not only the fermented material itself, but also a culture medium of the strain in which the strain and the culture coexist, a fermentation product obtained by filtering the strain from the culture medium, a supernatant obtained by centrifuging the culture medium to remove the strain, a fermentation product obtained by sterilizing the strain from the culture medium and filtering it, an extract obtained by extracting the fermentation product or the culture medium containing it, a diluted solution obtained by diluting the fermentation product or its extract, a dried product obtained by drying the fermentation product or its extract, and a lysate obtained by capturing and crushing the cells of the strain.

[0099] In the present invention, the term "extract" refers to the resultant product, such as a liquid component obtained by immersing a target substance in various solvents and then extracting it for a certain period of time at room temperature, low temperature, or heated conditions, or a solid component obtained by removing the solvent from the liquid component. In addition, it can be comprehensively interpreted to include, in addition to the resultant product, a dilution of the resultant product, a concentrate thereof, a controlled product thereof, a purified product thereof, etc.

[0100] In the present invention, the term "dry product" refers to a strain or culture from which moisture has been removed, and may include a fungus and culture dried by a known drying method such as spray drying, freeze drying, or vacuum drying.

[0101] In the present invention, the term "culture" refers to a product obtained by culturing the strain in a medium. For example, the culture of the present invention may include a component remaining in the medium remaining after harvesting the strain from the culture medium of Saccharomyces cerevisiae mutant strain HSY-NGM-029 (Accession No. KCTC 15779BP), or may include a component of a culture medium containing the strain. The term "culture solution" in the present invention refers to a portion of the culture excluding the strain. For example, the culture solution of the present invention may include a portion of the culture supernatant excluding the strain after centrifugation from the culture medium of Saccharomyces cerevisiae mutant strain HSY-NGM-029 (Accession No. KCTC 15779BP), or a portion of the culture supernatant excluding the lysate.

[0102] The culture may be a Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP), a whole culture of the strain, a culture medium, fractions thereof, etc. The culture solution may be a culture supernatant of the strain, etc. In this case, the culture supernatant may be obtained by centrifuging a culture of the strain, and the fraction may be obtained by subjecting the strain, the culture, the culture supernatant, etc. to a method such as centrifugation or chromatography.

[0103] The term "food composition" as used herein includes all forms of functional food, nutritional supplement, health food, and food additives, and the above types of food compositions can be manufactured in various forms according to conventional methods known in the art.

[0104] In the present invention, the food composition may contain 0.02 to 80 wt%, preferably 0.02 to 50 wt%, of the strain, a culture of the strain, a crude protein produced from the strain, or a crude protein isolated from a culture of the strain, based on the total weight of the composition, but is not limited thereto, and the content thereof may be appropriately changed depending on the type of food, the food consumption target, the consumption purpose, etc.

[0105] In the present invention, the food may include, but is not limited to, beverages, bakery products, pasta, cereals, cereal bars, confectionery, sauces, soups, dairy substitutes, frozen desserts, ice cream, yogurt, smoothies, creams, spreads, salad dressings, mayonnaise, food decorations, seasonings, candy, gum, jellies, liquid e-cigarettes, and the like. Alternatively, the food may include, but is not limited to, artificial meat for vegetarians, protein bars, or nutritional drinks.

[0106] The above meat may be, but is not limited to, artificial meat or a meat substitute. In the present invention, the artificial meat or meat substitute refers to a meat imitation that has a texture, flavor, and / or aroma similar to that of meat.

[0107] The food composition is not particularly limited as long as it is a drinkable food. Non-limiting examples of the food include hot dogs, burgers, mincemeat, sausages, steaks, fillets, grilled meats, breasts, thighs, wings, meatballs, meatloaf, bacon, strips, fingers, nuggets, cutlets, or cubes of animal-based or non-animal-based (e.g., plant-based) food products, or a combination of animal-based or non-animal-based food products.

[0108] The food may in another embodiment be provided in the form of a soup or stew base, a bouillon, for example, as a powder or cube, a flavor packet, or a food additive such as a seasoning packet or shaker.

[0109] As used herein, the term "feed composition" refers to food fed to animals. The feed composition refers to a substance that supplies organic or inorganic nutrients necessary for sustaining the life of an animal or producing meat, milk, etc. The feed composition may additionally contain nutrients necessary for sustaining the life of an animal or producing meat, milk, etc. The feed composition may be manufactured using various types of feed known in the art, and specifically may include concentrate feed, roughage, and / or special feed.

[0110] The term "feed additive" as used herein includes substances added to feed for various purposes such as nutrient supplementation and weight loss prevention, increasing the digestibility and availability of fiber in feed, improving milk quality, preventing reproductive disorders and improving conception rates, and preventing summer heat stress. The feed additive according to the present invention corresponds to supplementary feed under the Feed Management Act, and may additionally include mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals; mineral preparations that are trace minerals such as zinc, copper, cobalt, and selenium; vitamins such as carotene, vitamin E, vitamins A, D, E, nicotinic acid, and vitamin B complex; protected amino acids such as methionine and lysine; protected fatty acids such as fatty acid calcium salts; probiotics (lactic acid bacteria), yeast cultures, and mold fermentations; and yeast agents.

[0111] The composition of the present invention may further include grains, such as ground or crushed wheat, oats, barley, corn and rice; plant-based protein feeds, such as feeds mainly composed of soybeans and sunflower; animal-based protein feeds, such as blood meal, meat meal, bone meal and fish meal; dry ingredients composed of sugars and dairy products, such as various types of milk powder and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters and the like.

[0112] The composition of the present invention may be administered to an animal alone or in combination with other feed additives in an edible carrier. Furthermore, the composition may be readily administered to an animal as a top dressing, by mixing it directly into feed, or as an oral formulation separate from the feed. When administered separately from feed, the composition may be prepared as an immediate-release or sustained-release formulation by combining it with a pharmaceutically acceptable edible carrier, as is well known in the art. Such edible carriers may be solid or liquid, such as cornstarch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil, and propylene glycol. When a solid carrier is used, the composition may be in the form of a tablet, capsule, powder, troche, or sugar tablet, or a top dressing in the form of a microdispersible. When a liquid carrier is used, the composition may be in the form of a gelatin soft capsule, or a syrup, suspension, emulsion, or solution.

[0113] The composition of the present invention may contain, for example, a preservative, a stabilizer, a wetting or emulsifying agent, a cryoprotectant, or an excipient. The cryoprotectant may be at least one selected from the group consisting of glycerol, trehalose, maltodextrin, skimmed milk powder, and starch. The preservative, stabilizer, or excipient may be included in the composition in an effective amount sufficient to reduce deterioration of the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (Accession No. KCTC 15779BP), a culture of the strain, or a crude protein produced from the strain. In addition, the cryoprotectant may be included in the composition in an effective amount sufficient to reduce degradation of the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP), a culture of the strain, or a crude protein produced from the strain when the composition is in a dried state.

[0114] The above composition can be used by adding it to animal feed by immersion, spraying, or mixing.

[0115] The composition according to the present invention can be applied to a number of animal diets, including, but not limited to, mammals, birds, fish, crustaceans, cephalopods, reptiles, and amphibians. For example, the mammals can include pigs, cattle, sheep, goats, laboratory rodents, or pets, and the birds can include poultry, and the poultry can include, but is not limited to, chickens, turkeys, ducks, geese, pheasants, or quail. In addition, the fish can include commercially farmed fish and their fry, ornamental fish, and the crustaceans can include, but are not limited to, shrimp, barnacles, and the like. In addition, the composition can also be applied to the diet of rotifers, which are zooplankton.

[0116] Hereinafter, the present invention will be described in more detail through examples. However, the present invention can be modified in various ways and can take various forms. Therefore, the specific examples and descriptions described below are only intended to aid in understanding the present invention and are not intended to limit the present invention to a specific disclosed form. It should be understood that the scope of the present invention includes all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention.

[0117] [Example 1]

[0118] Selection of yeast parent strains and optimization of UV irradiation conditions

[0119] 1-1. Selection of parent strain

[0120] To produce a high-protein yeast strain that can be directly applied to the food industry, Saccharomyces cerevisiae KCCM12638, a wild type of Saccharomyces cerevisiae used in whiskey production, was obtained from the Korea Microbiological Conservation Center and used.

[0121] 1-2. Optimization of UV irradiation conditions

[0122] After liquid culture of Saccharomyces cerevisiae KCCM12638 at the test tube level, the parent strain was exposed to 3000 to 9000 mW / cm at a vertical distance of 5-30 cm from a 254 nm UV lamp for UV random mutation production. 2 Treatment was performed every 2 hours for 0 to 24 hours with the dose of 10 5 After diluting the culture medium and spreading it on a medium containing 1% yeast extract, 2% peptone, 2% glucose, and 1.5% agar, colonies were obtained at a time point where a survival rate of 5% compared to the parent strain was achieved, as confirmed in Figure 1.

[0123] [Example 2]

[0124] Tertiary screening of mutant strains

[0125] 2-1. Primary screening of mutant strains

[0126] Mutant strains with a crude protein content increased by 20% or more compared to the wild type were first selected through yeast culture at the 96-well plate level. Yeast colonies formed on YPD solid medium (10 g / L yeast extract, 20 g / L bacto peptone, 15 g / L bacto agar, 20 g / L glucose) were picked with a sterile toothpick and inoculated into 200 μL YP50D liquid medium (10 g / L yeast extract, 20 g / L bacto peptone, 50 g / L glucose). After inoculation, yeast cells were cultured with shaking at 30°C and 250 rpm for 48 h and then centrifuged at 5,000 rpm for 10 minutes to recover.

[0127] After preparing a cell lysate by adding Y-PER™ yeast protein extraction reagent (Thermo Fisher Scientific Inc.), the relative protein content (%) compared to the parent strain was determined using the Bradford protein assay. As shown in Fig. 2, when the parent strain was used as a control, mutant strains with a crude protein content increased by 20% or more were first selected.

[0128] 2-2. Secondary screening of mutant strains

[0129] After culturing the mutant strains selected in the first stage to the test tube level, mutant strains with a crude protein content increased by 20% or more compared to the mutant strain HS-Y001 selected in the first stage were selected in the same manner as in Example 2-2. The results of the second selection of mutant strains are shown in Fig. 3.

[0130] 2-3. Tertiary screening of mutant strains

[0131] The above-mentioned secondary selected mutant strains were cultured at the 500 mL flask level, and the protein concentration (mg / L) was determined using the Bradford protein assay. Yeast culture at the flask level was performed as follows. 100 μL of yeast glycerol stock stored in an ultra-low temperature deep freezer was inoculated into 5 mL YPD liquid medium (10 g / L yeast extract, 20 g / L bacto peptone, 20 g / L glucose) and cultured with shaking at 30 °C and 250 rpm for 48 hours. The pre-cultured culture was inoculated into a flask containing 100 mL YP50D liquid medium so that the optical density at 600 nm (OD600) became 1.0. The main culture was performed at 30 °C and 250 rpm for 48 hours. To measure protein concentration, 200 μL of the culture solution was transferred to a 96-well plate and centrifuged at 5,000 rpm for 10 minutes to recover yeast cells.

[0132] As shown in Fig. 4, the mutant strain HSY-NGM-029 (HS-Y001-98) with a crude protein content increased by more than 20% compared to the second-selected mutant strain HS-NGM-013 (HS-Y001-10) was selected for the third time. The results of the third selection of the mutant strains are shown in Fig. 4.

[0133] [Example 3]

[0134] Measurement of crude protein content of UV mutant strains

[0135] Using HS-Y001, which was selected in the first round, as a control, the crude protein contents of HSY-NGM-023 (HS-Y001-62) confirmed in the third round of screening and HSY-NGM-029 (HS-Y001-98), which was selected in the final round, were measured. The crude protein contents of the three strains were measured using a crude protein analyzer (FOSS, KJELTEC 8400) using cultures at the level of a 5 L fermenter in duplicate. As a result, as shown in Table 1, it was confirmed that the crude protein content of the finally selected strain HSY-NGM-029 (HS-Y001-98) was improved by approximately 29% compared to the control strain HS-Y001. Since the results are obtained by measuring the crude protein content using a 5L fermenter-level culture, the improved protein content compared to the control group may be less than the total protein content results of the 96-well plate, test tube, and flask-level cultures in the first, second, and third selection processes of Examples 1 and 2. However, it can be seen that the finally selected strain HSY-NGM-029 (HS-Y001-98) still contains crude protein that is improved by at least about 29% compared to the first selected strain HS-Y001 even in the 5L fermenter-level culture.

[0136] Yeast strain HS-Y001 (control) HSY-NGM-023 HSY-NGM-029 Crude protein content 56.1±0.3% 64.8±0% 72.2±0.2%

[0137] [Example 4]

[0138] Identification and SNP analysis of UV mutant strains

[0139] We commissioned Macrogen to perform whole genome resequencing, using Saccharomyces cerevisiae S288C as the whole genome, to obtain SNP information between the parent strain KCCM12638 and the final selected UV mutant strain HSY-NGM-029. Whole genome resequencing is a method of analyzing mutations between specific individuals in a species with a whole genome (reference genome) and comparing the base sequences of individuals or populations. Whole genome resequencing can be used to identify the distribution and pattern of mutations at the genome level.

[0140] First, we extracted the ITS1, 5.8S rRNA, and ITS2 sequences (hereinafter, ITS portion) from the base sequences obtained through whole genome resequencing to confirm whether the HSY-NGM-029 strain was the same Saccharomyces cerevisiae as the parent strain KCCM12638.

[0141] To compare the ITS region, BLAST (https: / blast.ncbi.nlm.nih.gov / ) was performed to compare the S288C of the whole genome from the whole genome resequencing with the ITS region of Saccharomyces cerevisiae KCCM12638, the parent strain of the mutant. As confirmed in the comparison results in Fig. 5, 841 of the 848 KCCM12638 ITS regions were identical to S288C.

[0142] Next, the ITS region of the parent strain Saccharomyces cerevisiae KCCM12638 and the UV mutant strain HSY-NGM-029 were compared. As shown in Fig. 6, the ITS region of the Saccharomyces cerevisiae KCCM12638 strain and the HSY-NGM-029 strain were identical in 484 out of 484 base sequences, confirming that the HSY-NGM-029 strain was also a Saccharomyces cerevisiae strain derived from KCCM12638.

[0143] Next, to confirm whether the HSY-NGM-029 strain is an independently produced strain modified from KCCM12638, SNPs from whole-genome resequencing analysis were used. The results of comparing SNPs between the KCCM12638 and HSY-NGM-029 strains based on the chromosomal sequence of S288C are shown in Table 2. The chromosomal sequence information of the reference S288C is publicly available on NCBI, and the NCBI ID information for each chromosome in Table 2 is shown in Table 3.

[0144] Chromosome locationS288CKCCM12638HSY-NGM-029126171TGC1194499TAC2813110ATG34090TAC331650 2ATG8224264GTC8364680CTA9248948ATC10502139GAC11432073GCA121064798ATG121064 817ATG121064832GTA121064858TAC121064858GAT13354374CGT13908306TGA13908390GC T146714TCA14783656CGA14783659CTG14783791GCT14783810AGT1530149GTAMT19717TCA

[0145] Chromosome#ChromosomeGenbank IDRefSeq ID1NC_001133.9BK006935.2NC_001133.92NC_001134.8BK006936.2NC_001134.83NC_001135.5BK006937.2NC_001135.53NC_001135.5BK0069 37.2NC_001135.58NC_001140.6BK006934.2NC_001140.69NC_001141.2BK006942.2NC_001141.210NC_001142.9BK006943.2NC_001142.911NC _001143.9BK006944.2NC_001143.912NC_001144.5BK006945.2NC_001144.513NC_001145.3BK006946.2NC_001145.313NC_001145.3BK006946.2NC_001145.314NC_001146.8BK006947.3NC_001146.815NC_001147.6BK006948.2NC_001147.6MitochondrialNC_001224.1AJ011856.1NC_001224.1

[0146] As shown in Table 2, the HSY-NGM-029 strain has at least 25 SNPs compared to the KCCM12638 strain. Specifically, the HSY-NGM-029 strain has 26171G>C, 194499A>C mutations on chromosome 1, 813110T>G mutations on chromosome 2, 4090A>C, 316502T>G mutations on chromosome 3, 224264T>C, 364680T>A mutations on chromosome 8, 248948T>C mutation on chromosome 9, 502139A>C mutation on chromosome 10, 432073C>A mutation on chromosome 11, 1064798T>G, 1064817T>G, 1064832T>A, 1064858A>C, 1064858A>T mutations on chromosome 12, and It has the SNPs 354374G>T, 908306G>A, 908390C>T mutations, 6714C>A, 783656G>A, 783659T>G, 783791C>T, 783810G>T mutations on chromosome 14, 30149T>A mutation on chromosome 15, and 19717C>A mutation on mitochondrial chromosome.

[0147] Accordingly, the third-selected Saccharomyces cerevisiae HSY-NGM-029 was deposited in the Korea Center for Bioscience and Biotechnology (KCTC) Gene Bank on January 18, 2024, and was assigned the accession number KCTC 15779BP.

[0148] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0149] [Accession number]

[0150] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0151] Accession number: KCTC15779BP

[0152] Date of acceptance: 20240118

[0153]

Claims

Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) with improved crude protein content compared to the wild type. In claim 1, the mutant strain HSY-NGM-029 is a strain derived from Saccharomyces cerevisiae KCCM12638, a yeast strain for producing American whiskey. In the second paragraph, the mutant strain HSY-NGM-029 is Saccharomyces cerevisiae KCCM12638, and is exposed to ultraviolet rays of 250 to 260 nm wavelength at a distance of 5 to 50 cm in the vertical direction at a dose of 3000 to 9000 mW / cm. 2 A strain manufactured by irradiating with a dose of 8 to 10 hours. In claim 1, the mutant strain HSY-NGM-029 has a crude protein content increased by 29 to 60% compared to the wild type. In the first paragraph, the mutant strain HSY-NGM-029, compared to Saccharomyces cerevisiae KCCM12638, has 26171G>C, 194499A>C mutations in chromosome 1, 813110T>G mutations in chromosome 2, 4090A>C, 316502T>G mutations in chromosome 3, 224264T>C, 364680T>A mutations in chromosome 8, 248948T>C mutations in chromosome 9, 502139A>C mutations in chromosome 10, 432073C>A mutations in chromosome 11, and 1064798T>G, 1064817T>G, 1064832T>A in chromosome 12. A strain having single nucleotide sequence mutations of 1064858A>C, 1064858A>T mutations, 354374G>T, 908306G>A, 908390C>T mutations in chromosome 13, 6714C>A, 783656G>A, 783659T>G, 783791C>T, 783810G>T mutations in chromosome 14, 30149T>A mutation in chromosome 15, and 19717C>A mutation in mitochondrial chromosome. a) a step of pre-cultivating Saccharomyces cerevisiae KCCM12638; and b) A step of irradiating the above-mentioned pre-cultured Saccharomyces cerevisiae KCCM12638 with ultraviolet rays; A method for producing a Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) having an improved crude protein content compared to the wild type. A manufacturing method in claim 6, further comprising, between steps a) and b), a step a') of diluting the pre-cultured strain so that the optical density at 600 nm becomes 0.5 to 1.

5. In the fifth paragraph, step b) is performed by irradiating Saccharomyces cerevisiae KCCM12638 with ultraviolet rays having a wavelength of 250 to 260 nm at a distance of 5 to 50 cm in the vertical direction at a wavelength of 3000 to 9000 mW / cm. 2 A manufacturing method, which is performed by irradiating for 8 to 10 hours with an amount of . In the 6th paragraph, after step b), compared to Saccharomyces cerevisiae KCCM12638, 26171G>C, 194499A>C mutations in chromosome 1, 813110T>G mutations in chromosome 2, 4090A>C, 316502T>G mutations in chromosome 3, 224264T>C, 364680T>A mutations in chromosome 8, 248948T>C mutations in chromosome 9, 502139A>C mutations in chromosome 10, 432073C>A mutations in chromosome 11, 1064798T>G, 1064817T>G, 1064832T>A in chromosome 12, A manufacturing method further comprising a step of selecting a mutant strain having the SNPs of 1064858A>C, 1064858A>T mutations, 354374G>T, 908306G>A, 908390C>T mutations in chromosome 13, 6714C>A, 783656G>A, 783659T>G, 783791C>T, 783810G>T mutations in chromosome 14, 30149T>A mutation in chromosome 15, and 19717C>A mutation in mitochondrial chromosome. A method for producing crude protein using the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) of Article 1. In Article 10, a) a step of culturing Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP); and b) a step of recovering crude protein from the strain; A method for producing crude protein comprising: A food or food additive composition comprising the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) of the first clause, a culture of the strain, or a crude protein produced from the strain. A composition for feed or feed additive comprising the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) of the first clause, a culture of the strain, or crude protein produced from the strain. A method for producing feed or feed additive using the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) of Article 1, a culture of the strain, or crude protein produced from the strain. A method for producing a food or food additive using the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) of Article 1, or a culture of the strain, or a crude protein produced from the strain. Use of the mutant strain HSY-NGM-029 (accession number KCTC 15779BP) of Saccharomyces cerevisiae of the first paragraph for improving crude protein production. Use of the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) of claim 1 or a culture of the strain for use in the manufacture of feed or feed additive with improved crude protein content. Use of the Saccharomyces cerevisiae mutant strain HSY-NGM-029 (accession number KCTC 15779BP) of claim 1 or a culture of the strain for use in the manufacture of a food or food additive with improved crude protein content.

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