Primer set or combination of primer sets for identifying strains of baker's yeast, and method for identifying strains of baker's yeast

Abstract

To provide a primer set or a combination of primer sets that enables identification of whether a strain of baker's yeast corresponds to a specific strain of baker's yeast, and a method for identifying strains of baker's yeast.SOLUTION: A primer set or a combination of primer sets for identifying strains of baker's yeast comprises at least one primer set selected from the group consisting of primer sets 1 to 7.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a primer set or a combination of primer sets for identifying strains of baker's yeast, and a method for identifying strains of baker's yeast.

Background Art

[0002] In recent years, baker's yeast required for fermenting bread dough has more diverse and higher functions. Specifically, functions such as freeze tolerance, fermentation ability in low-sugar / high-sugar dough, and production of mold inhibitors during fermentation are required, and development mainly by cross-breeding, mutation treatment, or cell fusion has become active. In addition, as the number of diverse and high-function baker's yeasts increases, there is an increasing demand for a technique for identifying whether a baker's yeast is developed and produced in-house.

[0003] As a general method for identifying strains in microbiology, next-generation sequencers have been used so far, but there is a problem that it takes a great deal of cost and labor to clarify the DNA base sequences of strains worldwide.

[0004] In addition, as a method for analyzing polymorphisms in DNA base sequences, there is a method using SSR markers. SSR is a base sequence consisting of repeats of a unit sequence of several bases, and its sequence has the characteristic that the number of repeats differs between strain lines. Utilizing this characteristic, SSR has been used as a marker for identifying varieties such as crops. A specific region having SSR in a DNA base sequence is called a microsatellite.

[0005] Usually, detection of SSR markers is performed by PCR using a primer set that amplifies SSR markers, separating the amplified DNA fragments by electrophoresis, and detecting the DNA fragments with a fluorescent dye. And identification is performed by comparing the difference in the length of the detected DNA fragments with a reference strain.

[0006] Patent Document 1 discloses the use of SSR markers for discriminating specific strains of soybeans from other soybean varieties, and Patent Document 2 discloses the use of SSR markers for discriminating varieties of beech fern. As shown in Patent Documents 1 and 2, usually, even for variety discrimination, it is necessary to use a plurality of SSR markers. Non-Patent Document 1 reports the construction of a phylogenetic tree of baker's yeast based on microsatellites. However, more techniques and ingenuity than the construction of a phylogenetic tree are required for strain discrimination of baker's yeast, and SSR markers have never been used for strain discrimination of yeast.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0008]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0009] An object of the present disclosure is to provide a primer set or a combination of primer sets capable of discriminating whether a strain of baker's yeast is a specific strain of baker's yeast, and a method for discriminating a strain of baker's yeast.

Means for Solving the Problems

[0010] As a result of intensive studies to solve the above problems, the present inventors have found that it is possible to discriminate whether a strain of baker's yeast is a specific strain of baker's yeast based on the length of a DNA fragment of baker's yeast amplified using at least one of specific primer sets.

[0011] That is, the present invention relates to a primer set or a combination of primer sets for identifying strains of baker's yeast, which is composed of at least one primer set selected from the group consisting of the following primer sets 1 to 7. Primer set 1: A primer set composed of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 2; Primer set 2: A primer set composed of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 4; Primer set 3: A primer set composed of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 5 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 6; Primer set 4: A primer set composed of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 7 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 8; Primer set 5: A primer set composed of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 9 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 10; Primer set 6: A primer set composed of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 11 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 12; Primer set 7: A primer set composed of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 13 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 14

Advantages of the Invention

[0012] According to the present disclosure, it is possible to provide a primer set or a combination of primer sets capable of identifying whether a strain of baker's yeast is a specific strain of baker's yeast, and a method for identifying strains of baker's yeast.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Modes for Carrying Out the Invention

[0014] Embodiments will be described below, but the present invention is not limited to the following embodiments. The primer set or combination of primer sets for identifying strains of baker's yeast according to the present disclosure is composed of at least one primer set selected from the group consisting of the following primer sets 1 to 7. Primer set 1 is a primer set composed of a primer having the nucleotide sequence shown in SEQ ID NO: 1 and a primer having the nucleotide sequence shown in SEQ ID NO: 2; Primer set 2 is a primer set composed of a primer having the nucleotide sequence shown in SEQ ID NO: 3 and a primer having the nucleotide sequence shown in SEQ ID NO: 4; Primer set 3 is a primer set composed of a primer having the nucleotide sequence shown in SEQ ID NO: 5 and a primer having the nucleotide sequence shown in SEQ ID NO: 6; Primer set 4 is a primer set composed of a primer having the nucleotide sequence shown in SEQ ID NO: 7 and a primer having the nucleotide sequence shown in SEQ ID NO: 8; Primer set 5 is a primer set composed of a primer having the nucleotide sequence shown in SEQ ID NO: 9 and a primer having the nucleotide sequence shown in SEQ ID NO: 10; Primer set 6 is a primer set composed of a primer having the nucleotide sequence shown in SEQ ID NO: 11 and a primer having the nucleotide sequence shown in SEQ ID NO: 12; and Primer set 7 is a primer set composed of a primer having the nucleotide sequence shown in SEQ ID NO: 13 and a primer having the nucleotide sequence shown in SEQ ID NO: 14. And according to the primer set or combination of primer sets, it is possible to identify whether the strain of baker's yeast is a specific strain of baker's yeast. The primer set or combination of primer sets is particularly useful for identifying a specific strain of baker's yeast from among the main groups of baker's yeast strains currently on the market.

[0015] <Primer set> In the present disclosure, a primer set refers to a combination of a primer complementary to one end of a target base sequence and a primer complementary to the other end of the target base sequence, which can amplify the target base sequence by the PCR method. One primer is referred to as the forward primer, and the other primer is referred to as the reverse primer.

[0016] As is understood by those skilled in the art, a forward primer is a primer that anneals to the antisense strand of DNA and provides a starting point for the extension reaction from the 5'-end to the 3'-end by DNA polymerase when amplifying a target base sequence by the PCR method. A reverse primer is a primer that anneals to the sense strand of DNA and provides a starting point for the extension reaction from the 5'-end to the 3'-end by DNA polymerase when amplifying a target base sequence by the PCR method. Any primer is a short single-stranded oligonucleotide. As the primer constituting the primer set for identifying strains of Saccharomyces cerevisiae in the present disclosure, 19 to 27 bases are preferred.

[0017] The primer set for identifying strains of Saccharomyces cerevisiae in the present disclosure or a combination of primer sets may be composed of at least one primer set selected from the group consisting of primer sets 1 to 7. Any two, three, four, five, six, or seven of these primer sets can be combined and used for identifying strains of Saccharomyces cerevisiae.

[0018] The primer base sequences (SEQ ID NOs: 1 to 14) constituting primer sets 1 to 7 are as shown in Table 1.

[0019]

Table 1

[0020] Here, Primer Set 1 is designed to amplify a sequence having a microsatellite II-CAACGA [i.e., a repeating sequence of CAACGA] as a target base sequence, Primer Set 2 is designed to amplify a sequence having a microsatellite II-CAAb [i.e., a repeating sequence of CAA] as a target base sequence, Primer Set 3 is designed to amplify a sequence having a microsatellite C5 [i.e., a repeating sequence of GT] as a target base sequence, Primer Set 4 is designed to amplify a sequence having a microsatellite C4 [i.e., a repeating sequence of TAA+TAG] as a target base sequence, Primer Set 5 is designed to amplify a sequence having a microsatellite SCYOR267c [i.e., a repeating sequence of TGT] as a target base sequence, Primer Set 6 is designed to amplify a sequence having a microsatellite XVI-TAAc [i.e., a repeating sequence of AAT] as a target base sequence, and Primer Set 7 is designed to amplify a sequence having a microsatellite XI-AGG [i.e., a repeating sequence of AGC+AGG] as a target base sequence, respectively.

[0021] <Baker's yeast> In the present disclosure, baker's yeast refers to yeast used in the production of bread that assimilates sugar to produce carbon dioxide and alcohol, and also produces organic acids and aroma components. For example, Saccharomyces cerevisiae, Saccharomyces exiguus, Kluyveromyces lactis, Torulaspora delbrueckii, Candida utilis, Candida kefyr, and other yeasts commonly used in bread production can be mentioned.

[0022] The primer set for identifying strains of baker's yeast of the present disclosure is used to identify whether a strain of baker's yeast is a strain of a specific baker's yeast. As the specific strain of baker's yeast, since it is commonly used industrially, it is preferably a strain belonging to Saccharomyces cerevisiae.

[0023] Examples of strains belonging to Saccharomyces cerevisiae include strains used as baker's yeasts having high maltose fermenting power and high sugar tolerance, such as CFB27-1 (Accession No.: FERM P-15903); strains used as mold-inhibiting baker's yeasts, such as KGLY59 (Accession No.: FERM P-20635) and KSY290 (Accession No.: FERM P-18863); strains used as baker's yeasts having excellent fermentation power preservation stability after long-term refrigerated or frozen storage in a fresh yeast state, such as KCY1217 (Accession No.: NITE BP-1058) and KCY1222 (Accession No.: NITE BP-1059); strains used as baker's yeasts having a function of enhancing the tensile strength of dough, a mold-inhibiting function, and a high-sugar fermentation power function in high-sugar dough, such as KCY1240 (Accession No.: NITE BP-1269), KCY1249 (Accession No.: NITE BP-1270), KCY1251 (Accession No.: NITE BP-1272), and KCY1254 (Accession No.: NITE BP-1396); and strains used as baker's yeasts having high fermentation power under high-ethanol conditions, such as KCY1275 (Accession No.: NITE BP-02461), KCY1278 (Accession No.: NITE BP-02462), KCY1279 (Accession No.: NITE BP-02463), and KCY1280 (Accession No.: NITE BP-02464), etc.

[0024] Furthermore, among these strains belonging to Saccharomyces cerevisiae, the strain identification of CFB27-1 (Accession No.: FERM P-15903), KGLY59 (Accession No.: FERM P-20635), KCY1217 (Accession No.: NITE BP-1058), KCY1222 (Accession No.: NITE BP-1059), KCY1254 (Accession No.: NITE BP-1396), or KCY1278 (Accession No.: NITE BP-02462) is particularly useful.

[0025] <Strain Identification> According to the primer set or combination of primer sets of the present disclosure, it is possible to identify whether a strain of baker's yeast is a specific strain of baker's yeast. The ability to identify strains of baker's yeast means that differences among multiple strains used as baker's yeast can be found.

[0026] <Method for identifying strains of baker's yeast> The method for identifying strains of baker's yeast of the present disclosure includes a step of amplifying the DNA of the baker's yeast to be identified and the DNA of the baker's yeast to be compared using the primer set by PCR method respectively, a step of detecting the DNA of the baker's yeast to be identified and the DNA of the baker's yeast to be compared amplified in the amplifying step respectively, and a step of comparing the length of the DNA fragment of the baker's yeast to be identified detected by the detecting step with the length of the DNA fragment of the baker's yeast to be compared, thereby determining the identity of the strain of the baker's yeast to be identified with respect to the strain of the baker's yeast to be compared. According to this method, it is possible to identify whether a strain of baker's yeast is a specific strain of baker's yeast.

[0027] (DNA extraction or purification step) Prior to the DNA amplification step, in order to increase the DNA concentration of the sample, it is preferable to have a step of extracting or purifying DNA from a sample containing the DNA of the baker's yeast to be identified and a sample containing the DNA of the baker's yeast to be compared respectively. In the subsequent DNA amplification step, the amplification of the DNA of the baker's yeast to be identified and the DNA of the baker's yeast to be compared can be performed efficiently.

[0028] As samples, baker's yeast and bakery dough containing baker's yeast can be used. Examples of the baker's yeast include fresh yeast, semi-dry yeast, dry yeast, and frozen yeast. The bakery dough containing the baker's yeast may be, for example, bakery dough before fermentation, bakery dough after fermentation and before baking, or bakery dough after baking. Examples of the bakery dough include dough for breads such as sandwich bread (square sandwich bread, British bread, etc.), roll bread, sweet bread (bean jam bread, cream bread, etc.), vegetable bread (curry bread, etc.), hard breads (French bread, German bread, etc.), croissant, Danish, Italian bread, nan, pita bread, etc.; dough for pizza; dough for Chinese buns and steamed buns; dough for baked confectioneries such as panettone, kouign-amann, stollen, waffle, muffin, madeleine, financier, cookie, cracker, biscuit, etc.; and dough for fried confectioneries such as doughnuts including yeast doughnuts. Among these, as bakery dough containing a fermentation step, dough for breads and dough for fermented confectioneries such as panettone, kouign-amann, stollen, waffle, and yeast doughnuts are preferable. Further, the bakery dough may be stored refrigerated or frozen.

[0029] Examples of the method for extracting DNA include the phenol-chloroform extraction method and the silica membrane method, etc., and extraction and purification can also be carried out using commercially available DNA extraction and purification kits such as DNeasy Blood&Tissue Kit (manufactured by QIAGEN) and NucleoSpin Tissue (manufactured by Takara Bio Inc.). The DNA concentration of the DNA solution after the DNA extraction or purification step is preferably 0.01 to 200 ng / μL for use in the DNA amplification step.

[0030] (DNA Amplification Step) In the DNA amplification step, the DNA of the baker's yeast to be identified and the DNA of the baker's yeast for comparison are each amplified by the PCR method using one kind of primer set selected from the group consisting of primer sets 1 to 7.

[0031] The PCR method can be carried out under the commonly used conditions, and appropriate reagents and reaction conditions can be set for each primer set. Examples of the reagents in the PCR method include a primer set, DNA polymerase, and buffer, etc. The primer set is as shown in Table 1, and as the DNA polymerase and buffer, commercially available kits such as Go Taq (Premix) (manufactured by Promega) can also be used. Also, as the reaction conditions, for example, first heat-denature at 95°C for 2 minutes, and then repeat the reactions of denaturation: 94°C, 20 - 40 seconds; annealing: 53°C, 20 - 40 seconds; extension: 72°C, 20 - 40 seconds for 30 - 40 cycles, and finally react at 72°C for 5 minutes to terminate the PCR.

[0032] The primer set is composed of a pair of a forward primer and a reverse primer, and the forward primer and the reverse primer are set so as to sandwich the region of the DNA to be amplified. Due to heat denaturation in the PCR method, dissociation of double-stranded DNA occurs, the forward primer anneals to one single-stranded DNA, and the reverse primer anneals to the other single-stranded DNA. Starting from each primer, DNA is extended by DNA polymerase.

[0033] The parts of the DNA to which the forward primer and the reverse primer anneal are due to the specificity of each primer, and the number of bases in the region of the DNA sandwiched by the forward primer and the reverse primer varies depending on the sequence of the original DNA. Therefore, when two or more types of DNA fragments with different numbers of bases are obtained from a single chromosome, and when DNA fragments with different numbers of bases are obtained for each chromosome, these situations may occur.

[0034] (Step of detecting amplified DNA) In the step of detecting the amplified DNA, the DNA of the target baker's yeast and the DNA of the comparative baker's yeast amplified by the PCR method are detected respectively. Examples of the detection method include separating and detecting the amplified DNA fragments according to their lengths.

[0035] As a method for separating a DNA fragment, there is a method of performing electrophoresis using a gel and a buffer according to the length of the DNA fragment under conditions where the DNA fragment can be separated. Examples of the gel include an agarose gel and a polyacrylamide gel, and examples of the buffer include a tris-borate-EDTA (TBE) buffer and a tris-acetate-EDTA (TAE) buffer. As a method for detecting the separated DNA fragment, there is a method of directly or indirectly visualizing the DNA fragment by staining it with a nucleic acid staining reagent.

[0036] Examples of the method for staining a DNA fragment with a nucleic acid staining reagent include a method of adding a staining reagent to a gel or a sample to be electrophoresed before performing electrophoresis, and a method of immersing the gel after electrophoresis in a staining reagent. However, the method of immersing the gel after electrophoresis in a staining reagent is preferred. Examples of the nucleic acid staining reagent include ethidium bromide, 4',6-diamidino-2-phenylindole, propidium iodide, and acridine orange.

[0037] (Step for determining the identity of baker's yeast) In the step for determining the identity, the identity of the strain of the baker's yeast to be identified with respect to the strain of the baker's yeast to be compared is determined by comparing the length of the DNA fragment of the baker's yeast to be identified detected in the detecting step with the length of the DNA fragment of the baker's yeast to be compared.

[0038] The determination of identity may be performed under conditions that allow comparison between the distribution of the DNA fragments of the baker's yeast to be identified separated according to their lengths and the distribution of the DNA fragments of the baker's yeast to be compared separated according to their lengths. In electrophoresis, based on the identity of the distribution of bands that can be directly or indirectly visually recognized and appear on the same gel, the identity of the baker's yeast to be identified with respect to the strain of the baker's yeast to be compared can be determined.

[0039] Using any one primer set selected from the group consisting of primer sets 1 to 7, the method for identifying strains of baker's yeast of the present disclosure is carried out, and the result of comparing the DNA of the baker's yeast to be identified and the DNA of the baker's yeast as a comparison target is combined with the result of carrying out the method for identifying strains of baker's yeast of the present disclosure using a primer set other than the above-mentioned any one, and comparing the DNA of the baker's yeast to be identified and the DNA of the baker's yeast as a comparison target, so as to determine the identity of the strain of the baker's yeast to be identified with respect to the strain of the baker's yeast as the comparison target.

[0040] When combining a plurality of results, the results of carrying out the method for identifying strains of baker's yeast of the present disclosure using any two, three, four, five, six, or seven primer sets can be combined, and there is no particular limitation on the order in which those results are used. However, from the viewpoint of efficiently identifying a specific strain of baker's yeast, it is preferable to first select primer set 1, primer set 4, primer set 6, or primer set 7. Also, the more results are combined, the higher the accuracy of strain identification of baker's yeast, but depending on the type of specific strain of baker's yeast, it may be time-consuming and unnecessary.

[0041] As described above, in the method for identifying strains of baker's yeast of the present disclosure, by comparing the DNA of the baker's yeast to be identified and the DNA of the baker's yeast as a comparison target using a primer set composed of at least one primer set selected from the group consisting of primer sets 1 to 7, the identity of the strain of the baker's yeast to be identified with respect to the strain of the baker's yeast as the comparison target is determined, and based on that determination, it is possible to identify whether the strain of the baker's yeast to be identified is a specific strain of baker's yeast.

Examples

[0042] Examples are shown below to more specifically explain the present invention, but the present invention is not limited to these examples in any way. (1) DNA extraction and purification step Yeast 5×10 -4DNA extraction and purification were performed from g. Yeasts used were six types of yeasts A, B, C, D, E, and F belonging to Saccharomyces cerevisiae shown in Table 2 (all manufactured by Kaneka Corporation), and 35 types of yeasts 1 to 35 available on the market (all yeasts other than those manufactured by Kaneka Corporation). For DNA extraction and purification, DNeasy Blood & Tissue Kit (manufactured by QIAGEN) was used.

[0043]

Table 2

[0044] (2) DNA amplification step 0.5 μL of the 100 ng / μL DNA solution obtained in the DNA extraction and purification step was combined with all the reagents used shown in Table 3, and the DNA in the solution was amplified by the PCR method using TP-600 (manufactured by Takara Bio Inc.) under the reaction conditions shown in Table 4. The relationship between the primer sets used and the microsatellites targeted for amplification by each primer set, and the nucleotide sequences of the primers constituting the primer sets are as shown in Table 1. As the DNA polymerase (including buffer), Go Taq (Premix) (manufactured by Promega) was used.

[0045]

Table 3

[0046]

Table 4

[0047] (3) Detection step of amplified DNA For the DNA amplified by the PCR method, electrophoresis was performed using a 6% polyacrylamide gel with a constant-temperature slab electrophoresis apparatus NA-1112 (manufactured by Nippon Eido Co., Ltd.) to which 0.5×TBE buffer was added. After electrophoresis, the gel was immersed in a staining reagent for staining. The composition of the polyacrylamide gel is shown in Table 5. As the staining reagent, 0.5 μg / mL ethidium bromide was used.

[0048]

Table 5

[0049] For yeasts A to F and yeasts 1 to 35, the electrophoretograms of the DNA amplified by the PCR method using primer set 1 are shown in FIGS. 1A and 1B; the electrophoretograms of the DNA amplified by the PCR method using primer set 2 are shown in FIGS. 2A and 2B; the electrophoretograms of the DNA amplified by the PCR method using primer set 3 are shown in FIGS. 3A and 3B; the electrophoretograms of the DNA amplified by the PCR method using primer set 4 are shown in FIGS. 4A and 4B; the electrophoretograms of the DNA amplified by the PCR method using primer set 5 are shown in FIGS. 5A and 5B; the electrophoretograms of the DNA amplified by the PCR method using primer set 6 are shown in FIGS. 6A and 6B; and the electrophoretograms of the DNA amplified by the PCR method using primer set 7 are shown in FIGS. 7A and 7B. Also, in each figure, "M" indicates a ladder marker [DNA ladder marker 100 bp (manufactured by WATSON)], "A to F" indicate samples of yeasts A to F, and "1 to 35" indicate samples of yeasts 1 to 35, respectively.

[0050] (4) Step for determining the identity of baker's yeast · When primer set 1 is used as the first-choice primer set As shown in FIGS. 1A and 1B, for example, when yeast A is compared with the bands of yeasts B to F and yeasts 1 to 35, yeast A has a unique band distribution. Therefore, it can be seen that by performing the method for identifying strains of baker's yeast of the present disclosure using primer set 1, the strain of yeast A can be identified from the group of strains consisting of yeasts A to F and yeasts 1 to 35. In addition, since yeasts 1 to 35 are currently the main strains of baker's yeast circulating in the market, when the same band distribution as that of yeast A, which is the baker's yeast to be compared, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is a strain of yeast A. Note that the band distribution refers to the positions and the number of detectable bands, and if all of the positions and the number of bands are common, it can be determined that they have the same band distribution.

[0051] Similarly, when yeast C or E among yeasts A to F and yeasts 1 to 35 is compared with the bands of the other yeasts, respectively, yeast C or E has a unique band distribution. Therefore, it can be seen that by performing the method for identifying strains of baker's yeast of the present disclosure using primer set 1, the strains of yeasts C and E can be identified from the group of strains consisting of yeasts A to F and yeasts 1 to 35, respectively. In addition, since yeasts 1 to 35 are currently the main strains of baker's yeast circulating in the market, when the same band distribution as that of yeast C, which is the baker's yeast to be compared, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is a strain of yeast C, and when the same band distribution as that of yeast E, which is the baker's yeast to be compared, is obtained, it can be substantially determined that the baker's yeast to be identified is a strain of yeast E.

[0052] In addition, for the DNA of the baker's yeast to be identified, when the same band distribution as that of yeast B or F, which is the baker's yeast for comparison, is obtained by performing the baker's yeast strain identification method of the present disclosure using primer set 1, for the DNA of the baker's yeast to be identified, further, the primer set 2, primer set 3, primer set 4, primer set 5, primer set 6, or primer set 7 that can obtain the unique band distribution of yeast B or F may be used to perform the baker's yeast strain identification method of the present disclosure. Thus, it can be seen that the strains of yeast B and F can be identified from the strain group consisting of yeast A to F and yeast 1 to 35. In addition, since yeast 1 to 35 are currently the main strains of baker's yeast circulating in the market, when the same band distribution as that of yeast B, which is the baker's yeast for comparison, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is a strain of yeast B, and when the same band distribution as that of yeast F, which is the baker's yeast for comparison, is obtained, it can be substantially determined that the baker's yeast to be identified is a strain of yeast F.

[0053] Furthermore, for the DNA of the baker's yeast to be identified, when the same band distribution as that of yeast D, which is the baker's yeast for comparison, is obtained by performing the baker's yeast strain identification method of the present disclosure using primer set 1, for the DNA of the baker's yeast to be identified, further, whether the unique band distribution of yeast D can be obtained among yeast A to F and yeast 1 to 35, or the primer set 4, primer set 5, primer set 6, or primer set 7 that can identify at least (i), (ii), or (iii) among (i) yeast A or yeast D, (ii) yeast C or yeast D, and (iii) yeast D or yeast E may be used to perform the baker's yeast strain identification method of the present disclosure. Thus, it can be seen that the strain of yeast D can be identified from the strain group consisting of yeast A to F and yeast 1 to 35. In addition, since yeast 1 to 35 are currently the main strains of baker's yeast circulating in the market, when the same band distribution as that of yeast D, which is the baker's yeast for comparison, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is a strain of yeast D.

[0054] · When primer set 2 is the first-choice primer set As shown in FIGS. 2A and 2B, for example, when yeast E is compared with the bands of yeasts A to D, F, and yeasts 1 to 35, yeast E has a unique band distribution. Therefore, it can be seen that by performing the method for identifying strains of baker's yeast of the present disclosure using primer set 2, the strain of yeast E can be identified from the group of strains consisting of yeasts A to F and yeasts 1 to 35. Further, since yeasts 1 to 35 are the main strains of baker's yeast currently distributed in the market, when the same band distribution as that of yeast E, which is the comparative baker's yeast, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is a strain of yeast E.

[0055] Similarly, when yeast F is compared with other yeasts, yeast F has a unique band distribution. Therefore, it can be seen that by performing the method for identifying strains of baker's yeast of the present disclosure using primer set 2, the strain of yeast F can be identified from the group of strains consisting of yeasts A to F and yeasts 1 to 35. Further, since yeasts 1 to 35 are the main strains of baker's yeast currently distributed in the market, when the same band distribution as that of yeast F, which is the comparative baker's yeast, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is a strain of yeast F.

[0056] In addition, for the DNA of the baker's yeast to be identified, when the strain identification method of the baker's yeast of the present disclosure is performed using primer set 2, if the same band distribution as that of yeast A, B, C, or D, which is the baker's yeast for comparison, is obtained, then for the DNA of the baker's yeast to be identified, the strain identification method of the baker's yeast of the present disclosure may be further performed using primer set 1, primer set 4, primer set 5, primer set 6, or primer set 7 that can obtain the unique band distribution of yeast A, B, C, or D. From this, it can be seen that the strains of yeast A, B, C, and D can be respectively identified from the strain group consisting of yeast A to F and yeast 1 to 35. Also, since yeast 1 to 35 are currently the main strains of baker's yeast circulating in the market, when the same band distribution as that of yeast A, B, C, or D, which is the baker's yeast for comparison, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is the strain of yeast A, B, C, or D respectively.

[0057] · When primer set 3 is used as the first-choice primer set As shown in FIGS. 3A and 3B, for example, when yeast F is compared with the bands of yeast A to E and yeast 1 to 35, yeast F has a unique band distribution. Therefore, it can be seen that the strain of yeast F can be identified from the strain group consisting of yeast A to F and yeast 1 to 35 by performing the strain identification method of the baker's yeast of the present disclosure using primer set 3. Also, since yeast 1 to 35 are currently the main strains of baker's yeast circulating in the market, when the same band distribution as that of yeast F, which is the baker's yeast for comparison, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is the strain of yeast F.

[0058] In addition, for the DNA of the baker's yeast to be identified, when the strain identification method of the baker's yeast of the present disclosure is performed using primer set 3, if the same band distribution as that of yeast A, B, C, D, or E, which are the baker's yeasts for comparison, is obtained, then for the DNA of the baker's yeast to be identified, further, the strain identification method of the baker's yeast of the present disclosure may be performed using primer set 1, primer set 2, primer set 4, primer set 5, primer set 6, or primer set 7 that can obtain the unique band distribution of yeast A, B, C, D, or E. Thus, it can be seen that the strains of yeast A, B, C, D, and E can be identified from the strain group consisting of yeast A to F and yeast 1 to 35. Also, since yeast 1 to 35 are the main strains of baker's yeast currently circulating in the market, when the same band distribution as that of yeast A, B, C, D, or E, which are the baker's yeasts for comparison, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is the strain of yeast A, B, C, D, or E, respectively.

[0059] · When primer set 4 is used as the first-choice primer set As shown in FIGS. 4A and 4B, for example, when yeast A is compared with the bands of yeast B to F and yeast 1 to 35, yeast A has a unique band distribution. Therefore, it can be seen that the strain of yeast A can be identified from the strain group consisting of yeast A to F and yeast 1 to 35 by performing the strain identification method of the baker's yeast of the present disclosure using primer set 4. Also, since yeast 1 to 35 are the main strains of baker's yeast currently circulating in the market, when the same band distribution as that of yeast A, which is the baker's yeast for comparison, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is the strain of yeast A.

[0060] Among yeasts A to F and yeasts 1 to 35, when yeast B or D is compared with the bands of other yeasts, yeast B or D also has its own unique band distribution. Therefore, by performing the method for identifying strains of baker's yeast of the present disclosure using primer set 4, it can be seen that the strains of yeast B and D can be identified respectively from the group of strains consisting of yeasts A to F and yeasts 1 to 35. In addition, since yeasts 1 to 35 are currently the main strains of baker's yeast circulating in the market, for the baker's yeast to be identified, when the same band distribution as that of yeast B, which is the baker's yeast for comparison, is obtained, it can be substantially determined that the baker's yeast to be identified is the strain of yeast B, and when the same band distribution as that of yeast D, which is the baker's yeast for comparison, is obtained, it can be substantially determined that the baker's yeast to be identified is the strain of yeast D.

[0061] In addition, for the DNA of the baker's yeast to be identified, by performing the method for identifying strains of baker's yeast of the present disclosure using primer set 4, when the same band distribution as that of yeast C, E, or F, which is the baker's yeast for comparison, is obtained, for the DNA of the baker's yeast to be identified, further, the method for identifying strains of baker's yeast of the present disclosure may be performed using primer set 1, primer set 2, primer set 3, primer set 5, primer set 6, or primer set 7 that can obtain the unique band distribution of yeast C, E, or F. Thus, it can be seen that the strains of yeast C, E, and F can be identified respectively from the group of strains consisting of yeasts A to F and yeasts 1 to 35. In addition, since yeasts 1 to 35 are currently the main strains of baker's yeast circulating in the market, for the baker's yeast to be identified, when the same band distribution as that of yeast C, which is the baker's yeast for comparison, is obtained, it can be substantially determined that the baker's yeast to be identified is the strain of yeast C, when the same band distribution as that of yeast E, which is the baker's yeast for comparison, is obtained, it can be substantially determined that the baker's yeast to be identified is the strain of yeast E, and when the same band distribution as that of yeast F, which is the baker's yeast for comparison, is obtained, it can be substantially determined that the baker's yeast to be identified is the strain of yeast F.

[0062] · When primer set 5 is used as the first-choice primer set As shown in FIGS. 5A and 5B, for example, when yeast B is compared with the bands of yeasts A, C to F, and yeasts 1 to 35, yeast B has a unique band distribution. Therefore, it can be seen that by performing the method for identifying strains of baker's yeast of the present disclosure using primer set 5, the strain of yeast B can be identified from the group of strains consisting of yeasts A to F and yeasts 1 to 35. In addition, since yeasts 1 to 35 are currently the main strains of baker's yeast circulating in the market, when the same band distribution as that of yeast B, which is the baker's yeast for comparison, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is the strain of yeast B.

[0063] Similarly, when yeast F among yeasts A to F and yeasts 1 to 35 is compared with the bands of other yeasts, yeast F has a unique band distribution. Therefore, it can be seen that by performing the method for identifying strains of baker's yeast of the present disclosure using primer set 5, the strain of yeast F can be identified from the group of strains consisting of yeasts A to F and yeasts 1 to 35. In addition, since yeasts 1 to 35 are currently the main strains of baker's yeast circulating in the market, when the same band distribution as that of yeast F, which is the baker's yeast for comparison, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is the strain of yeast F.

[0064] In addition, for the DNA of the baker's yeast to be identified, when the same band distribution as that of yeast C or D, which is the baker's yeast for comparison, is obtained by performing the baker's yeast strain identification method of the present disclosure using primer set 5, for the DNA of the baker's yeast to be identified, further, the primer set 1, primer set 4, primer set 6, or primer set 7 that can obtain the unique band distribution of yeast C or D can be used to perform the baker's yeast strain identification method of the present disclosure. From this, it can be seen that the strains of yeast C and D can be identified respectively from the strain group consisting of yeast A to F and yeast 1 to 35. In addition, since yeast 1 to 35 are the main strains of baker's yeast currently circulating in the market, when the same band distribution as that of yeast C, which is the baker's yeast for comparison, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is the strain of yeast C. When the same band distribution as that of yeast D, which is the baker's yeast for comparison, is obtained, it can be substantially determined that the baker's yeast to be identified is the strain of yeast D.

[0065] Furthermore, for the DNA of the baker's yeast to be identified, when the same band distribution as that of yeast A or E, which is the baker's yeast for comparison, is obtained by performing the baker's yeast strain identification method of the present disclosure using primer set 5, for the DNA of the baker's yeast to be identified, further, the primer set 1, primer set 2, or primer set 4 that can obtain the unique band distribution of yeast A or E among yeast A to F and yeast 1 to 35 can be used to perform the baker's yeast strain identification method of the present disclosure. From this, it can be seen that the strains of yeast A and E can be identified respectively from the strain group consisting of yeast A to F and yeast 1 to 35. In addition, since yeast 1 to 35 are the main strains of baker's yeast currently circulating in the market, when the same band distribution as that of yeast A, which is the baker's yeast for comparison, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is the strain of yeast A. When the same band distribution as that of yeast E, which is the baker's yeast for comparison, is obtained, it can be substantially determined that the baker's yeast to be identified is the strain of yeast E.

[0066] · When primer set 6 is used as the first-choice primer set As shown in FIGS. 6A and 6B, for example, when yeast B is compared with the bands of yeast A, C to F, and yeast 1 to 35, yeast B has a unique band distribution. Therefore, it can be seen that by performing the method for identifying strains of baker's yeast of the present disclosure using primer set 6, the strain of yeast B can be identified from the group of strains consisting of yeast A to F and yeast 1 to 35. Further, since yeast 1 to 35 are the main strains of baker's yeast currently distributed in the market, when the same band distribution as that of yeast B, which is the baker's yeast for comparison, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is a strain of yeast B.

[0067] Similarly, when yeast D or F among yeast A to F and yeast 1 to 35 is compared with the bands of the other yeasts respectively, yeast D or F has a unique band distribution respectively. Therefore, it can be seen that by performing the method for identifying strains of baker's yeast of the present disclosure using primer set 6, the strains of yeast D and F can be identified from the group of strains consisting of yeast A to F and yeast 1 to 35 respectively. Further, since yeast 1 to 35 are the main strains of baker's yeast currently distributed in the market, when the same band distribution as that of yeast D, which is the baker's yeast for comparison, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is a strain of yeast D, and when the same band distribution as that of yeast F, which is the baker's yeast for comparison, is obtained, it can be substantially determined that the baker's yeast to be identified is a strain of yeast F.

[0068] In addition, for the DNA of the baker's yeast to be identified, when the strain identification method of the baker's yeast of the present disclosure is performed using primer set 6, if the same band distribution as that of yeast A, C, or E, which is the baker's yeast for comparison, is obtained, then for the DNA of the baker's yeast to be identified, further, the strain identification method of the baker's yeast of the present disclosure may be performed using primer set 1, primer set 2, primer set 4, primer set 5, or primer set 7 that can obtain the unique band distribution of yeast A, C, or E. Thus, it can be seen that the strains of yeast A, C, and E can be identified from the strain group consisting of yeast A to F and yeast 1 to 35. Also, since yeast 1 to 35 are the main strains of baker's yeast currently circulating in the market, when the same band distribution as that of yeast A, which is the baker's yeast for comparison, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is the strain of yeast A. When the same band distribution as that of yeast C, which is the baker's yeast for comparison, is obtained, it can be substantially determined that the baker's yeast to be identified is the strain of yeast C. When the same band distribution as that of yeast E, which is the baker's yeast for comparison, is obtained, it can be substantially determined that the baker's yeast to be identified is the strain of yeast E.

[0069] · When primer set 7 is used as the first-choice primer set As shown in FIGS. 7A and 7B, for example, when yeast B is compared with the bands of yeast A, C to F, and yeast 1 to 35, yeast B has a unique band distribution. Therefore, it can be seen that the strain of yeast B can be identified from the strain group consisting of yeast A to F and yeast 1 to 35 by performing the strain identification method of the baker's yeast of the present disclosure using primer set 7. Also, since yeast 1 to 35 are the main strains of baker's yeast currently circulating in the market, when the same band distribution as that of yeast B, which is the baker's yeast for comparison, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is the strain of yeast B.

[0070] Among yeasts A to F and yeasts 1 to 35, when yeast C or D is compared with the bands of other yeasts respectively, yeast C or D has its own unique band distribution. Therefore, it can be seen that by performing the method for identifying strains of baker's yeast of the present disclosure using primer set 7, the strains of yeast C and D can be identified respectively from the group of strains consisting of yeasts A to F and yeasts 1 to 35. Also, since yeasts 1 to 35 are the main strains of baker's yeast currently on the market, for the baker's yeast to be identified, when the same band distribution as that of yeast C, which is the baker's yeast for comparison, is obtained, it can be substantially determined that the baker's yeast to be identified is a strain of yeast C, and when the same band distribution as that of yeast D, which is the baker's yeast for comparison, is obtained, it can be substantially determined that the baker's yeast to be identified is a strain of yeast D.

[0071] Also, for the DNA of the baker's yeast to be identified, by performing the method for identifying strains of baker's yeast of the present disclosure using primer set 7, when the same band distribution as that of yeast E or F, which is the baker's yeast for comparison, is obtained, for the DNA of the baker's yeast to be identified, further, the method for identifying strains of baker's yeast of the present disclosure may be performed using primer set 1, primer set 2, primer set 3, primer set 5, or primer set 6 that can obtain the unique band distribution of yeast E or F. Thus, it can be seen that the strains of yeast E and F can be identified respectively from the group of strains consisting of yeasts A to F and yeasts 1 to 35. Also, since yeasts 1 to 35 are the main strains of baker's yeast currently on the market, for the baker's yeast to be identified, when the same band distribution as that of yeast E, which is the baker's yeast for comparison, is obtained, it can be substantially determined that the baker's yeast to be identified is a strain of yeast E, and when the same band distribution as that of yeast F, which is the baker's yeast for comparison, is obtained, it can be substantially determined that the baker's yeast to be identified is a strain of yeast F.

[0072] Furthermore, for the DNA of the baker's yeast to be identified, when the same band distribution as that of yeast A, which is the baker's yeast for comparison, is obtained by performing the baker's yeast strain identification method of the present disclosure using primer set 7, for the DNA of the baker's yeast to be identified, the baker's yeast strain identification method of the present disclosure may be further performed using primer set 1 or primer set 4 that can obtain the unique band distribution of yeast A. From this, it can be understood that the strain of yeast A can be identified from the strain group consisting of yeasts A to F and yeasts 1 to 35. In addition, since yeasts 1 to 35 are currently the main strains of baker's yeast distributed in the market, when the same band distribution as that of yeast A, which is the baker's yeast for comparison, is obtained for the baker's yeast to be identified, it can be substantially determined that the baker's yeast to be identified is the strain of yeast A.

[0073] As described above, by performing the baker's yeast strain identification method of the present disclosure using only primer set 1, it is possible to substantially determine whether the strain is any of strains A, C, or E of baker's yeast. By using only primer set 2, it is possible to substantially determine whether the strain is any of strains E or F of baker's yeast. By using only primer set 3, it is possible to substantially determine whether the strain is the strain of yeast F. By using only primer set 4, it is possible to substantially determine whether the strain is any of strains A, B, or D of baker's yeast. By using only primer set 5, it is possible to substantially determine whether the strain is any of strains B or F of baker's yeast. By using only primer set 6, it is possible to substantially determine whether the strain is any of strains B, D, or F of baker's yeast. By using only primer set 7, it is possible to substantially determine whether the strain is any of strains B, C, or D of baker's yeast.

[0074] Furthermore, even when it is not possible to determine which strain of the baker's yeast for comparison the strain of the baker's yeast to be identified is by any one primer set, by combining the results obtained using one or more primer sets other than the any one primer set, it is possible to substantially determine which strain of the baker's yeast for comparison the strain of the baker's yeast to be identified is.

[0075] Therefore, a primer set or a combination of primer sets containing at least one primer set selected from primer sets 1 to 7 is currently useful for identifying a specific strain of baker's yeast from among the main groups of baker's yeast strains circulating in the market, and it can be seen that it is possible to identify whether a baker's yeast strain is a specific strain of baker's yeast.

Claims

1. A primer set or combination of primer sets for identifying strains of baker's yeast, composed of at least one primer set selected from the group consisting of the following primer sets 1 to 7. Primer set 1: A primer set composed of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 2; Primer set 2: A primer set composed of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 4; Primer set 3: A primer set composed of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 5 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 6; Primer set 4: A primer set composed of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 7 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 8; Primer set 5: A primer set composed of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 9 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 10; Primer set 6: A primer set composed of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 11 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 12; Primer set 7: A primer set composed of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 13 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 14

2. The primer set or combination of primer sets according to claim 1, wherein the strain of baker's yeast is CFB27-1 (deposit number: FERM P-15903), KGLY59 (deposit number: FERM P-20635), KCY1217 (deposit number: NITE BP-1058), KCY1222 (deposit number: NITE BP-1059), KCY1254 (deposit number: NITE BP-1396), or KCY1278 (deposit number: NITE BP-02462) belonging to Saccharomyces cerevisiae.

3. A step of amplifying the DNA of the baker's yeast to be identified and the DNA of the baker's yeast to be compared by the PCR method using the primer set according to claim 1, A step of detecting the DNA of the baker's yeast to be identified and the DNA of the baker's yeast to be compared amplified in the step of amplification, and A method for identifying strains of baker's yeast, comprising a step of determining the identity of the strain of the baker's yeast to be identified with respect to the strain of the baker's yeast to be compared by comparing the length of the DNA fragment of the baker's yeast to be identified detected in the detecting step with the length of the DNA fragment of the baker's yeast to be compared.

4. The method for identifying strains of baker's yeast according to claim 3, wherein the strain of the baker's yeast to be compared is CFB27-1 (Accession No.: FERM P-15903), KGLY59 (Accession No.: FERM P-20635), KCY1217 (Accession No.: NITE BP-1058), KCY1222 (Accession No.: NITE BP-1059), KCY1254 (Accession No.: NITE BP-1396), or KCY1278 (Accession No.: NITE BP-02462) belonging to Saccharomyces cerevisiae.

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