JPH1156366A - Identification of yeast - Google Patents

Abstract

PROBLEM TO BE SOLVED: To carry out identification whether a yeast is a yeast for brewing or a yeast for non-brewing and is a yeast belonging to the genus Saccharomyces or a yeast belonging to another genus, by utilizing a polymerase chain reaction and a restriction fragment length polymorophism. SOLUTION: A primer so as to amplify a part of FLO1 gene of a yeast of the genus Saccharomyces is synthesized and the yeast of the genus Saccharomyces is identified whether it is a yeast except the genus Saccharomyces by the amplification result by PCR method. A primer prepared so as to amplify a part of rDNA is synthesized and a yeast is identified whether it is a yeast for brewing or a yeast except the yeast by utilizing a restriction fragment length polymorphism for comparing fragments obtained by amplified by PCR method with restriction enzymes.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to a brewer's yeast or a non-brewer's yeast, and to Saccharomyces.
Identification of a genus yeast or a yeast belonging to another genus is performed by polymerase chain reaction (Polymerase Chain Reaction (hereinafter, referred to as PCR)) and restriction enzyme polymorphism (Restriction Fragment Le
ngth Polymorphism (hereinafter referred to as RFLP)).

[0002]

BACKGROUND OF THE INVENTION In beer brewing, it is important to clearly distinguish brewing yeast from non-brewing yeast in order to maintain fermentation characteristics and overall product characteristics. For example, genus Hansenula, genus Brettanomyces, Candid
a) Saccharomyces other than genus and brewer's yeast
Incorporation of non-brewing yeasts of the genus yces reduces the quality of the beer, causing cloudiness and off-flavor.

[0003] On the other hand, detection, identification and characterization of yeast have traditionally been performed physiologically, biochemically, and morphologically, but are time-consuming and provide only incomplete and incomplete information. Often. Further, since the brewing yeast belongs to the genus Saccharomyces, yeasts belonging to the genus Saccharomyces other than the brewing yeast, such as Saccharomyces cerevisiae (S.
cerevisiae), Saccharomyces bayanas (S. bayan)
us), Saccharomyces ellipsoidus (S. ellip)
soideus) is difficult to separate and identify by phenotype. In particular, Saccharomyces diastaticus (S. diastaticus) is a serious beer-harmful yeast, and it is important that it can be reliably detected.

[0004] In view of the above background, a method for separating and identifying non-brewing yeast and brewing yeast belonging to the genus Saccharomyces has been desired. On the other hand, as prior art related to the present invention,
FEMS Microbiol. Lett., Vol. 108, p. 259-264, (1993)
And J. Am. Soc. Brew. Chem., Vol. 54, p. 97-102,
(1996). These documents disclose a method of amplifying a part of the yeast rDNA gene by a PCR method and identifying the amplified rDNA gene by using RFLP. However, although these methods can identify some genera of yeast, there is no report that it was possible to identify yeast for brewing and non-brewing reliably.

[0005]

Means for Solving the Problems The present invention relates to a conventional yeast.
In addition to identification using RFLP, which creates primers that can amplify a portion of rDNA and compares fragments generated by the action of restriction enzymes on PCR amplification products, yeast for brewing and yeast for non-brewing The present invention relates to a method for identifying a yeast in which a primer is synthesized based on a part of a gene having a difference between and a PCR is performed.

[0006] More specifically, (1) a PC capable of amplifying a part of a gene having characteristics as a brewing yeast capable of distinguishing a brewing yeast from a non-brewing yeast.
A method for preparing primers for R and identifying Saccharomyces yeast or non-Saccharomyces yeast based on the results of amplification by PCR, and (2) PCR primers capable of amplifying a part of yeast rDNA A method for identifying yeasts for brewing or other yeasts by creating and comparing fragments generated by the action of restriction enzymes on amplified products by the PCR method, so that yeast identification can be performed effectively. It is to be.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Further, the present invention will be specifically described. Conventional DNA extraction (Gene Saccharomyces) from typical Saccharomyces brewing yeasts and non-brewing yeasts (including Saccharomyces yeasts) for which the genus species to be identified is unclear and identification results are to be compared. , Vol.57, p267-27
2, (1987)).

[0008] On the other hand, the DNA sequences of brewer's yeast and non-brewer's yeast, which have already been reported or independently analyzed, are compared, and a gene having a difference between brewer's yeast and non-brewer's yeast is selected. For example, the FLO1 gene related to the cohesiveness of yeast reported previously (Watari et al., Yeast, Vol.
p.211-225, (1994)). FLO1
Since a gene has many repetitive sequences and polymorphism of the gene is expected, primers are synthesized so that the repetitive sequence portion can be amplified by PCR.

[0009] Various yeasts from which the synthesized primers have been extracted
Perform PCR in addition to DNA. PCR method is FEMS Microbiol. Let
t., Vol. 108, p. 259-264, (1993). A part of the amplified product is taken out and subjected to agarose gel electrophoresis. The results of the electrophoresis are compared, and it is determined whether the yeast belonging to the genus Saccharomyces depends on whether the electrophoresis pattern of the yeast to be identified is a yeast belonging to the genus Saccharomyces or a yeast other than the genus Saccharomyces.

On the other hand, primers for partially amplifying rDNA can be independently synthesized, but have been reported previously in FEMS Microbiol. Lett., Vol. 108, p. 259-264, (19)
93), J. Am. Soc. Brew. Chem., Vol. 54, p. 97-102, (1
996) can be synthesized and used. Those restriction enzymes used for RFLP is also introduced in this document, for example, it can be used Msp I, Scr FI, a Bst UI like.

Next, in the same manner as described above, the extracted yeast
A primer that amplifies a part of rDNA acts on DNA, and P
Amplify by the CR method and use for agarose gel electrophoresis. Further, a part of the amplification product is taken and subjected to a restriction enzyme, and then subjected to agarose electrophoresis. Thereafter, the results of the electrophoresis of the amplified product or the product treated with the restriction enzyme are compared, and among the Saccharomyces, it is possible to determine whether the yeast is a brewing yeast or a non-brewing yeast by an electrophoresis pattern.

As described above, by implementing the two methods,
It is possible to determine whether the yeast to be identified is a yeast belonging to the genus Saccharomyces, and whether or not the yeast belonging to the genus Saccharomyces is a yeast for brewing. In addition, by accumulating these data in a computer or the like, by comparing the experimental results of the yeast to be identified with the accumulated data,
It can be easily identified.

[0013]

The present invention will be specifically described below with reference to examples. Extraction of yeast DNA Four strains of brewing yeast of the genus Saccharomyces, 12 strains of non-brewing yeast of the genus Saccharomyces and 15 strains of non-brewing yeast other than the genus Saccharomyces were each added to 10 ml of YPD medium (1% Ye
(Asts extract, 2% Peptone, 2% Glucose), and the cells were collected by stationary centrifugation. Then, wash with distilled water, 0.2 ml of 2% Triton X-100, 1% SDS, 100 mM
The suspension was suspended in NaCl, 10 mM Tris-HCl (pH 8), and 1 mM Na ₂ EDTA. Further, 0.2 ml of phenol / chloroform / isoamyl alcohol (25: 24: 1) and 0.3 g of acid-washed glass beads were added, and the mixture was thoroughly stirred for 2 minutes and centrifuged for 5 minutes. After separating the aqueous layer, 1 ml of ethanol was added and 1
After leaving still at −20 ° C. for 0 minutes or more, the mixture was centrifuged again to collect the precipitate. The resulting precipitate is washed with 0.4 ml of TE (10 mM Tris-
HCl (pH7.5), 1mM EDTA) and 10mg / ml Rnase A
After adding 3 ml of the solution and heating at 37 ° C. for 30 minutes, 1 ml of ethanol was added, the mixture was allowed to stand at −20 ° C. for at least 10 minutes, and centrifuged to collect a precipitate. The resulting precipitate is combined with 50 ml of TE (10
mM Tris-HCl (pH 7.5), 1 mM EDTA), and a part of the solution was used for the test.

[0014] To determine the site of primers used in yeast of the genus Saccharomyces whether identification PCR method by PCR using a part of the FLO1 gene, using FLO1 gene. The FLO1 gene was referred to the report of Watari et al. (Yeast, Vol. 10, p. 211-225, (1994)). From the sequence of the FLO1 gene, a portion having a large number of repeated sequences and expected to be polymorphic in the gene was determined, and the following primers were synthesized so as to amplify the portion. 5'-CCAAAAATGACAATGCCTCATCGCTAT-3 '5'-CCATTGCTAGGATAGAATGGGGTAATAATTGGACG-3' PCR condition is 94 ° C after hot start for 5 minutes, 94 ° C
Thirty cycles of 2 minutes and 30 seconds, 55 ° C. for 45 seconds, and 72 ° C. for 1 minute and 30 seconds were repeated, and finally, the temperature was kept at 72 ° C. for 10 minutes. A part of this was taken out and subjected to agarose gel electrophoresis.

The results are shown in FIG. As a result, the genus Hansenula other than the genus Saccharomyces,
No band was observed in Candida, but only in Saccharomyces. Also, the characteristics of the band pattern made it possible to some extent to discriminate among yeasts for brewing or non-brewing among Saccharomyces.

Identification of Saccharomyces yeast for brewing or non-brewing Saccharomyces yeast Primers for amplifying a part of rDNA were prepared by the method of Molina et al. (FEMS Microbiol. Lett., Vol. 108, p. 259-264, ( 199
According to 3)), the following two primers were synthesized. 5'-CACCGTTTCCCGTCCGATC-3 '5'-CAGAACGCCTCTAAGTC-3' PCR conditions were FEMS Microbiol. Lett., Vol. 108, p. 256.
-264, (1993)). That is, 94 ° C
After 5 minutes hot start, 94 ° C for 2 minutes 30 seconds, 55 ° C 1
25 cycles of 30 minutes at 72 ° C for 3 minutes
Incubated at 10 ° C for 10 minutes.

The process of restriction enzyme A portion of the PCR amplification products was performed by treatment with Msp I and Scr FI. The results are shown in FIGS. As a result, in Saccharomyces yeast, different band patterns were observed between brewer's yeast and non-brewer's yeast.

[0018]

According to the present invention, identification of yeast for brewing or non-brewing, or yeast of the genus Saccharomyces or other genus can be performed effectively by combining PCR and RFLP. .

[Brief description of the drawings]

FIG. 1 is an electrophoretogram of the results of PCR performed with primers prepared from the DNA sequence of the FLO1 gene.

FIG. 2 is an electrophoretogram showing the results of PCR performed with primers prepared from the DNA sequence of rDNA.

FIG. 3 is an electrophoretogram when a PCR product obtained by a primer prepared from a DNA sequence of rDNA is treated with a restriction enzyme MspI .

FIG. 4 is an electrophoretogram when a PCR product obtained by a primer prepared from a DNA sequence of rDNA is treated with a restriction enzyme ScrFI .

 ──────────────────────────────────────────────────の Continued on the front page (72) Inventor Wataru Funabashi 2-13-1 Omorikita, Ota-ku, Tokyo Asahi Breweries, Ltd.

Claims (2)

[Claims] 1. A step of synthesizing a primer prepared to amplify a part of the FLO1 gene of Saccharomyces yeast and performing a PCR method to classify the yeast, and a method of amplifying a part of the rDNA gene. A method for identifying a yeast by combining a step of synthesizing a primer, performing a PCR method, and further performing a classification using a restriction enzyme fragment length polymorphism. 2. The method for identifying a yeast according to claim 1, wherein the primer prepared to amplify a part of the FLO1 gene has the following sequence: 5′-CCAAAAATGACAATGCCTCATCGCTAT-3 ′ 5′-CCATTGCTAGGATAGAATGGGGTAATAATTGGACG-3 ′