JP3498137B2 - Breeding yeast without foam - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to yeast breeding, and more particularly, to yeast breeding in which bubbles generated in mash of a sake-making process are low or absent. Further, the present invention, in addition to the breeding of such yeast, yeasts separated by it and alcoholic beverages by using the yeast (sake, grape wine, various brewed sake, liquor for producing distilled liquor such as shochu, moromi for producing vinegar) , Moromi for alcohol production, etc.).

[0002]

2. Description of the Related Art So far, there is a breeding example in which a non-foaming yeast is separated by separating a yeast having a low affinity for foam from a normal yeast having a foam having a high affinity for foam. However, a satisfactory breeding method capable of rapidly and surely selecting a target yeast by a method other than this has not yet been established.

[0003]

In the production of sake and other alcoholic beverages, in view of the fact that the efficiency of alcohol production is more important than ever before, the present inventors have focused on the problem of foam. Usually, moromi often produces bubbles during the fermentation process, and it is not uncommon for bubbles to overflow from the fermentation tank.

In order to prevent this point, a tank having a capacity much larger than the amount of lumps is used to prevent bubbles from overflowing, and a foam eraser or the like is used, but these are a great burden. , Far from being efficient. In addition, although the use of other antifoaming agents for soybean oil has been used in some areas, the use of additives does not meet the needs of modern consumers, and the development of an efficient antifoaming system is not possible. It is sought after.

[0005]

[Means for Solving the Problems] As described above, in the manufacturing process of sake and other alcoholic beverages, the amount of raw material that can be added to the fermentation tank is equal to the volume of the tank minus the amount of foam generated during fermentation. Become. If bubbles generated during the fermentation process are expected to overflow from the fermentation tank, it is necessary to deal with them using a foam eraser or the like.

Therefore, if moromi with low or no bubbles can be produced, the amount of raw materials to be added to the tank can be increased and the need for a bubble eliminator is eliminated, so that labor can be saved.

[0007] As described above, the present invention has been made in order to achieve the efficiency of alcohol production by producing moromi which has a lower foam than the conventional one or has no foam.

[0008] Therefore, the inventors of the present invention aim to improve the efficiency of alcohol production by producing moromi that has low foam or no foam, and to achieve the purpose, the yeast of moromi that produces low or no foam A new technical issue of breeding was set. That is, as described above, completely different from the conventional method of improving the apparatus and using additives, paying attention to the yeast itself, the generation or generation of bubbles is low,
Alternatively, yeast that can produce moromi without it (hereinafter,
It was decided to newly breed the yeast.

[0009] That is, the inventors of the present invention are aimed at the yeast breeding that newly produces moromi from brewer's yeast in which bubbles are unlikely to be generated in the mash process of sake, and as a result of studies from various aspects, genetic engineering techniques have been applied. As a result of various experiments, we developed a yeast isolation method that produces moromi that is less likely to cause bubbles than conventional yeasts by expressing specific DNA. By this method, Saccharomyces (Sacc
It has succeeded in breeding cerevisiae of the genus haromyces.

That is, the present invention is basically characterized in that a yeast for producing a mash that hardly causes bubbles is isolated and bred from yeasts belonging to the genus Saccharomyces cerevisiae using a specific DNA. This is a technical idea, and the specific DNA is MNN.
There is a DNA of a specific gene called 4 genes, and its nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing.

It is known that the MNN4 gene is involved in the synthesis of yeast cell wall, but they are involved in the bubble-free property of sake yeast, and the breeding of the bubble-free sake yeast using the same was successful. No examples have hitherto been reported, and the present invention is the first.

That is, the present invention was newly discovered that genes involved in yeast cell wall synthesis greatly contribute to the bubble-free property, and was completed as a result of further studies based on these useful new findings. And efficient selection of yeast that produces a mash that does not easily cause bubbles due to introduction of the MNN4 gene,
It is also involved in a total system for creating alcoholic beverages, including creation of the yeast.

In the present invention, the target foam-free yeast may be separated from the yeast into which the MNN4 gene has been introduced (positive selection). Therefore, the separation work is easy and simple. The method is excellent.

The foam-free yeast according to the present invention may be obtained by artificially mutating the parent strain or separating the one in which the charge of the cell wall is negatively changed without artificially mutating the parent strain.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION To carry out the present invention, cerevisiae of the genus Saccharomyces.
Using the yeast belonging to e), the MNN4 gene is introduced, and the strain having a negative cell wall charge is isolated. In this way, the target foam-free yeast is obtained. Further, if desired, a small-capacity test may be performed using these yeasts to isolate yeasts having a good fermenting power, and foam-free practical yeasts having a good fermenting power can be selected and bred.

The practice of the present invention may be carried out according to a conventional method of genetic engineering. For example, the MNN4 gene is amplified by the PCR method or the like and then introduced into a vector, and the obtained expression plasmid is subjected to the lithium chloride method, the protoplast method, It may be introduced into a host bacterium such as yeast according to a conventional method such as PEG method and electroporation method, and the obtained transformant may be screened using a marker in the vector.

In this way, the present inventors succeeded in breeding excellent foam-free yeast, one of which was K9-
It was named MNN4 and deposited as FERM P-17941 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

The foam-free yeast according to the present invention produces 50% of foam when compared to the parent strain when producing mash using the foam-free yeast.
Hereinafter, it is preferably 30% to 20% or less, and 10% or less, or almost no bubbles may be generated in some cases.

In the present invention, as yeast, cerevisiae of the genus Saccharomyces
Any yeast can be used as long as it is a yeast belonging to e), and for example, sake yeast (Kyoto No. 7 yeast, Kyokai 9 yeast,
Association No. 10 yeast, Meiki Ogawa yeast, etc.), wine yeast (Grape wine No. 1 yeast (Japan Brewing Society grape wine No. 1 yeast), grape wine No. 3, yeast 4 etc.), beer yeast, baker's yeast, etc. All yeasts can be advantageously used as long as they are yeasts belonging to the genus Saccharomyces cerevisiae, including the practical yeasts mentioned above and other yeasts commonly used for alcoholic fermentation. As the yeast, the present invention can be applied to yeast with bubbles, and further excellent effects without bubbles can be obtained by applying the present invention to yeast without bubbles.

[0020]

Example 1 (Isolation of MNN4 transgenic yeast) (1) Saccharomyces cerevisiae
cerevisiae) from No. 9 yeast (hereinafter also referred to as K-9) genomic DNA, using primer A (SEQ ID NO: 2, FIG. 5) and primer B (SEQ ID NO: 3, FIG. 6). Gene (SEQ ID NO: 1, Figure 1
4) was amplified by the PCR method and then the plasmid vector p
AUR101 (purchased from Takara Shuzo Co., Ltd.) and the amplified MNN4 gene were cleaved with restriction enzymes SphI and KpnI, and then ligated to give M in pAUR101.
A vector having the NN4 gene inserted therein was selected to obtain a vector pAUR101-MNN4 containing the MNN4 gene (its restriction enzyme map is shown in FIG. 7).

(2) Saccharomyce
s) Association No. 9 sake yeast (K-9) belonging to the genus cerevisiae, YPD medium (1% yeast extract, 2% peptone, 2% glucose) were inoculated and grown, and then aseptically collected. The cells were washed and transformed with the vector pAUR101-MNN4 containing the MNN4 gene (FIG. 7) to obtain aureobasidin A (0.0005 mg).
(/ Ml) containing YPD agar medium and culturing at 30 ° C., and growing colonies of aureobasidin A (0.00
(05 mg / ml) was further subcultured on YPD agar medium.

(3) That is, after the expression vector pAUR101-MNN4 containing the MNN4 gene obtained above was cleaved with the restriction enzyme XbaI, Aureob of Takara Shuzo Co., Ltd.
According to the instruction of the asidinA resistant yeast transformation system, K-9 was transformed by the lithium chloride method, and the transgenic yeast was selected on the plate containing aureobasidin A.

[0023]

Example 2 (Isolation of yeast expressing MNN4 gene from transformed yeast) The aureobasidin A resistant yeast strain isolated in Example 1 was cultured in YPD medium,
After collecting and washing the cells, Alcian blue solution pH 2.5 is applied to the cells.
(Wako Pure Chemical Industries, Ltd.) is added and suspended, and it is left to stand for 10 minutes. Then, it was washed twice with distilled water and a blue-stained strain was selected.

[0024]

Example 3 (Further Separation of Yeast Having Excellent Fermenting Power from Yeast Expressing MNN4 Gene) Regarding the yeast strain isolated in Example 2, 70% polished rice was used as the following table. Three-stage preparation was carried out with the preparation composition shown in 1. Yeast suspension was used for yeast (5 × 10 per 1 ml of feed water)
⁶ pieces), spices at 15 degrees, relations at 12 degrees, stays at 9 degrees,
From the next day, the product temperature was raised at 1 ° C / day until it reached 15 ° C, and thereafter the fermentation was continued at 15 ° C. Ethanol in the fermentation broth was measured using a vibration densitometer (Kyoto Denshi Co., Ltd.), acidity was 0.1N NaOH, amino acidity was 0.1N NaOH, and formalin solution (50% aqueous solution) was used for analysis prescribed by the National Tax Agency. We conducted a lawful analysis. The components of the resulting sake (in the upper tank) are shown in Table 2 below.

[0025] Lactic acid, etc .: Lactic acid 6 ml ・ KH ₂ PO ₄ 0.65 g ・ NaCl 0.25 g ・ MgSO ₄ 0.66 g / 100 mH ₂ O

[0026]

The generation state of bubbles is shown in FIG. 8 (photograph). As shown in this drawing-substitute photograph, for the parent strain K-9,
A strain showing a bubble-free property was obtained. The arrow indicates the liquid surface of mash. The conventional yeast with foam, Society No. 9, is covered with a large amount of foam after a while even if the foam on the liquid surface is erased, but the strain with the MNN4 gene introduced is not covered with foam from the beginning.

In this way, it was possible to breed yeast having a foam-free property and an excellent fermenting power. This strain was named K9-MNN4, and FER was established at the Institute of Life Science.
Deposited as MP-17941.

Although K-9 is described as an example, the MNN4 gene-introduced yeast can be isolated from other yeasts by the same method. According to the present invention, excellent foam-free yeast can be bred. For example, when the moromi is placed in a vertically long 300 ml beaker and heated at 30 ° C. for 30 minutes on the 6th day after the completion of the preparation test, compared to the parent strain. It is possible to screen for bacteria that have a foam height of 50% or less, preferably 30 to 20% or less from the liquid surface, and 10% or less or almost no foam is generated.

[0030]

According to the present invention, the MNN4 gene is introduced,
By expressing, bubble-free yeast can be bred very efficiently. Then, by further selecting, as the MNN4 gene-expressing strain, a strain having excellent fermenting power, it is possible to obtain a highly practical yeast having not only foam-free property but also fermenting power.

The foam-free yeast according to the present invention can suppress the generation of foam or completely suppress the generation of foam. For example, the amount of foam generated in the parent strain is 50% or less, further 30%, further 20. % Or less or 10% or less. Further, if the screening is further carried out, it can be expected to breed a complete foam-free yeast. Therefore, according to the present invention, it is not only possible to prevent bubbles from overflowing from the tank during the fermentation process, and since it is not necessary to consider the generation of bubbles, a very large capacity compared to the amount of mash. There is no need to use a tank, space and energy savings are achieved, and there is no need to use a defoamer or defoaming agent.

[0032]

[Sequence list]                 SEQUENCE LISTING <110> National Research Institute of Brewing <120> Breeding Method of Non-foaming Yeast <130> 6342 <141> 2000-8-15 <160> 3 <210> 1 <211> 4193 <212> DNA <213> Saccharomyces cerevisiae <400> 1 tgccctctga cctacgtagt tacttgcgtg gcttcaaatt tgatatcagc 50 tctcgagacg tcctgatctc tataccttat tgcttgcttt aggttgaggc 100 aataaagaaa taattacatc tttggttctt tccccctttt tatatatctg 150 caacaatatt aaacaattaa attaaagaag cacgtttccg ttttttatca 200 tataccattc ccctaaccgc cgaactcacc gcatcacaac gtcactattc 250 cttcacacaa ataaactaat tagttatgct tcagcgaata tcatctaaac 300 ttcacaggcg gttcttatct ggcctgctgc gtgtcaagca ctacccatta 350 aggcgcattc tccttccact gattctactg cagatcatca ttataacgtt 400 tatctggtca aattcaccgc agcgtaacgg acttgggcgg gacgctgatt 450 accttctacc aaattacaac gaacttgaca gtgatgatga ttcctggtat 500 agcatcctga cttcgtcttt caaaaacgat cgcaagatcc agttcgctaa 550 gacattatac gaaaatttaa aattcggcac caaccctaaa tgggtcaatg 600 aatatactct gcaaaatgac ctgctctcgg tcaaaatggg ccctcgaaag 650 ggcagtaagc tcgaatccgt ggatgagttg aagttttacg acttcgaccc 700 tcgtctcacg tggtccgttg tgctgaacca tttgcaaaat aatgacgcag 750 atcagccaga aaagttaccc ttttcatggt acgactggac aaccttccac 800 gagctgaata agctgatttc catagataaa actgttctgc cctgcaattt 850 tcttttccag tccgctttcg acaaagagtc tttagaggcc attgagacag 900 agctcggcga acctttgttc ctatacgaaa gaccaaagta cgcgcagaaa 950 ctgtggtaca aggccgctag aaaccaggac agaatcaaag actcaaagga 1000 actaaaaaag cattgttcca agctattcac tccagacggg catggctctc 1050 ctaagggttt aagatttaat acgcaatttc aaataaagga gctgtatgat 1100 aaagttagac ccgaagttta ccaattgcag gcaagaaact acattttgac 1150 tacacagtcg catccactat ccatttccat catcgaatca gataattcca 1200 cgtatcaagt ccccttgcaa actgaaaaat caaaaaactt ggtgcaatcc 1250 ggcctgttgc aggaatatat taatgataac attaattcta cgaacaagag 1300 aaagaaaaat aaacaggacg tagaattcaa ccataacagg cttttccagg 1350 aattcgtcaa taacgaccaa gttaactccc tatacaaact ggaaattgaa 1400 gaaactgata aattcacttt tgataaagat ttggtttatt tatccccttc 1450 ggatttcaag ttcgatgcct ccaaaaaaat tgaagagtta gaggaacaga 1500 agaaactcta tccggacaaa ttttccgctc ataatgagaa ttatctgaac 1550 agtttgaaga attccgtaaa gacaagccct gcattgcaaa gaaagttctt 1600 ctatgaggct ggtgccgtga accaatataa aggtatgggg ttccatcgtg 1650 acaagaggtt cttcaatgtt gatacattaa tcaatgataa acaagaatac 1700 caggctagat tgaactcaat gatcagaaca ttccaaaagt ttactaaagc 1750 caacggcatc atatcttggt tgtctcacgg aacgctgtac ggctatcttt 1800 acaatggaat ggctttccct tgggataacg atttcgactt gcaaatgccc 1850 attaagcatt tacaattgct cagtcaatac ttcaaccaat ctcttatatt 1900 ggaagaccca agacagggta atggacgtta tttcctagac gtcagcgact 1950 ccttgacagt aagaattaac ggtaacggta aaaacaatat cgatgcaaga 2000 ttcattgacg tcgacaccgg cctttacatt gatattaccg gtctagctag 2050 cacttctgcc cctagtaggg attacttgaa ttcttatatt gaagaccggt 2100 tgcaagagga acatttggat atcaataata tccctgaatc gaacggtgag 2150 accgctactt tgcccgacaa agtagatgat gggttagtca atatggctac 2200 actaaacatc actgagctac gtgattacat taccagcgac gaaaataaaa 2250 atcataaaag agtccccact gatactgatt tgaaagatct tttgaaaaag 2300 gaactggaag agttaccaaa gtctaagacc attgaaaaca agttgaatcc 2350 taaacaaaga tattttctca acgaaaaact taaactttac aattgtagaa 2400 acaaccattt taactcgttc gaggaactat ctcccttaat caatactgtt 2450 ttccatggtg tgccagcgtt gattcctcac agacatacct actgcttgca 2500 caatgaatat catgtacctg atagatatgc atttgatgct tacaaaaata 2550 ctgcttattt gcccgaattt agattttggt tcgactatga cgggttaaag 2600 aaatgcagta atattaattc atggtatcca aacatcccca gtattaattc 2650 atggaatccg aacctcttga aagaaatatc gtctacgaaa tttgagtcga 2700 aactttttga ttccaacaaa gtctctgaat actctttcaa aaacctatcc 2750 atggatgatg ttcgcttaat ttataaaaat attccaaaag ctggctttat 2800 cgaggtattt actaacttgt acaattcctt caatgtcact gcatataggc 2850 aaaaggaatt ggaaattcaa tactgccaaa acctgacatt tattgaaaaa 2900 aagaaattat tacatcaatt gcgcattaat gttgctccta agttaagctc 2950 ccctgcaaag gacccatttc tttttggtta tgaaaaagct atgtggaagg 3000 atttatcaaa atctatgaac cagactacat tagatcaagt taccaagatt 3050 gttcatgaag aatatgtccg aaaaattatt gatctgtccg aaagtttgaa 3100 atacaggaat ttttcacttt tcaacattac ttttgatgaa actggaacaa 3150 ctctagatga taacacagaa gattatactc ctgctaatac tgttgaagta 3200 aatcctgtgg attttaaatc aaatttaaac tttagtagca actccttttt 3250 ggatttaaat tcatatggtt tagacctttt tgcgccaact ttatccgacg 3300 ttaacagaaa gggtattcaa atgtttgata aggaccctat tattgtatac 3350 gaggactatg cttatgccaa gttacttgaa gaaagaaagc ggagggagaa 3400 gaagaagaag gaggaagagg agaagaagaa gaaggaagaa gaggaaaaga 3450 aaaagaagga agaagaagaa aagaaaaaga aggaagagga agagaagaaa 3500 aagaaggaag aagaagagaa gaaaaagaag gaagaagaag aaaagaagaa 3550 gcagcaggaa gaggagaaaa agaagaagga agaagaagag aagaagaagc 3600 aggaagaagg agaaaagatg aagaatgaag atgaagaaaa taagaagaat 3650 gaagatgaag aaaagaacaa gaacgaagaa gaggaaaaaa agaagcagga 3700 agagaaaaac aagaagaatg aagatgaaga aaagaagaag caggaagagg 3750 aagaaaagaa gaagaacgaa gaagaggaaa aaaagaagca ggaggagggg 3800 cacagcaatt aaaagtcgga gaacctgact gaaaattcat gaatctcttc 3850 atttctatag cctttcctct atgcatttgt attatatatt tattaccgtc 3900 attttttaca tactgctgca ttttggcgcc agtgataagt ggcaaacaat 3950 tcgacggaat cgtggtaatt ataccacgtt actctataac atcatgatat 4000 tgcaattaat caaacataca tttaatctta atgctattag cttactacaa 4050 ctcttttctt taagttatat cgtatatttc ttgggcgatg tcagaatatt 4100 tacccggata ttccttttta agcactgaat atgtttgaat agagactgac 4150 atatatggca gcaattaaaa ttggaagaaa tgtaatgaca gta 4193 <210> 2 <211> 30 <212> DNA <213> Artificial sequence <400> 2 tgcgcatgcc ctctgaccta cgtagttact 30 <210> 3 <211> 30 <212> DNA <213> Artificial sequence <400> 3 accggtacct actgtcatta catttcttcc 30

[Brief description of drawings]

FIG. 1 shows the DNA sequence of the MNN4 gene.

FIG. 2 shows the continuation of the above.

FIG. 3 shows the continuation of the above.

FIG. 4 shows the continuation of the above.

FIG. 5 shows primer A.

FIG. 6 shows primer B.

FIG. 7 shows a restriction map of plasmid pAUR101-MNN4.

FIG. 8: Association No. 9 yeast (yeast with foam) and the MNN4 transgenic strain newly bred according to the present invention (yeast without foam)
7 is a photograph as a substitute for a drawing, which compares the occurrence of bubbles in FIG.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI // (C12N 1/19 C12N 15/00 ZNAA C12R 1: 865) (C12N 15/09 ZNA C12R 1: 865) (58) Survey Fields (Int.Cl. ⁷ , DB name) C12N 15/09-15/90 C12G 1/00-1/12 C12G 3/00-3/14 C12N 1/19 SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / Gene Seq BIOSIS / MEDLINE / WPID S (STN)

Claims (5)

(57) [Claims] 1. A method of transforming a yeast commonly used for alcohol fermentation with a plasmid vector containing the DNA represented by the nucleotide sequence of SEQ ID NO: 1 in the sequence listing, and isolating the bubble-free yeast from the transformants, A method for breeding a bubble-free yeast characterized by: 2. A pAUR101- comprising a plasmid vector containing the DNA represented by the nucleotide sequence of SEQ ID NO: 1.
The breeding method according to claim 1, wherein the breeding method is MNN4. 3. A DNA represented by the base sequence of SEQ ID NO: 1.
Sake yeast that is a plasmid vector containing
Y9, a foam-free yeast transformed with No. 9 yeast
MNN4 (FERM P-17941). 4. A method for producing alcoholic beverages, comprising producing sake using the foam-free yeast bred and separated by the breeding method according to claim 1. 5. A method for producing alcoholic beverages, which comprises producing alcoholic beverages using the transformed foam-free yeast according to claim 3.