JP3426633B2 - Beer production method - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a high-concentration brewing method for beer. More specifically, the present invention relates to a method for producing high-concentration beer obtained by breeding maltose highly fermentable yeast and fermenting wort having a high sugar content using the yeast. In the present invention, "high-concentration beer" refers to a beer obtained by fermenting high-concentration wort. Also, "high concentration wort"
"Wort" refers to wort in which the wort extract concentration is higher than usual in each beer production method. [0002] Conventionally, in the production of beer, raw wort having an extract concentration of about 11% is fermented to obtain an alcohol concentration of 4.
About 5-5% of beers are obtained. Many beer companies have adopted high-concentration brewing methods to improve beer productivity. What is high gravity brewing?
This method involves fermenting wort that gives a beer having a higher wort concentration than the beer to be produced, and then diluting it with water to obtain a beer having a predetermined wort concentration (The BREWERS DI
GEST, Vol. 51, p. 34 (1976)). The extract concentration of the raw wort represents the specific gravity of the raw wort measured with a hydrometer as the concentration of sugar having the same specific gravity (weight / weight%). [0003] As concrete measures, (1) employ a temperature higher than the normal fermentation temperature, (2) increase the aeration rate to wort, (3) increase the amount of yeast added, and It is a combination of It is said that in the production of ordinary beer, brewing with a high concentration of about 15% of raw wort extract is the limit in these methods. [0004] One of the problems that arises when high-concentration brewing with a raw wort extract concentration of 15% or more is carried out is that the fermentation rate in the middle to late stages of fermentation is remarkably reduced. Carbohydrates contained in wort are mainly maltose, maltotriose, glucose, fructose,
Sucrose. Yeast first assimilates glucose, fructose and sucrose, and then assimilate maltose and maltotriose. Therefore, from the middle stage of the fermentation to the latter stage of the fermentation, only assimilable sugars in the mash are maltose and maltotriose. Moreover, the abundance ratio is overwhelmingly high at 3: 1 with maltose. [0005] Maltose is taken up into cells by maltose permease of yeast, then hydrolyzed to two molecules of glucose by maltase, and is converted into carbon dioxide and ethanol via the pathway of Embden-Meyerhof. Is converted to These two enzymes are
At the transcriptional level, it is known to be induced by maltose and suppressed by glucose. [0006] Since wort contains glucose at about 10 % of the assimilating sugar of yeast, the maltose metabolism gene of yeast is suppressed by glucose at an early stage of fermentation.
Maltose assimilation will be greatly delayed. This phenomenon becomes even more pronounced during high-concentration brewing, not only delaying maltose fermentation, but also leaving a large amount of maltose as assimilated sugar at the end of fermentation. Due to such problems, high-concentration brewing, for example, fermenting wort at twice the normal concentration, has been incomplete with conventional techniques. Japanese Patent Laid-Open No. 1-153082 discloses a baker's yeast in which a plasmid containing a promoter of an alcohol dehydrogenase gene which is not inhibited by glucose, a maltase gene and a maltose permease gene is introduced in order to improve dough fermentation by baker's yeast. Is disclosed. However, there is no specific description for utilizing this for high-concentration brewing of beer. SUMMARY OF THE INVENTION An object of the present invention is to improve maltose metabolism genes in yeast to promote maltose fermentation and realize high-concentration brewing. In order to achieve the above object, the present invention provides a glyceraldehyde triphosphate dehydrogenase (GAPDH) gene promoter and the structure of maltose permease under the control of the promoter. Provided is a method for producing beer, characterized in that high-concentration wort is fermented using yeast containing a gene to obtain high-concentration beer. In the present invention, a brewer's yeast into which a promoter of the GAPDH gene which is not suppressed by glucose and a structural gene of maltose permease under the control of the promoter is used, is used. However, the assimilation of maltose is not suppressed, and as a result, maltose is assimilated and fermented without waiting for the disappearance of glucose, so that the fermentation rate increases, and beer brewing with high-concentration wort becomes possible. [0011] DETAILED DESCRIPTION yeast Saccharomyces My Seth cerevisiae
(Saccharomyces cerevisiae ) has maltose metabolism genes as MAL1, MAL2, MAL3, and MAL.
Four overlapping genes of 4, MAL6 are known, and if any one of them is an active allele (locus), it becomes maltose fermentable. Each locus encodes three genes: maltose permease, maltase, and a positive regulator required for expression of these structural genes. As mentioned above, this maltose permease and maltase are induced at the transcriptional level by maltose and are suppressed by glucose. Therefore, in the early stage of fermentation, the presence of glucose in wort suppresses these enzymes, which significantly delays the utilization of maltose. The present inventors have proposed maltose permease (MAL6T), maltase (MAL6
S), and a positive control factor (MAL6R) was cloned by replacing the promoter of these genes to the promoter of the GAPDH (Guriseruarudehi de triphosphate Sande hydrogenase) gene, even in the presence of glucose, undergo silencing There is no mechanism, each,
Alternatively, in combination, multi-copy episomes are introduced into yeast cells, for example, in the form of a plasmid, or chromosome recombination is carried out with a chromosome-introduced plasmid to breed maltose-high-fermentable yeast, and for the first time actually produce brewer's yeast. It was confirmed that it could be applied to the production of the used beer. The present inventors bred the above maltose highly fermentable yeast in brewer's yeast, and brewed it at a high concentration under the conditions of actual brewing. In the above-mentioned Japanese Patent Application Laid-Open No. 01-153082, the assimilation rate of maltose is determined to be maltase (MAL6S).
The strain in which both the promoters of the maltose permease gene (MAL6T) and the promoter of the maltose permease gene (MAL6T) were replaced with higher expression was faster than the strain in which the promoter of the maltose permease gene (MAL6T) alone was replaced, but in the present invention, The maltose assimilation rate under brewing conditions was higher in the strain in which the promoter of the maltose permease gene (MAL6T) alone was replaced than in the maltase (MAL6S) and maltose permease gene (MAL6T).
L6T) was faster than the strain in which both promoters were replaced. From the above, it has become clear that maltose fermentation is more dependent on maltose uptake activity than maltase activity under actual beer brewing conditions. Furthermore, the strain of the present invention in which only the maltose permease gene (MAL6T) is highly expressed is a normal 2 strain.
Even in the wort of double concentration, the time to rapidly assimilate maltose and reach the appearance fermentation degree (% of the extract lost by fermentation with respect to the original wort extract) of 65% is reduced by about 30% compared to the parent strain. Was done. In order to increase the concentration of wort, it is possible to add glucose syrup and perform fermentation. According to the present invention, beer can generally be brewed using raw wort having an extract concentration higher than the extract concentration in the conventional method. Specifically, according to the present invention, beer brewing can be performed using raw wort having an extract concentration of 20% or more. More preferably, according to the present invention, beer brewing can be performed using raw wort having an extract concentration of 24% or more. The following experimental data is provided to illustrate the present invention. These methods are based on other beer brewing yeasts such as Saccharomyces cerevisiae IF O 951, I
It can be similarly used in FO1 952, IF O1 953, IF O1 954 and the like. Embodiment 1 Cloning of MAL6 gene Each gene of MAL6S, MAL6T and MAL6R has been cloned and its nucleotide sequence has been reported (SH Hong and J Marmur, Gene, Vol. 41, p75-8).
4, 1986, B Yao, P Sollitti and J Marmur, Gene, Vo
l.79, P189-197,1989, P Sollitti and J Marmur Mol G
en Gent Vol.213, P56-62 1988). Each gene was cloned based on the information. The research yeast NCYC74-CB11 (MATa,
adel, MAL6) (National Collection of Yeast Cultures
(UK). This yeast NCYC74-CB11 was obtained from Saccharomyces cerevisiae.
Named SAM 2034, it has been deposited with the Agency Fermentation Research Institute Bikoken Article nearest No. 4198 as (FERM BP-4198). ], The chromosomal DNA was extracted, partially digested with the restriction enzyme Sau3AI, and then a DNA fragment of about 10 Kb was separated by a sucrose density gradient method. Plasmid pBR for E. coli
After cutting 322 (manufactured by Takara Shuzo) with BamHI, it was enzymatically dephosphorylated using alkaline phosphatase (manufactured by Takara Shuzo), mixed with a DNA fragment, and ligated. E. FIG. E. coli JM109 strain (Takara Shuzo) was transformed into a gene bank. Cloning of the maltose metabolism gene was performed by colony hybridization using synthetic DNA. The probe was A291
(MAL6S; 5'-CGATGGCTGGGGTGATTTAAAAGGTATC-
3 ') (SEQ ID NO: 1) and A341 (MAL6R; 5'
-GGTATTGCGAAACAGTCTTGCG-3 ') (SEQ ID NO: 2) was synthesized and used. Plasmids were extracted from colonies hybridizing to A291 and A341, and as a result of restriction enzyme analysis and Southern hybridization, clones pMAL6S containing MAL6S (FIG. 1), MAL6T and MAL6T were cloned.
The clone pMAL6TR containing 6R (FIG. 3) was selected. Further, MAL6S has a Hind III of about 4.5 Kb.
-Since this fragment is contained in the SalI fragment,
PMAL6SHS was prepared by subcloning into HindIII-SalI site of No. 19 (Takara Shuzo). Further, in order to remove the 5 'untranslated portion of MAL6S, pMAL6SHS was cut with BglII and SmaI, followed by agarose gel electrophoresis to separate the largest fragment. Further, after pMAL6SHS was digested with BglII and SalI, the minimum fragment was separated, further digested with RsaI, and the BglII-SmaI fragment described above was ligated, and pU
C19MAL6SΔBgl II was obtained. PUC19MAL6SΔBgl II was converted to Bg
about 3.2 obtained by digestion with lII and HindIII
The Kb fragment and pMAL6SHS were replaced with BglII and XbaI.
Approximately 1.2 Kb fragment obtained by digestion with pMAL
6SHS was cleaved with XbaI-HindIII, and three of about 2.6 Kb fragments obtained were ligated simultaneously.
pUC19MAL6S was obtained (see FIG. 1). Embodiment 2 FIG. Construction of plasmid (1) pYMS222G This plasmid is a YEp type plasmid having a replication origin of yeast 2 μm plasmid, capable of autonomous replication in yeast, and having a GAPDH gene promoter (JP
Holland, MJ Holland, J. Biol. Chem. Vol. 254, p9839-9
845,1976) and a gene encoding maltase (MAL6S) linked to TRP as a transformation marker.
1 gene and G418 resistance gene which is a drug resistance gene (A. Oka, H. Sugisaki, and M. Takanami, J. Mol. Biol. Vol.
147, p217-226, 1981). Specifically, pUC19MA is inserted into the EcoRI site of the multicloning site of the yeast expression plasmid pYE22m containing the GAPDH gene promoter.
The structural gene of maltase (MAL6S, about 2.0 Kb) obtained by partially decomposing L6S with EcoRI was ligated to p
YMS222 was created. Yeast expression plasmid pYE
The method of making the 22m is described in detail in JP-A-04-228078. On the other hand, plasmid pGR1 containing a G418-resistant structural gene was obtained from Agric. Biol. Chem. Vol. 54, NO.
p2689-2696.1990, which is obtained by digesting with EcoRI and PvulI.
418 resistant structural gene with SalI linker
A coRI-SalI fragment, which was used as described above for pYE22
mGAPDH gene was inserted between the promoter and terminator to prepare pYG1. This is Hind I
A BamHI linker was attached to each end of the approximately 2.5 Kb fragment obtained by partial cleavage with II, and the BamHI of pYMS222 was inserted.
This was inserted into the HI cleavage site to create pYMS222G (see FIG. 2). (2) pYMT221G This plasmid comprises a gene encoding maltose permease (MAL6T) instead of MAL6S in the plasmid of (1), which is linked to the promoter of the GAPDH gene and inserted. Is exactly the same as (1). Specifically, pMAL6TR was replaced with ScaI
And inserted into the SmaI site of pUC19 to insert pUC19.
A MALT is created and this is EcoRI and BamHI.
Maltose permease structural gene (MAL6T, about 2.0 Kb) obtained by digestion with yeast expression plasmid p
Ligation into the EcoRI-BamHI site of YE22m
YMT221 was prepared, and G41 ligated to the GAPDH gene promoter in the same manner as pYMS222G.
8 resistance genes were ligated to prepare pYMT221G (see FIG. 3). (3) pYMST222G This plasmid comprises a gene encoding maltase (MAL6S) linked to the promoter of the GAPDH gene instead of the MAL6S of the plasmid of (1).
And a gene encoding maltose permease (MA
L6T). Other configurations are exactly the same as (1). Specifically, pYMT221 is converted to Hind I
The fragment of about 9.8 Kb obtained by partial digestion with II contains pYM
Approximately 3.1 obtained by cutting S222 with HindIII.
The Kb fragment was ligated to prepare pYMST222, and the G418 resistance gene linked to the GAPDH gene promoter was ligated to pYMST222 similarly to pYMS222G.
ST222G was produced (see FIG. 4). (4) pYMR222G In this plasmid, the gene (MAL6R) encoding a positive regulator of maltase and maltose permease instead of MAL6S of the plasmid of (1) is GAPDH.
It is inserted after being linked to the promoter of the gene, and the other configuration is exactly the same as (1). Specifically, the yeast expression plasmid pYE
After cutting the 22m multicloning site with SmaI and further cutting with SalI, pMAL6TR was
A gene (MAL6R, about 2.4 Kb) encoding a positive regulatory element obtained by digestion with maI and SalI was ligated to prepare pYMR222, to which pYMS222G was added.
In the same manner as described above, the G418 resistance gene linked to the GAPDH gene promoter was ligated to prepare pYMR222G (see FIG. 5). Embodiment 3 FIG. Transformation of yeast Beer yeast strain BH84 was treated with lithium acetate, and a plasmid solution and polyethylene glycol 4000 were added.
After holding at 0 ° C. for 30 minutes, treatment at 42 ° C. for 5 minutes, YPD
After adding 2 ml of a liquid medium (1% yeast extract, 2% polypeptone, 2% glucose) and culturing with shaking at 30 ° C. for 6 hours, the cells were centrifuged and the cells were concentrated 10-fold.
m on a YPD plate medium (YPD liquid medium + 2% agar), and culture at 30 ° C. for 3 to 5 days to pick out the growing colonies and use them as transformants. The names of the transformed yeast strains are as follows. BH8 transformed with pYMS222G
Four strains were transformed with BH-S, pYMT221G.
The H84 strain is designated as BH-T, the BH84 strain transformed with pYMST222G is designated as BH-ST, and the BH84 strain transformed with pYMR222G is designated as BH-R. Embodiment 4 FIG. Maltase Activity of Transformed Yeast Maltase activity is determined by Halvorson (Methods Enzymo
l, vol. 7, p559, 1966). That is, YP
Inoculate one loopful of cells into 10 ml of D liquid medium,
After culturing for 7 hours, centrifuging, collecting cells, washing with 0.01 M phosphate buffer, suspending in 0.7 ml of the same buffer,
Add 0.4g of glass beads and add 1 with bead beater.
Break the cells by treating for a minute. 100 ml of this cell lysate
After centrifugation at 00 × g for 10 minutes, the supernatant is used as a crude enzyme solution. A 0.01 M phosphate buffer (pH 7.
0) 2.8 ml (20 ° C) dissolved in the same buffer.
100 μl (20 ° C.) of 01M PNPG (ρ-nitrophenyl-α-D-glucopyranoside) and 20 μl of the above crude enzyme solution were mixed in a cuvette, and the enzyme activity was measured by increasing the absorbance at 400 nm for 1 minute. 17 with glucose
FIG. 6 shows the maltase activity of the parent strain BH84 and the transformants BH-S, BH-ST and BH-R after culturing for an hour. BH-S is about 19 times the parent strain, BH-S
As for T, maltase activity was increased about 5-fold. BH-R
Under these conditions, maltase activity was not different from that of the parent strain. Embodiment 5 FIG. Transformed yeast maltaux
Spermase activity Maltose permease activity was determined by R. Serrano (Eu
rJBiochem., vol. 80, p97-102, 1977). That is, Yeast Nitrogen Base containing 2% glucose
(Difco) 10 ml was pre-incubated overnight at 30 ° C., 2.5 ml of the bacterial solution was transferred to 50 ml of fresh medium, and cultured at 30 ° C. for 8 hours. After culturing, cells are collected, washed cells, OD660
= 1.0, 5 ml to 1 ml of 0.1 mM maltose (0.2
μCi / ml containing ¹⁴ C maltose) /0.1M Tartarate-
The cells were suspended in Tris (pH 4.3), sampled at 100 μl every 20 seconds, and the radioactivity incorporated into the cells was measured. The parent strain BH8 when cultured for 8 hours on glucose
4 strains, and transformants BHT, BH-ST and BH
The maltose permease activity of -R is shown in FIG. BH
Maltose permease activity of -T was about 90 times that of the parent strain and BH-ST was about 20 times that of the parent strain. Under these conditions, BH-R had the same maltose permease activity as the parent strain. Embodiment 6 FIG. Malter in the presence of glucose
Ze and (MAL6S), Marutosupa Miaze (MAL
6T), positive regulator (MAL6R), the mR NA of the origination
Analysis of current amount Yeast Nitrogen Base (Difco) containing 2% glucose 1
The yeast cells cultured in 0 ml at 30 ° C. for 17 hours are collected,
After washing, 0.2 ml of LETS buffer (0.1 M L
iCl, 1% (W / V) SDS, 0.2M Tris-
The suspension is suspended in HCl (pH 7.4, 0.01 M EDTA), added with 0.4 g of glass beads, and treated with a bead beater for 2.5 minutes to disrupt the cells. The cell lysate was centrifuged at 10000 × g for 10 minutes to obtain a supernatant, subjected to phenol treatment three times, and then added with ethanol to give -20.
Precipitate the RNA at ° C. After centrifugation, the precipitate is rinsed with 70% ethanol, dried and dissolved in 55 μl of distilled water to obtain a total RNA solution. This was treated with oligotex dT30 (manufactured by Daiichi Kagaku) to obtain an mRNA solution. The obtained mRNA was subjected to formaldehyde gel electrophoresis as usual, and transferred to a membrane to obtain MAL6S, MAL6T , MAL.
The 6R fragment was hybridized with a biotin-labeled probe, and the expression levels were compared. Detection of biotin label and Northern blotting of the probe
Kit (BioNick Labelling System # 8247SA, Phot
oGene NucleicAcid Detection System # 8192SA). Table 1 shows the results. Table 1 {MALS MALT MLR} ────────────────── BH-84 ++ ++ BH-S ++ ++ BH-T ++ ++ BH-ST ++ ++ BH-R ++ ++ ── ──────────────────────────── Note + : Weakly expressed ++: Strongly expressed NT: Not tested. When the parent strain is maltase (MAL6S) and maltose permease (MAL6
T), while the expression of the mRNA of the positive regulator (MAL6R) was kept low, whereas BHS - S, BH-T , B
In H-ST and BH-R , the introduced genes were each highly expressed. Embodiment 7 FIG. Beer brewing test Parent strain BH84, and transformants BH-S, BH-
Using T, BH-ST and BH-R, a fermentation test with high-concentration wort was performed under the conditions shown in Table 2. Table 2. Fermentation conditions ① Raw wort extract concentration 24% wort volume 2L wort dissolved oxygen concentration 9ppm Fermentation temperature 15 ℃ Constant yeast input amount 15 g wet yeast cells / 2 L wort ─────────────────────────── [0042] Yeast growth during fermentation ( OD660) (FIG. 8), the assimilation process of glucose, maltose and maltotriose (FIGS. 9, 10, and 11), the maltose consumption rate (FIG. 12), and the consumption process of the extract (FIG. 13). BH-T and BH-ST have a higher rate of assimilation of maltose and a higher rate of consumption of the extract as compared with the parent strain, and the time required to reach a fermentation degree of 65% is as follows.
215 hours in BH-ST compared to 265 hours of parent strain,
In the case of BH-T, a remarkable shortening of the fermentation time was observed at 180 hours. In particular, BH-T was a very effective yeast for high-concentration brewing. From these results, maltose fermentation in beer brewing depends more on maltose uptake activity than maltase activity, and by significantly expressing the maltose permease gene (MAL6T), it is possible to significantly shorten the fermentation time. It became clear that we could do it. [Sequence List] SEQ ID NO: 1 Sequence length: 28 Sequence type: Number of nucleic acid strands: Single-stranded topology: Type of linear sequence: Synthetic DNA sequence CGATGGCTGG GGTGATTTAA AAGGTATC 28 : 2 Sequence length: 22 Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence: Synthetic DNA sequence GGTATTGCGA AACAGTCTTG CG 22

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the configuration of pUC19MAL6S. FIG. 2 shows the construction of plasmid pYMS222G. Arrows indicate the transcription direction of the gene in the figure. Symbol: G4
18 ^r , G4 under the control of GAPDH gene promoter
A gene that confers resistance to 18. PGAP, GAPDH gene promoter. TGAP, GAPDH gene terminator. Ap ^r , ampicillin resistance gene. TRP1,
Tryptophan auxotrophy (trp1) restoration gene. IR,
Inverted repeats in yeast 2 μm DNA. FIG. 3 shows the structure of plasmid pYMT221G. FIG. 4 shows the structure of plasmid pYMST222G. FIG. 5 shows the construction of plasmid pYMR222G. FIG. 6 shows the maltase activity of a parent strain and a transformant when cultured on glucose. FIG. 7 shows maltose permease activities of a parent strain and a transformant when cultured on glucose. FIG. 8 shows the amount of yeast growth during fermentation (OD660). FIG. 9 shows the assimilation process of glucose. FIG. 10 shows the assimilation process of maltose. FIG. 11 shows the process of assimilation of maltotriose. FIG. 12 shows the consumption rate of maltose. FIG. 13 shows the consumption progress of the extract.

────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Toshihiko Ashikari 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka Prefecture Suntory Limited Basic Research Laboratories (56) References JP-A-1-130882 (JP, A) JP, A 60-256388 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12C 1/00-13/10 C12N 15/00-15/90 JICST (JOIS) BIOSIS / MEDLINE / WPID S (STN)

Claims (1)

(57) [Claim 1] It contains only the maltose permease structural gene as a structural gene artificially placed under the control of the promoter of the glyceraldehyde triphosphate dehydrogenase gene and the promoter. d by using the yeast
A method for producing beer, comprising fermenting high-concentration wort having a kiss content of 15% or more to obtain high-concentration beer.