JP4178176B2 - Barley lipoxygenase-1 gene, method for selecting barley, raw material for malt alcoholic beverage, and method for producing malt alcoholic beverage - Google Patents

Description

  The present invention relates to a barley lipoxygenase-1 gene, a method for selecting barley, a raw material for malt alcoholic beverages, and a method for producing a malt alcoholic beverage.

  Barley lipoxygenase-1 (hereinafter referred to as “LOX-1”), which is an enzyme contained in malt, oxidizes malt-derived linoleic acid in the preparation step when producing a malt alcoholic beverage, and 9-hydroperoxyoctadecadienoic acid Is generated (Non-patent Document 1). 9-hydroperoxyoctadecadienoic acid is further converted into trihydroxyoctadecenoic acid (THOD) by peroxygenase-like activity (Non-patent Document 2). This THOD is known to reduce beer foaminess, to give an astringent taste, to worsen cutting (Non-Patent Documents 3 and 4), and to reduce the quality of malt alcoholic beverages. Yes. Moreover, it is known that 9-hydroperoxyoctadecadienoic acid is also converted into trans-2-nonenal, which is a causative substance of the cardboard odor of an aged malt alcoholic beverage (Non-patent Document 5).

  In order to improve the flavor durability and foam retention of malt alcoholic beverages, a method for producing a malt alcoholic beverage using malt having low LOX-1 activity has been proposed as a technique for suppressing the production of THOD and trans-2-nonenal. (Non-patent document 6).

  Also, Douma et al. Induced an induced mutation by applying mutagen (drug) treatment to barley and created an induced mutant line in which LOX-1 activity was reduced to 9% of the control, and was used to produce a malt alcoholic beverage. (Patent Document 1).

However, even when such barley is used, reduction of the trans-2-nonenal concentration of the resulting malt alcoholic beverage is insufficient, and the flavor durability is not sufficiently improved. Moreover, nothing is made clear about the reduction of THOD amount or improvement of foam retention.
International Publication No. 02/053721 Pamphlet Kobayashi, N. et al., J. Ferment. Bioeng., 76, 371-375, 1993 Kuroda, H., et al., J. Biosci. Bioeng., 93, 73-77, 2002 Kobyashi, N., J. Am. Soc. Brew. Chem. 60: 37-41. 2002 Kaneda, H. et al., J. Biosci. Bioeng., 92, 221-226. 2001 Yasui Brewing Association Journal 96: 94-99 (2001) Drost, J. Am. Soc. Brew. Chem. 48: 124-131 (1990)

  The present invention has been made in view of the above-mentioned problems of the prior art, and is useful for producing a malt alcoholic beverage with improved flavor durability and foam retention without genetic manipulation. A gene, a method for selecting LOX-1-deficient barley, a raw material for malt alcoholic beverages derived from barley obtained by the selection, and a method for producing a malt alcoholic beverage using the raw materials for malt alcoholic beverages are provided. For the purpose.

  As a result of intensive studies to achieve the above object, the present inventors have found a conventional barley variety completely lacking LOX-1 activity, and have found a novel LOX-1 mutant gene from the barley variety. The invention has been completed.

  That is, the LOX-1 mutant gene of the present invention is characterized in that the guanine at the splicing donor site (5′-GT-3 ′) of the fifth intron of the known LOX-1 gene is mutated to another base. To do. Here, the other base is preferably adenine.

  The method for selecting LOX-1 deficient barley according to the present invention discriminates barley LOX-1 deficient barley depending on whether or not the guanine at the splicing donor site of the fifth intron of the LOX-1 gene has been mutated to another base. It is characterized by doing. Here, the other base is preferably adenine.

  Furthermore, the method for selecting LOX-1 deficient barley of the present invention comprises a genomic DNA extraction step for extracting genomic DNA from barley to be tested, and a splicing donor site for the fifth intron of LOX-1 gene from the extracted genomic DNA. A DNA fragment amplification step of amplifying a DNA fragment containing a DNA fragment, and a DNA fragment containing a splicing donor site of the 5th intron of the LOX-1 gene amplified in the DNA fragment amplification step, and cleaved with a restriction enzyme, A DNA fragment detection step of detecting a fragment and discriminating barley LOX-1 deficient barley depending on whether or not the guanine at the splicing donor site has been mutated to another base.

  Here, the restriction enzyme used in the DNA fragment detection step is preferably AfaI and / or RsaI that recognizes the base sequence 5'-GTAC-3 '.

  According to the said invention, it becomes possible to select whether it is a barley variety which has a LOX-1 activity deficient character based on the presence or absence of a guanine mutation at the splicing donor site of the fifth intron of the LOX-1 gene. .

  As a result, barley varieties lacking LOX-1 activity can be easily selected by gene-level analysis without directly measuring LOX-1 activity. In particular, the enzyme activity is affected by the growth stage of the individual, the environment, etc., and accurate measurement may be difficult. According to this method, unlike the enzyme measurement, the LOX-1 activity is not affected by the environment. Deleted barley varieties can be selected. Furthermore, enzyme activity cannot be measured until the seeds are ripened, but DNA can be determined before flowering, so it can be determined early whether or not it is an activity-deficient trait and is also effective for continuous backcrossing. .

  Moreover, the raw material for malt alcoholic beverages of the present invention is characterized in that it is seed, malt, malt extract, barley degradation product or processed barley product derived only from barley having the LOX-1 mutant gene of the present invention.

  Moreover, the raw material for malt alcoholic beverages of this invention is a seed derived only from the barley selected by the selection method of the LOX-1 deficient barley of this invention, malt, a malt extract, a barley decomposition product, or a barley processed material. It is characterized by.

  Furthermore, the manufacturing method of the malt alcoholic beverage of this invention uses the raw material for malt alcoholic beverages of this invention, It is characterized by the above-mentioned.

  According to the present invention, since LOX-1 is not included in the raw material, 9-hydroperoxyoctadecadienoic acid is hardly generated from linoleic acid in the production process of malt alcoholic beverage, and therefore THOD and trans-2-nonenal are not produced. Since it becomes difficult to produce | generate, the malt alcoholic beverage which the flavor durability and the foam retention improved can be obtained.

  Hereinafter, preferred embodiments of the present invention will be described in detail.

  First, the LOX-1 mutant gene according to the present invention will be described.

  The LOX-1 mutant gene according to the present invention is a novel gene found by the present inventors, and is compared with the known LOX-1 gene (SEQ ID NO: 1) in the 60th position of the fifth intron of the LOX-1 gene. The base G is substituted with A (SEQ ID NO: 2). Since the 60th-61st position of the 5th intron of the LOX-1 gene is a splicing donor site (5′-GT-3 ′), substitution of this base causes LOX-1 to be spliced abnormally and active LOX-1 Cannot be expressed.

  In the sequence listing, SEQ ID NO: 1 shows the nucleotide sequence of the fifth intron region of the known LOX-1 gene, and SEQ ID NO: 2 shows the nucleotide sequence of the fifth intron region of the LOX-1 mutant gene of the present invention. The base sequence of the corresponding part is shown.

  Next, the selection method of the LOX-1 deletion barley of this invention is demonstrated.

  The method for selecting LOX-1 deficient barley of the present invention discriminates barley LOX-1 deficient barley depending on whether or not the guanine at the splicing donor site of the fifth intron of the LOX-1 gene has been mutated to another base. It is characterized by that.

  As a method for selecting LOX-1-deleted barley using the above-mentioned base mutation, for example, DNA amplification is performed using a primer containing the above mutation site in the 3 ′ end of the primer sequence or in the primer sequence. Detecting base mutations by detecting the presence or absence of amplification and amplification efficiency, selecting LOX-1-deleted barley, or amplifying a DNA fragment containing the mutation site and determining the base sequence It is possible to use a method of selecting LOX-1 deficient barley.

  Detection of these base mutations is not particularly limited as long as a DNA fragment can be detected, but agarose gel electrophoresis or polyacrylamide gel electrophoresis may be used. In addition, in the case of detecting a DNA mutation based on the presence or absence of amplification or amplification efficiency, a quantitative PCR method such as TAQMAN method can be used in addition to the above electrophoresis.

In addition, the method for selecting LOX-1 deficient barley of the present invention preferably comprises:
A genomic DNA extraction step of extracting genomic DNA from barley to be tested;
A DNA fragment amplification step of amplifying a DNA fragment containing a splicing donor site of LOX-1 gene 5th intron from the extracted genomic DNA;
The DNA fragment containing the splicing donor site of the 5th intron of the LOX-1 gene amplified in the DNA fragment amplification step is cleaved with a restriction enzyme to detect a DNA fragment having a predetermined number of bases. A DNA fragment detection step of discriminating barley LOX-1 deficient barley depending on whether or not it has been mutated to a base;
It is characterized by including.

  First, the genomic DNA extraction process according to the present invention will be described.

  The method for extracting genomic DNA from barley as a test target is not particularly limited and can be performed by a known method. Specifically, for example, the CTAB method (Murray et al., 1980, Nucleic Acids Res. 8: 4321-4325) and Ethidium bromide method (Varadarajanand Prakash 1991, Plant Mol. Biol. Rep. 9: 6-12). Here, not only barley seeds but also leaves, stems, roots, and the like can be used as tissues for extracting genomic DNA. For example, by using leaves, it is possible to select a large number of individuals during the backcross generation.

  Next, the DNA fragment amplification step according to the present invention will be described.

  A method for amplifying a DNA fragment is not particularly limited, and can be performed by, for example, a PCR method (Polymerase Chain Reaction Method). Here, the primer used in the PCR method is not particularly limited as long as it is set in a region capable of amplifying a DNA fragment containing the splicing donor site of the LOX-1 gene fifth intron. Specifically, for example, the LOX-1 gene preferably has 10 to 60 consecutive bases, more preferably 15 to 30 consecutive bases. In general, the GC content in the primer base sequence is preferably 40 to 60%. Furthermore, it is preferable that there is no difference or little difference in Tm values between the primers of the two primers used in the PCR method. Moreover, it is preferable not to take a secondary structure within a primer.

  Next, the DNA fragment detection process according to the present invention will be described.

  Since the LOX-1 mutant gene according to the present invention has a difference in base sequence from known LOX-1 as described above, the amplification product can be cleaved using a restriction enzyme that recognizes or cleaves the difference. There are differences in the size of the DNA fragments obtained. The restriction enzyme according to the present invention is not particularly limited as long as it recognizes or cleaves the above-mentioned difference portion, but the restriction enzyme AfaI and / or already known to have such an action. RsaI is preferred.

  That is, in the LOX-1 mutant gene of the present invention, the guanine at the splicing donor site is mutated to another base, so that the restriction enzyme AfaI and RsaI cleavage sites (5 '-GTAC-3': 5th intron 60-63) has disappeared. As a result, since the cleavage pattern when the gene amplification product containing this cleavage site is cleaved with AfaI and / or RsaI is different from that of the known LOX-1 gene, it can be determined whether or not it is a LOX-1 mutant gene. Is possible.

  In addition, a DNA fragment having a predetermined number of bases is a DNA fragment in which the difference is present, so that the size of the DNA fragment obtained by cleaving the amplified product with a restriction enzyme is different. The number of bases is not particularly limited.

  The detection in this step is not particularly limited as long as it can detect a DNA fragment cleaved by a restriction enzyme. Specifically, for example, detection by agarose gel electrophoresis or polyacrylamide gel electrophoresis may be used. That's fine.

  Next, the raw material for malt alcoholic beverages of this invention is demonstrated.

  The raw material for malt alcoholic beverage of the present invention is a seed derived from only barley having the LOX-1 mutant gene of the present invention, malt, malt extract, barley degradation product or processed barley product, and the present invention. It is characterized in that it is a seed derived from only barley selected by the method for selecting barley, malt, malt extract, barley decomposition product or processed barley product.

Here, the malt extract refers to an extract of malt, and examples thereof include extracts of sugar components and protein components in the malt. The barley decomposition product refers to a product obtained by decomposing barley with an enzyme or the like, and barley saccharified liquid corresponds to this. The barley processed product corresponds to the barley pulverized product used as an auxiliary material for beer and sparkling liquor.
Since the raw material for malt alcoholic beverages of the present invention does not contain LOX-1, it is difficult to produce 9-hydroperoxyoctadecadienoic acid from linoleic acid in the production process of malt alcoholic beverages, and therefore THOD and trans-2-nonenal are also produced. Generation | occurence | production suppression is anticipated and it can anticipate obtaining the malt alcoholic beverage with which flavor durability and foam | bubble persistence improved.

  Finally, the manufacturing method of the malt alcoholic beverage of this invention is demonstrated.

  The manufacturing method of the malt alcoholic beverage of this invention uses the raw material for malt alcoholic beverages of this invention, It is characterized by the above-mentioned.

  First, the malting process according to the present invention will be described.

  The malting process according to the present invention is a process for obtaining malt characterized by using LOX-1 deficient barley, and the malting method is not particularly limited and may be performed by a known method. Specifically, for example, after malting until the degree of soaking reaches 40% to 45%, germination can be performed at 10 to 20 ° C. for 3 to 6 days and dried to obtain malt.

  Next, the preparation process according to the present invention will be described.

  The preparation process according to the present invention is a process for obtaining wort by saccharifying the malt. Specifically, it is further divided into the following first to fourth steps.

  That is, the first step is a charging step of mixing the raw material containing malt and water for charging, heating the resulting mixture to saccharify the malt, and collecting wort from the saccharified malt. is there.

  It is desirable that the malt used in this step is one obtained by germinating barley with moisture and air and drying to remove radicles. Malt is an enzyme source necessary for wort production and at the same time, a major starch source as a raw material for saccharification. Moreover, in order to give the flavor and pigment peculiar to a malt alcoholic beverage, what dried the germinated malt is used for wort manufacture. Further, as raw materials, in addition to the malt, auxiliary raw materials such as LOX-1 deficient barley or general barley, corn starch, corn grits, rice and sugars according to the present invention may be added.

  In addition, in the wort production process, the malt extract or barley degradation product prepared from the LOX-1 deficient barley or general barley according to the present invention, or a barley processed product is mixed with water for charging, and if necessary, The wort can be obtained by adding raw materials.

  The malt is added to the feed water and then mixed. What is necessary is just to mix here, when adding the said auxiliary material. In the case of saccharides, it may be added before boiling as described below. Moreover, the said water for preparation is not restrict | limited in particular, What is necessary is just to use suitable water according to the malt alcoholic beverage to manufacture. Saccharification may be basically performed under known conditions. After filtering the malted saccharified solution thus obtained, a raw material that can impart aroma, bitterness, etc., such as hops or herbs, is boiled, and cooled to obtain cold wort.

  The second step is a step in which yeast is added to the cold wort and fermented to obtain a malt alcoholic beverage intermediate product.

  Here, the yeast used may be any alcoholic yeast that performs so-called alcoholic fermentation that metabolizes the sugar in the wort obtained by saccharification of the malt to produce alcohol, carbon dioxide, etc. Examples include Saccharomyces cerevisiae and Saccharomyces ubalum.

  Fermentation is performed by cooling the wort obtained in the above charging step and adding the yeast thereto. The fermentation conditions are basically the same as the known conditions. For example, the fermentation temperature is usually 15 ° C. or lower, preferably 8 to 10 ° C., and the fermentation time is preferably 8 to 10 days.

  Furthermore, a 3rd process is a liquor storage process which stores the malt alcoholic beverage intermediate product obtained at the said fermentation process.

  In this step, the fermented liquor after alcohol fermentation is transferred to a closed tank and stored. The storage conditions are basically the same as the known conditions. For example, the storage temperature is preferably 0 to 2 ° C., and the storage time is preferably 20 to 90 days. By storing the fermented liquid, the remaining extract is refermented and aged.

And the 4th process is a filtration process which filters the malt alcoholic beverage intermediate product obtained at the above-mentioned alcohol storage process, and obtains a malt alcoholic beverage. Diatomaceous earth, PVPP (polyvinyl polypyrrolidone), silica gel, cellulose powder, etc. are used as the material, and the temperature is 0 ± 1 ° C.

  A malt alcoholic beverage is thus obtained. The filtered malt alcoholic beverage is either as it is or after being subjected to aseptic filtration or heat treatment, and then tanked, barreled, bottled or canned and shipped to the market.

  The malt alcoholic beverage is not particularly limited in the proportion of malt used in its production, and may be any alcoholic beverage produced using malt as a raw material. Specific examples include beer and sparkling liquor. In addition, so-called non-alcohol beer and non-alcohol sparkling liquor are malt alcoholic beverages because they use the same manufacturing method as malt alcoholic beverages such as beer.

  According to the present invention, since LOX-1 is not included in the raw material, 9-hydroperoxyoctadecadienoic acid is hardly generated from linoleic acid in the production process of malt alcoholic beverage, and therefore THOD and trans-2-nonenal are not produced. It can be expected that a malt alcoholic beverage with improved flavor durability and foam durability can be obtained.

  EXAMPLES Hereinafter, although the content of this invention is demonstrated more concretely by an Example, this invention is not limited to these Examples at all.

Exploratory test 1
(Search for LOX-1 deficient barley by measuring LOX-1 enzyme activity)
LOX-1 enzyme activity was measured by the following method, and LOX-1-deficient barley was searched from barley genetic resources.

  First, a crude enzyme solution was extracted from barley seeds by the following method. One ripe barley seed was crushed with a hammer and extracted with 500 μL of extraction buffer (0.1 M sodium acetate buffer (pH 5.5)) by shaking at 4 ° C. for 30 minutes. The obtained extract was centrifuged at 15000 rpm for 10 minutes, and then the supernatant was used as a crude enzyme solution.

  Next, 5 μL of a substrate solution (40 mM linoleic acid, 1.0% (W / V) Tween 20 aqueous solution) and 85 μL of extraction buffer were added to 10 μL of the crude enzyme solution and mixed, and then reacted at 24 ° C. for 5 minutes. The reaction was stopped by adding and mixing 100 μL of a reaction stop solution (80 mM 2,6-di-t-butyl-p-cresol methanol solution). The reaction solution was allowed to stand at −20 ° C. for 30 minutes, and then centrifuged at 3000 rpm for 20 minutes, and the supernatant was used for the next color development reaction. To 20 μL of the obtained supernatant, 200 μL of color developing solution (4 mM 2,6-di-t-butyl-p-cresol, 25 mM sulfuric acid, 0.25 mM ammonium iron (II) sulfate hexahydrate, 100 mM xylenol orange, 90% methanol) Aqueous solution) was added and allowed to stand for 30 minutes, and then the absorbance at a wavelength of 550 nm was measured. In addition, as a negative control, the crude enzyme solution was heat-treated at 100 ° C. for 5 minutes and reacted in the same manner with LOX-1 inactivated. As a positive control, a crude enzyme solution of seeds of barley variety Kendall Was used.

  As a result of searching for genetic resources, as shown in FIG. 1, it was found that no significant LOX-1 activity was found in the seeds of SV73 (Okayama University Preserved OUI003). Since SV73 is a native species, it is not a mutagenized line but a natural mutant.

Certification test 1
(Confirmation that SV73 crude enzyme solution has no LOX-1 inhibitory activity)
Next, the presence or absence of LOX-1 inhibitory activity of the SV73 crude enzyme solution was examined.

  When the crude enzyme solution (10 μL, 20 μL, 50 μL) of SV73 was added to the crude enzyme solution showing LOX-1 activity (positive control: PC) and the change in LOX-1 activity was examined, the addition of the crude enzyme solution of SV73 Did not change the LOX-1 activity, and the SV73 crude enzyme solution showed no LOX-1 inhibitory activity (FIG. 2). From this, it is considered that the cause of the loss of LOX-1 activity in SV73 is not due to a LOX activity inhibitor.

Certification test 2
(Confirmation of LOX-1 protein expression level in SV73 seed)
Next, using an anti-LOX-1 antibody, it was examined whether or not LOX-1 protein was expressed in SV73 seeds.

  First, an anti-LOX-1 antibody was prepared. The LOX-1 protein used as an antigen was obtained by purifying the LOX-1 protein expressed in E. coli (Kuroda et. Al. (2002) J. Bioscience and Bioengineering 93: 73-77). The purified protein was immunized to rabbits to produce LOX-1 antibody. This antibody recognizes LOX-1 and LOX-2.

  Next, Western blotting was performed by the following method to examine LOX-1 protein expression in SV73 seeds. 3 μg of total soluble protein extracted from SV73 using 0.1 M sodium acetate buffer (pH 5.5) was fractionated by SDS polyacrylamide electrophoresis (SDS-PAGE) and then blotted onto a PVDF membrane (Millipore). After washing this membrane with TTBS (20 mM Tris-HCl (pH 7.5), 0.15 M sodium chloride, 0.05% (w / v) Tween 20, 0.05% (w / v) sodium azide)), The mixture was reacted for 30 minutes with LOX-1 antibody solution (diluted 1000 times / TTBS). The membrane was washed with TTBS three times for 5 minutes, and then reacted with an alkaline phosphatase-labeled goat anti-rabbit IgG antibody solution (manufactured by Santa Cruz, 1000-fold diluted TTBS solution) for 30 minutes. The membrane was washed with TTBS for 5 minutes × 2 times, and further washed with AP9.5 (10 mM Tris-HCl (pH 9.5), 0.1 M sodium chloride, 5 mM magnesium chloride) for 5 minutes × 1 time. Color was developed by reacting with alkaline phosphatase substrate solution (1 mg / ml nitro blue tetrazolium, 0.5 mg / ml BCIP, AP 9.5 solution). As a result, a strong band having a molecular weight of about 95 Kd was obtained in the control cultivar (Kendall), whereas very thin bands were detected in the molecular weight regions of about 95 Kd and about 57 Kd in the seeds of the SV73 line (FIG. 3A). ).

  In addition, this extracted sample was subjected to isoelectric focusing (IEF, pI3-9) using a PhastSystem (manufactured by Amersham Pharmacia), and then subjected to Western analysis in the same manner. As a result, in SV73, a band was detected at the pI position of LOX-2, but no clear band was observed at the pI position of LOX-1 (FIG. 3B). From this, it is thought that the band of about 95 Kd recognized by the seed extract protein of SV73 is LOX-2 protein.

  From the above results, it was confirmed that the LOX-1 protein was hardly expressed in the seeds of SV73.

Certification test 3
(Analysis of LOX-1 RNA in SV73 seeds)
RT-PCR was performed using as a template total RNA extracted from seeds at about 4 weeks ripening of SV73 and seeds from day 3 of germination. The reaction was performed using a commercially available kit (Roche Diagnostics, Perkin Elmer) according to the kit manual. Primers were designed to 5′-GGAGAGGAGGCCAAGAAACAAGATG-3 ′ (SEQ ID NO: 3) and 5′-GGTTGCCCGATGGGCTTAGAT-3 ′ (SEQ ID NO: 4) based on the previously reported sequence (DNA data bank: Accession L35931). In PCR, a reaction of 94 ° C. for 2 minutes once, 94 ° C. for 1 minute, 60 ° C. for 2 minutes and 72 ° C. for 4 minutes was repeated 31 times, and then an extension reaction was performed at 72 ° C. for 7 minutes.

  When the amplified DNA was electrophoresed, an amplification of a band of about 2.5 Kb was detected with respect to RNA during ripening and germination, although the amount of amplification was slightly smaller than that of the control (variety Kendall) (Fig. 4). This indicates that the LOX-1 gene is normally transcribed.

  As described above, in SV73 seeds, (1) LOX-1 activity was not observed, (2) antigen protein reacting with LOX-1 antibody was present only in a trace amount, and (3) molecular weight of about 57 Kd (4) LOX-1 mRNA was recognized, and thus the mechanism by which the LOX-1 activity of SV73 is lost is thought to be due to abnormalities after transcription.

Certification test 4
(Structural analysis of the LOX-1 gene intron region of SV73)
In order to analyze the structure of the intron and exon region of the LOX-1 gene, genomic DNA of the region containing all exons was isolated. SV73 total DNA was used as a template. Primers were designed based on a previously reported sequence (DNA data bank: Accession U83904, L35931) (5'-CACGTCGCCGTCCCATCCCATC-3 '(sequence table SEQ ID NO: 5), 5'-GGTTGCCCGATGGCTTTAGAT-3' (sequence table sequence) Number 4)). In PCR, a reaction at 94 ° C. for 1 minute, 65 ° C. for 2 minutes, and 72 ° C. for 3 minutes was repeated 31 times, and then an extension reaction was performed at 72 ° C. for 7 minutes. The obtained DNA fragment was cloned into pCR2.1 (pGLXABAL1) and used as a template for structural analysis. ABI sequencer was used for the structural analysis, and a dye terminator method was used for the sequence reaction.

  Sequence number 1 of a sequence table showed the structure of the area | region which has a 5th intron among the base sequences (WO02053721) of a reported LOX-1 gene. The splicing donor site is the 60-61th nucleotide sequence 5′-GT-3 ′.

  On the other hand, the nucleotide sequence of the corresponding region of SV73 found from the analysis results is shown in SEQ ID NO: 2. In SV73, it was revealed that the 60th guanine, which is a splicing donor site, was mutated to adenine.

  Further, by replacing the 60th base of SEQ ID NO: 2 with adenine, a new stop codon is formed (58-60th base sequence 5′-TGA-3 ′ of SEQ ID NO: 2), If there is no change 5 ′ upstream of the splicing site, translation of the LOX-1 protein is considered to end here (FIG. 5).

The exon region in the vicinity of the fifth intron is known to be the active center of plant LOX (Shibata and Axelrod (1995) J. Mol.
Lipid Mediaters and Cell Signaling 12: 213-228), the splicing abnormality is considered to have a great influence on the LOX-1 enzyme activity.

Certification test 5
(Analysis of splicing in the fifth intron)
In order to confirm that splicing abnormality actually occurred in the fifth intron, analysis by RT-PCR was performed.

  Total RNA was extracted from the germinated SV73 and Kendall, and cDNA was synthesized using a commercially available kit (Roche Diagnostics) as template DNA. In genomic DNA, two primers (5′-CCATCACGCAGGGGCATCCTG-3 ′ (SEQ ID NO: SEQ ID NO: SEQ ID NO: 5) designed so that the amplified fragment contains the third intron (106 bp), the fourth intron (132 bp) and the fifth intron (79 bp). 6), 5′-GCGTTGATGAGCGTCTCGCCG-3 ′ (SEQ ID NO: 7)) was used for PCR. In PCR, a reaction at 94 ° C. for 1 minute, 65 ° C. for 2 minutes, and 72 ° C. for 3 minutes was repeated 31 times, and then an extension reaction was performed at 72 ° C. for 7 minutes. When the amplified DNA fragment was subjected to agarose gel electrophoresis, the SV73 amplified fragment had about 80 bp more bases than the Kendall amplified fragment (FIG. 6A). In addition, a fragment of about 1.2 Kb is amplified with respect to SV73 genomic DNA, and the result of the RT-PCR is considered to be a result of the expressed RNA.

  Next, in order to investigate which intron of the third intron (106 bp), the fourth intron (132 bp), and the fifth intron (79 bp) is causing splicing abnormality, the amplified fragment is compared with the fourth intron (132 bp) and the fourth intron (132 bp). Digested at the restriction enzyme StuI site present in the exon region between 5 introns. As a result, since the DNA fragment containing the fifth intron is equal in mobility to the amplified fragment of the genomic DNA, the splicing of the fifth intron is abnormal and is not spliced or the splicing site is shifted to the 3 ′ side. (Fig. 6B). That is, it was clarified that the translation of SV73 LOX-1 protein was terminated at this codon due to the newly generated stop codon shown in FIG. 5, and LOX-1 activity was lost.

Certification test 6
(Mapping and selection of barley mating lines using the 5th intron mutation)
In the previously reported LOX-1 base sequence, the sequence containing the splicing donor site of the fifth intron (60th to 63rd positions in SEQ ID NO: 1: 5′-GTAC-3 ′) is a restriction enzyme AfaI that recognizes the GTAC sequence. Can be digested with (RsaI). On the other hand, in the SV73 strain, there is a mutation in the sequence of this region (SEQ ID NO: 2 at positions 60-63: 5′-ATAC-3 ′), and it cannot be digested with AfaI and / or RsaI (FIG. 5). ) To map the LOX-1 deletion gene. Two primers (5'-CCATCACGCAGGGGCATCCTG-3 '(Sequence Listing) were designed to extract DNA from the leaves of 144 hybrid second generation (F2) individuals of barley cultivars Kendall and SV73, and the amplified fragment was designed to contain this AfaI site. PCR was performed using SEQ ID NO: 6), 5′-GCGTTGATGAGCCGCTGCCG-3 ′ (SEQ ID NO: 7). In PCR, a reaction at 94 ° C. for 1 minute, 65 ° C. for 2 minutes, and 72 ° C. for 3 minutes was repeated 31 times, and then an extension reaction was performed at 72 ° C. for 7 minutes. The amplified fragment was cut with AfaI and analyzed by 2.5% agarose gel electrophoresis (the above is called AfaI method). As a result, SV73 type, Kendall type, and heterotype could be easily distinguished (FIG. 7). In addition to the AfaI method polymorphism survey, a polymorphism survey of each strain was also conducted for the DNA marker (JBC970) near the LOXA locus of the barley 4H chromosome on which the LOX-1 gene is located (FIG. 8).

  Next, the seed LOX activity was investigated using the seed which grew in this F2 individual | organism | solid. For lines in which LOX activity was not observed, the activity of multiple seeds (4-7 grains) was further measured (FIG. 8).

  As a result of the AfaI method polymorphism survey of the F2 generation and the LOX activity measurement of F3 seeds, the separation of the LOX-1 deletion trait of SV73 completely coincided with the separation of the AfaI method polymorphism. That is, it was revealed from these series of genetic investigations that the LOX-1 deletion gene of SV73 is in the LOXA locus.

  In addition, this result shows that the AfaI method, which is an example of a barley selection method using DNA mutation, can be used to select a LOX-1-deleted mating line at the early stage of barley growth and to produce seeds. Indicates that there is no need to wait.

Example 1
(AfaI polymorphism survey of other barley varieties)
AfaI polymorphism survey was conducted using common barley varieties / lines. The varieties used were Mikamo Golden, Golden Melon, Haruna Nijo, Myougi Nijo, Saitama Nijo, Wasedori Nijo, Agurimochi, Harpin Nijo, Ryofu, Kitaiku 33, Kitaiku 35, Prior, Schooner, Sloop, Lofty Nijo, Franklin, Betzes, Harlington, Manley, B1251, CDC Kendall, CDC Stratus, CDC Copeland, Hanna, Merit, AC Metcalfe, TR145, Chariot, Chariot. As a result of the AfaI method polymorphism investigation, these varieties tested were not SV73 type, but a restriction enzyme AfaI site containing the 5th intron splicing donor sequence (positions 60-63 of SEQ ID NO: 1: 5′-GTAC- 3 ′) was digested (FIG. 9). This indicates that in these breeding lines, no DNA mutation is observed in the AfaI site (60th to 63rd positions of SEQ ID NO: 1: 5′-GTAC-3 ′), and the LOX- This AfaI method can be used effectively for selection of 1-deleted genes.

Example 2
(Search for genetic resources)
We investigated the world genetic resources (conventional species) preserved by Okayama University using the AfaI method. As a result, in 5 new lines, a line was found in which the restriction enzyme AfaI site containing the splicing donor sequence of the 5th intron (60th to 63rd positions of SEQ ID NO: 1, 5′-GTAC-3 ′) was not digested ( Okayama University Preservation OUI001 (India), OUJ095 (Taiwan), OUJ695 (Taiwan), OUN345 (Nepal), OUN347 (Nepal)) (FIG. 10).

  Next, the LOX-1 activity of seeds of these lines was measured by the method described in Exploratory Test 1. For OUN345 and OUN347, activity was measured in 5 minutes of reaction (FIG. 11A), and for OUI001, OUJ095, and OUJ695, the activity was measured by extending the reaction time to 90 minutes so that the presence or absence of activity could be clarified (FIG. 11A). 11B). As a result, no significant activity was observed in all these lines (FIG. 11).

  Therefore, SV73, OUI001 (India), OUJ095 (Taiwan), OUJ695 (Taiwan), OUN345 (Nepal), OUN347 (Nepal) (SV73 type LOX-1 deficient barley) are LOX-1 deficient barley Became clear. Since these lines are both native species and not mutagenized lines, they are natural mutants with respect to the LOX-1 gene.

  Based on the above results, the AfaI method, which is an example of a barley selection method using DNA mutation, is effective not only in selecting LOX-1 deficient barley mating progeny, but also from barley genetic resources. It became clear that the technology enables the selection of barley.

Example 3
(Growing barley for test brewing)
In the F2 generation obtained by crossing barley varieties Taisho and SV73 and self-breeding the resulting hybrid first generation (F1), the LOX-1 enzyme activity measurement method described in Example 1 above and Example 7 above The LOX-1 deletion trait was confirmed by the AfaI method described above, and 6 LOX-1 deletion lines and 10 LOX-1 possession lines were used as a group for subsequent seed propagation.

  Seed multiplication was performed for each line (population), and was performed using a uniform field or greenhouse until F4 seeds were obtained. When LOX-1 enzyme activity was measured for F4 seeds, the presence or absence of LOX-1 activity determined in F2 individuals was maintained, and from this result, LOX-1 deletion traits were stably transmitted to progeny Became clear.

  This F4 seed was used for the subsequent malt production test and malt alcoholic beverage production test.

Example 4
(Manufacture of malt for test brewing)
LOX-1 deficient barley F4 population (LOX-F4) consisting of barley seeds not having LOX-1 activity, derived from the crossing of the above barley × SV73, and barley F4 possessing LOX activity from barley seeds having LOX-1 A group (LOX + F4) was created and used for malting.

  Barley is made using an Automatic Micromalizing System (manufactured by Phoenix Systems) at a total soaking time of 16 ° C. of 16 ° C. for 82 hours (5 hour WET / 7 hour DRY cycle), germination 15 ° C. 139 hours, and roasting 29 hours (55 ° C. 13. 5 hours, 65 ° C. for 8 hours, 75 ° C. for 3.5 hours, 83 ° C. for 4 hours).

Example 5
(Analysis of malt and wort)
The analysis of the malt was performed according to the EBC standard method (European Brewery Convention, Analytica EBC (4th Ed), 1987). As a result, it was clarified that malt using LOX-F4 and LOX + F4 had no significant difference and could be used without any problem for malt alcoholic beverage brewing for the purpose of comparing the presence or absence of LOX-1 activity (FIG. 12).

  Next, wort was produced using 50 g of malt by the congress method (European Brewery Convention, Analytica EBC (4th Ed), 1987), and lipid oxides in the wort were analyzed.

  First, the amount of trans-2-nonenal in wort was measured by the following method. A 8 ml wort sample was placed in a vial and 3 g NaCl was added and capped. Next, Spellco polydimethylsiloxane SPME fibers were inserted, incubated at 40 ° C. for 15 minutes, and then subjected to gas chromatography.

  Gas chromatography mass spectrometry, J & W Inc. DB-1 (30m X 0.25mm, film thickness 1 [mu] m) as a capillary column, helium (1 mL / min) was used as a carrier gas, the oven conditions were 225 ° C. from 60 ° C. ( The trans-2-nonenal was quantified in the select ion mode (m / z: 70). In addition, the standard addition method which used trans-2-nonenal made by Sigma as a standard product was used for quantification.

  As a result, the trans-2-nonenal concentrations of worts prepared using LOX-F4 and LOX + F4 were 0.36 ppb and 3.85 ppb, respectively. Therefore, when wort was manufactured using the malt concerning this invention, it became clear that trans-2-nonenal production amount can be suppressed to 1/10 or less compared with the case where the conventional malt is carried out.

  Moreover, wort nonnal potential was measured by the following method. First, the method of Drost et al. (Drost, BW, van den Berg, R., Freijee, FJM, van der Velde, EG, and Hollemans, M., J. Am. Soc. Brew. Chem., 48, 124-131 , 1990) boiled wort for 2 hours. Thereafter, the amount of trans-2-nonenal in the sample was measured according to the above trans-2-nonenal measurement method, and the nonenal potential in wort was calculated.

  As a result, whey produced using LOX-F4 and LOX + F4 had a nonnal potential of 2.74 ppb and 11.9 ppb, respectively. Nonenal potential is known as an index that can predict product aging (Drost, BW, et al., J. Am. Soc. Brew. Chem., 48, 124-131, 1990, Ueda et.al. (2001). EBC-processeds 55: p3 28th Congress) If malt beverages are brewed using malt prepared from barley according to the present invention, the flavor durability of malt alcoholic beverages can be greatly improved.

  FIG. 13 shows a comparison of the trans-2-nonenal concentration and the nonenal potential of wort prepared using LOX-F4 with those of wort prepared using commercially available malt. As is clear from the results shown in FIG. 13, the wort according to the present invention showed analytical values that could not be achieved with conventional barley.

  From the above results, it became clear that if the barley according to the present invention is used, malt having quality not found in conventional products can be produced.

  Furthermore, the THOD concentration in the wort was measured by high performance liquid chromatography mass spectrometry. The conditions for high performance liquid chromatography are as follows. The mobile phase flow rate was 0.3 mL / min, and a mixed solution of 0.5% acetic acid (liquid A) and acetonitrile (liquid B) was used as the mobile phase. Liquid A: Liquid B = 35: 65 (0 minute) − Liquid A: Liquid B = 5: 95 (30 minutes) The linear gradient was used. In addition, the column was Waters Assymetry (No.106005; C18, 3.5 μm: 2.1 × 150 mm), the column temperature was 50 ° C., and 5 μL was measured using a Hewlett Packard 1100 type high performance liquid chromatograph system. A wort or beer sample was isolated. Mass spectrometry used Waters ZQ and monitored the mass 329 in ES ionization negative mode. In addition, a beer extraction sample was used as a standard solution of THOD (Kobayshi, N., et al., J. Biosci. Bioeng., 90, 69-73, 2000).

  As a result, the THOD concentrations of worts prepared using LOX-F4 and LOX + F4 are 6.5 ppm and 14.7 ppm, respectively. If wort is produced using the malt according to the present invention, the wort THOD concentration is reduced. It became clear that it can be suppressed to 1/2 or less.

As described above, THOD is produced by conversion from linoleic acid by the action of malt LOX-1 and malt peroxygenase-like activity in the charging process, but malt peroxygenase-like activity is considered to be the rate-limiting step of THOD production ( Kuroda, H., et al., J. Biosci. Bioeng., 93, 73-77, 2002), it is not clear how much THOD production is suppressed when malt LOX-1 activity is reduced It was. However, from the results of this example, it was proved that the amount of THOD in wort was reduced when malt produced from barley seeds without LOX-1 activity was used. Since THOD moves to the final product without being metabolized by yeast (Kobayashi, N., et al., J. Inst. Brew., 106, 107-110 (2000)), it is derived from barley according to the present invention. Example 6 It was revealed that malt alcoholic beverages with good flavor quality and foam quality can be produced by using malt.
(Malt alcoholic beverage test brewing)
1. Production and analysis of cold wort About 2 points of LOX-F4 malt and LOX + F4 malt obtained in Example 11 above, charging was carried out with a 50 L scale charging equipment with a sparkling liquor specification (malt use rate 24%). The preparation conditions are as follows.

  Each 1.5kg of malt is charged with 15L of water for single use according to the diagram of 50 ° C, 20 minutes → 65 ° C, 30 minutes → 75 ° C, 3 minutes, filtered through wort with Reuters equipment, and finally 35L Of filtered wort was obtained.

  The obtained filtered wort is mixed with 5 kg of liquid sugar (sugar content 75%), added with 13 g of hop pellets (bitter taste analysis value 87.0BU (EBC)), boiled for 70 minutes, cooled to 10 ° C., and added with water By adjusting the extract, a cold wort with an extract content of 11.6 to 11.8% was obtained.

  The obtained cold wort was analyzed according to the EBC standard method (European Brewery Convention, Analytica EBC (4th Ed), 1987). The analytical values are shown in Table 1. As shown in Table 1, there was no clear difference between LOX-F4 and LOX + F4 with respect to general analysis items.

2. Production of Malt Alcohol Beverage (Happoshu) The cold wort obtained in 1 above was transferred to a steam-sterilized 30 L scale cylindrical type tank, yeast was added to an initial concentration of 30 million cells / mL, and 13 ° C. The main fermentation was carried out. When the fermented liquor extract was cut to 2.5%, it was transferred to the same type of tank and the alcohol storage process was performed. The alcohol storage process was performed at 13 ° C for the first 6 days and at 0 ° C for the following 2 weeks.

The fermented liquor after the liquor storage process filtered beer in a beer filtration facility and a filling facility, and filled in the koji.
3. Analysis of Happoshu The analysis of the Happoshu obtained in 2 above was performed as follows.

  First, an analysis was performed according to the EBC standard method (European Brewery Convention, Analytica EBC (4th Ed), 1987), and general analytical values other than lipid oxide analytical values were clearly between LOX-F4 and LOX + F4. No difference was observed (Table 2).

  Next, the foam retention of the sparkling liquor obtained in 2 above was analyzed by the following method.

  The bubble retention analysis utilized the NIBEM method. Haffmans' FOAM STABILITY TESTER was used to analyze foam retention (Table 3). LOX-F4 barley was 21 points higher than LOX + F4 barley and was found to have high foam retention. .

  Moreover, as a result of measuring the THOD concentration by the method described in Example 12, LOX-F4 was reduced to less than half compared with LOX + F4.

  From the above results, it was clarified that the malt alcoholic beverage produced by the method for producing a malt alcoholic beverage according to the present invention can suppress the accumulation of THOD and improve the foam retention of the product.

  Next, a sensory test using a panel of 13 persons was performed as follows, and the flavor durability of the sparkling liquor obtained in 2 above was compared.

  First, malt alcoholic beverages of LOX-F4 and LOX + F4 were stored at 37 ° C. for 1 week. Next, what was poured into a cup at a normal drinking temperature was subjected to a sensory test on the panel, and 0 to 4 for two items of aging odor and overall aging degree (assessed by considering aging odor and aging taste). Evaluation was made with a score (the score was higher as aging progressed) (Tables 4A and B).

  As a result, regarding aging odor, 10 out of 13 gave LOX-F4 a lower score, and LOX-F4 showed a lower score (average value) than LOX + F4. The difference was determined to be significant at a 5% risk by t-test (Table 4A).

  In addition, with regard to overall aging, 11 out of 13 gave LOX-F4 a lower score, LOX-F4 showed a lower score (average value) than LOX + F4, and the difference was t-test Was determined to be significant at a risk rate of 5% (Table 4B).

  From the above sensory test and statistical analysis, it became clear that LOX-F4 has a lower overall aging degree and lower aging odor than LOX + F4.

  In addition, as a result of measuring the trans-2-nonenal concentration of the happoshu obtained in 2 above before and after storage at 37 ° C. for 1 week, LOX-F4 has a trans-2-nonenal concentration higher than that of LOX + F4 even before storage. It was clarified that it was reduced to about 1/3 of LOX + F4 after storage (Table 5).

  From the results of the sensory test and the analysis result of the trans-2-nonenal concentration in the malt alcoholic beverage, if the malt alcoholic beverage is produced by the method for producing a malt alcoholic beverage of the present invention, malt alcohol with improved flavor durability It became clear that a drink was obtained.

  Finally, the darkness and sharpness of the sparkling liquor obtained in 2 above were analyzed by a sensory test and a lipid membrane sensor.

  First, sensory tests were conducted by 13 panels to compare flavor quality. Liquefied liquor of LOX-F4 and LOX + F4 were subjected to a sensory test, and evaluation was performed with a score of 0 to 4 (higher dark blue or better black marks) with respect to two items of dark blue and sharp ( Table 6).

  Regarding darkness, there was no significant difference (5% risk) between LOX-F4 and LOX + F4 (Table 6A).

  Regarding sharpness, 8 out of 13 gave LOX-F4 a higher score (Table 6B). Further, LOX-F4 showed a higher score (average value) than LOX + F4, and the difference was determined to be significant at a 5% risk by t-test.

  From the above results, it has been clarified that, when a malt alcoholic beverage is brewed using LOX-F4, the sharpness can be improved without affecting the darkness.

Furthermore, by the method of Kaneda et al. (Kaneda, H. et al., J. Biosci. Bioeng., 92,
221-226, 2001.) Using a lipid membrane sensor, the darkness and sharpness of the product were evaluated (Table 7).

  The darkness was evaluated by the adsorptivity to the lipid membrane. As a result, no statistically significant difference (risk rate 5% level) was observed between the adsorptivity of LOX-F4 and LOX + F4 (Table 7A). .

  On the other hand, cracks are evaluated by their persistence in lipid membranes (the worse they are, the higher the survivability is), but LOX-F4 is about 1/4 of the survivability compared to LOX + 4, with a risk rate of 1% level. A significant difference was observed (Table 7B).

  So far, there has been no correlation between malt LOX-1 activity and THOD production in the charging process (Kobayshi, N. et al., (2000). J. Biosci. Bioeng. 90: 69-73.) It is unknown how much THOD production is suppressed by suppression of LOX-1. In addition, as a result of the suppression, it has not been predicted by the prior art whether the sharpness can be improved. However, from the sensory test results of this example and the analysis results of the darkness and sharpness of the product using the lipid membrane sensor, if the malt using the claimed gene is used, the darkness of the product is not affected. For the first time, it was demonstrated to improve the sharpness of the product.

  LOX-1 mutant gene and LOX-1 deletion barley selection method useful for producing malt alcoholic beverages with improved flavor durability and foam retention without genetic manipulation, and obtained by selection It is possible to provide a raw material for malt alcoholic beverages derived from barley and a method for producing a malt alcoholic beverage using the raw materials for malt alcoholic beverages.

It is a graph which shows the result of the LOX-1 activity in the exploratory test 1. It is a graph which shows the result of the LOX-1 inhibitory activity in the verification test 1. It is an electrophoresis photograph which shows the result of the Western analysis of the LOX protein of the barley seed in the proof test 2. A shows the result of Western analysis after SDS-PAGE, and B shows the result of Western analysis after IEF. It is an electrophoresis photograph which shows the result of RT-PCR analysis of barley seed RNA in the verification test 3. It is a figure which shows the structure of a LOX-1 gene 5th intron splicing donor site. It is an electrophoresis photograph which shows the result analyzed about the splicing of the LOX-1 mutant gene in the verification test 5. A is an electrophoretic photograph of an amplified fragment containing the third intron to the fifth intron, and B is an electrophoretic photograph after digesting the same amplified fragment as A with StuI. 7 is an electrophoresis photograph showing a DNA polymorphism in the second generation of Kendall × SV73 hybrid in verification test 6. FIG. It is the figure which put together the DNA polymorphism in the hybrid 2nd generation of Kendall * SV73 in the verification test 6, and the LOX activity in the hybrid 3rd generation. 2 is an electrophoretogram showing the results of analysis of general barley varieties / lines in Example 1 by the AfaI method. It is an electrophoresis photograph which shows the analysis result by the AfaI method of LOX-1 deletion barley in Example 2. It is a figure which shows the result of the seed LOX activity of the LOX-1 deletion barley in Example 2. A is a diagram showing a result of an enzyme reaction time of 5 minutes and B is a result of an enzyme reaction time of 90 minutes. It is a figure which shows the result of the malt analysis of the seed of the LOX + F4 population in Example 5, and a LOX-F4 population. It is a figure which shows the trans-2-nenal concentration in wort in Example 5, and a nonenal potential.

Claims (4)

Barley lipoxygenase-1 gene 5th intron splicing donor site (5'-GT-3 ') guanine mutated to adenine mutated a barley lipoxygenase-1 mutant gene of barley mating progeny lipoxygenase-1 deletion A raw material for alcoholic beverages characterized by being seeds, malt, malt extract, barley degradation products or processed barley products derived only from the barley line. Barley lipoxygenase-1 gene 5th intron splicing donor site (5'-GT-3 ') guanine mutated to adenine mutated a barley lipoxygenase-1 mutant gene of barley mating progeny lipoxygenase-1 deletion A raw material for malt alcoholic beverages characterized by being seeds, malt, malt extract, barley degradation products or processed barley products derived only from barley lines. A method for producing an alcoholic beverage comprising using the raw material for an alcoholic beverage or the raw material for a malt alcoholic beverage according to claim 1 or 2. An alcoholic beverage comprising the alcoholic beverage material or malt alcoholic beverage material according to claim 1 or 2 .

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