JP4531782B2 - Barley selection method and selection marker - Google Patents

Description

  The present invention relates to a method for selecting barley and a selection marker.

  In addition to the method of growing and selecting the progeny of mating obtained by crossing excellent strains until the desired trait is expressed, the selection method of barley is not limited to the seed or seedling stage. And selecting using the presence or absence of a DNA region or protein serving as a selection marker as an index.

  Foam retention of malt fermented drinks is one of the important factors for judging the quality of malt fermented drinks, and barley that improves foam retention of malt fermented drinks is required. LTP-1 and hordein have been reported as proteins involved in foam retention of malt fermented beverages contained in barley (Non-patent Documents 1 and 2).

  Recently, proteomics has come to be used for plant selection, and attempts to comprehensively analyze the protein composition in barley seeds are involved in malt characteristics by comparing malt characteristics and two-dimensional electrophoresis patterns. Attempts have been made to identify proteins (Non-Patent Documents 3 to 5).

Bamforth et al., 2004, J. Am. Sci. Food Agric. 84, p. 1001-1004 Van Nierop et al., 2004, J. MoI. Agric. Food. Chem. 52, p. 3120-3129 Ostergaard et al., 2004, Proteomics, 4, p. 2437-2447 Sass Bak-Jensen et al., 2004, Proteomics, 4, p. 728-742 Finnie and Svensson, 2004, Proc. 9th International Barley Genetics Symposium, p. 431-436

  However, a selection marker that enables selection of barley that improves the foam retention of the malt fermented beverage has not been found, and there is no method for selecting barley that improves the foam retention of the malt fermented beverage.

  LTP-1 and hordein in barley have been reported to be related to the foam retention of malt fermented beverages, but the correlation with the NIBEM value, which is a criterion for the quality of foam retention, has not been clarified. Therefore, it is difficult to use it as a selection marker for barley used in the production of a malt fermented beverage with good foam.

  Then, the objective of this invention is providing the selection method of the barley which improves the foam retention of the malt fermented drink, and is contributing to manufacture of the malt fermented drink with good foam using the selected barley as a raw material. Another object of the present invention is to provide a barley selection marker that improves the foam retention of a malt fermented beverage.

  In order to achieve the above object, the present invention is a method for selecting barley using protein Z7 as a selection marker to improve the foam retention of a malt fermented beverage, comprising barley seeds, malt obtained from the seeds, and malt A step of measuring the concentration of protein Z7 contained in the wort obtained from the above, a malt fermented beverage obtained by fermenting the wort, or a raw material solution before fermentation or a raw material solution during fermentation of the malt fermented beverage, and this concentration When the barley is low, the selection method includes a determination step of determining that the barley is barley that improves the foam retention of the malt fermented beverage.

  The inventors of the present invention comprehensively analyzed proteins in beer using proteomics as a tool, and barley seeds used as a raw material in beer with high foamability, which has a high NIBEM value, which is a criterion for foaminess. And the concentration of protein Z7 in wort and the concentration of protein Z7 in the beer were clarified. Furthermore, as a result of statistically analyzing the correlation between the concentration of the protein Z7 and the NIBEM value, it was found that by examining the concentration of the protein Z7, the goodness of foaming of the malt fermented beverage can be determined and predicted.

  For this reason, if the density | concentration of the protein Z7 contained in the raw material liquid before fermentation of a barley seed, malt, wort, malt fermented drink or malt fermented drink or raw material liquid during fermentation is measured, NIBEM of the malt fermented drink which is a final product It is possible to select barley that improves foam retention without directly measuring the value. In addition, the NIBEM value is greatly influenced by temperature, weather, and human factors, so it is difficult to compare the NIBEM values measured under different conditions to determine the goodness of foam. The concentration of protein Z7 can be measured without the influence of these factors, and since there is a correlation between the concentration of protein Z7 and the NIBEM value, it can also be used to determine the goodness of foam.

  The measurement step includes barley seeds, malt obtained from the seeds, wort obtained from the malt, fermented malt drink obtained by fermenting the wort, or raw material liquid before fermentation or raw material liquid during fermentation Preferably, the method comprises an extraction step for extracting the protein from the protein and a quantitative step for quantifying the protein Z7 contained in the protein using an anti-protein Z7 antibody.

  Since protein Z7 is specifically recognized by the anti-protein Z7 antibody, protein Z7 can be detected with high sensitivity using an ELISA method, Western blotting method, protein chip, etc., and its concentration can be quantified easily.

  The present invention also relates to a method for selecting barley using protein Z7 as a selection marker to improve the foam retention of malt fermented beverages, and extracting RNA from barley seeds or malt obtained from the seeds; A quantification step for quantifying protein Z7 mRNA expressed in barley seeds or malt, and a determination step for judging that barley is a barley that improves the foam retention of malt fermented beverages when the expression level of protein Z7 mRNA is low; A selection method comprising:

  Even if an anti-protein Z7 antibody that specifically recognizes protein Z7 is not available, protein Z7 mRNA can be detected with high sensitivity using, for example, PCR, Northern blotting, DNA chip, etc. Can be easily quantified.

  Moreover, this invention provides the selection marker of the barley which improves the foam retention of the malt fermented drink which consists of protein Z7.

  Protein Z7 can be regarded as a negative factor for the foam retention of malt fermented beverages. Since the foam retention of malt fermented beverages made from barley seeds with low protein Z7 concentration is improved, the above selection marker Can be used to select barley varieties used as raw materials for malt fermented beverages with good foaminess.

  ADVANTAGE OF THE INVENTION According to this invention, the barley which improves the foam retention of a malt fermented drink can be selected, without measuring the NIBEM value of the malt fermented drink which is the final product which uses barley as a raw material. Protein Z7 concentration can be measured by eliminating the influence of temperature, weather, and human factors, and there is a correlation between protein Z7 concentration and NIBEM value. The selection method of the invention can also be used as a determination method for a fermented malt fermented beverage.

  Furthermore, the selection marker of the present invention can be used for selection of barley to be used in a fermented malt fermented beverage.

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

  The selection method of the present invention is a selection method of barley that uses protein Z7 as a selection marker to improve the foam retention of malt fermented beverages, and is a barley seed, malt obtained from the seed, and wheat obtained from the malt A measurement step for measuring the concentration of protein Z7 contained in a soup, a malt fermented beverage obtained by fermenting the wort, or a pre-fermentation raw material solution or a raw material solution during fermentation of the malt fermented beverage, and when this concentration is low The barley is characterized by comprising a judgment step for judging that the barley has improved foam retention of the malt fermented beverage.

  “Barley” in the present specification refers to a plant having the scientific name of Hordeum vulgare, and corresponds to barley varieties, barley lines, and individual barley individuals of barley.

  In addition, “foam” means the time required for foam to disappear when malted fermented beverage is poured into a glass, and “good foam” means that this time is long, “Poor foam” means that this time is short. “Barley for better foam retention” means that when a malt fermented beverage is produced using the barley as a raw material, it contributes to better foam retention compared to a malt fermented beverage produced from the same raw material other than barley. It refers to barley.

  The “malt fermented beverage” refers to a beverage brewed using malt as a raw material, and examples thereof include beer, happoshu or miscellaneous sake containing malt in the raw material. Beer is fermented using malt, hops and water as raw materials, or malt, hops, water and wheat and other articles specified by government ordinances (wheat, rice, corn, corn, potato, starch, sugar, or the Ministry of Finance) (Bitter taste or colorant determined) is fermented as a raw material and has a malt use rate of 2/3 or more. Happoshu is a liquor made from malt or wheat as part of the raw material. Which has a malt use rate of less than 2/3.

  “Pre-fermentation raw material liquid of malt fermented beverage” means that the raw material liquid before the wort is obtained from the preparation process in the middle of obtaining the wort from the malt, or the wort before boiling, or the wort It refers to the wort or cold wort until it is cooled and cold wort before yeast is added. The “raw material liquid during fermentation of malt fermented beverage” refers to all the fermented liquor until the malt fermented beverage is obtained through the fermentation process after yeast is added to the cold wort.

  “Protein Z7” is a serine protease inhibitor present in barley seeds or beer, and is a protein having a molecular weight of about 43 kDa (NCBI Access CAA 64599). In barley seeds, besides protein Z7, there is protein Z4 (NCBI Accession CAA66232), which belongs to the same barley serine protease inhibitor subfamily, and its amino acid sequence has about 73% homology with protein Z7. Yes.

  The measurement step includes barley seed, malt obtained from the seed, wort obtained from the malt, malt fermented beverage obtained by fermenting the wort, or raw material solution before fermentation or raw material solution during fermentation. Preferably, the method comprises an extraction step for extracting the protein from the protein and a quantitative step for quantifying the protein Z7 contained in the protein using an anti-protein Z7 antibody. For example, the concentration of protein Z7 can be quantified by ELISA, Western blotting, analysis by protein chip, quantitative analysis by affinity chromatography, or the like.

  The anti-protein Z7 antibody used in the ELISA method or Western blotting method is preferably an antibody capable of specifically recognizing barley protein Z7 used for fermentation. For example, a peptide synthesized based on the amino acid sequence of protein Z7 is used as an antigen. It can be created by immunizing rabbits and mice. These immunological techniques are well known to those skilled in the art and will not be described in detail in this specification. For textbooks describing such methods, see, for example, “Antibodies: a laboratory manual” (Harlow and Lane, 1988, Cold Spring Harbor Laboratory).

  The selection method of the present invention is a method for selecting barley that uses protein Z7 as a selection marker to improve the foam retention of malt fermented beverages, and extracts RNA from barley seeds or malt obtained from the seeds. When the extraction step, the quantification step for quantifying protein Z7 mRNA expressed in barley seeds or malt, and the expression level of protein Z7 mRNA are low, it is determined that this barley is a barley that improves the foam retention of the malt fermented beverage And a determining step.

In the extraction step, for example, RNA can be extracted according to the hot phenol method (a method of extracting RNA while dissolving barley seeds or malt with heated phenol and sodium dodecyl sulfate). In order to obtain it, it is more preferable to freeze barley seeds or malt with liquid nitrogen, and then grind them with a grinder and extract RNA with an RNA extraction reagent containing guanidine thiocyanate. Examples of the RNA extraction reagent include Isogen ^™ (manufactured by Nippon Gene).

  In the quantification step, protein Z7 mRNA contained in the barley-derived RNA extracted in the extraction step is detected with high sensitivity using, for example, RT-PCR, Northern blotting, DNA chip, etc., and the expression level is quantified. it can. These methods are described in, for example, “Molecular cloning” (Maniatis et al., 1989, Cold Spring Harbor Laboratory Press) and “Current Protocols in Molecular Biology” (F. M. Ausubel, 1988, John Wiley & , Inc.) and other genetic engineering protocols.

  Moreover, the selection marker of the barley which improves the foam retention of the malt fermented drink of this invention consists of protein Z7, It is characterized by the above-mentioned.

  Protein Z7 can be regarded as a negative factor for the foam retention of malt fermented beverages, and if the concentration of protein Z7 is low, the foam retention of malt fermented beverages will be improved. It can be used as a marker, and can be used as a selection marker for barley used as a raw material for a fermented malt fermented beverage. So far, it has not been known at all that protein Z7 can be used as a negative factor for determining the goodness of foaming of malt fermented drinks and for selecting barley used as a raw material for fermented malt fermented drinks.

  Here, the “marker for determining foam retention” means that the concentration of barley seeds, malt, wort, malt fermented beverage or malt fermented beverage in the raw material solution before fermentation or in the raw material solution during fermentation correlates with the NIBEM value. It refers to the protein that is shown, and refers to a protein that can be used as an index for determining or predicting the goodness of foaming of a malt fermented beverage.

  Protein Z7 is present in barley seeds, malt, wort, malt fermented beverages or pre-fermentation raw material liquids or fermented raw material liquids, for example, ELISA method, Western blotting method, protein chip analysis, affinity It can be measured by quantitative analysis by chromatography.

Furthermore, if the selection marker of barley which improves the foam retention of the malt fermented drink of this invention is used, the determination method of the good foam retention of the malt fermented drink can be provided. That is, a method for determining the foamability of a malt fermented beverage, which is obtained by fermenting barley seeds used in a malt fermented beverage, malt obtained from the seeds, wort obtained from the malt, and wort A determination method for determining the goodness of foaming, using the concentration of protein Z7 (M _Z7 ) contained in the pre-fermented raw material liquid or the raw material liquid during fermentation of the malted fermented beverage as a negative factor for foaming it can.

  By measuring the concentration of protein Z7 contained in the pre-fermentation raw material liquid or in-fermentation raw material liquid of barley seeds, malt, wort, malt fermented beverage or malt fermented beverage, the goodness of foam can be obtained without measuring the NIBEM value. Since the measurement of the concentration of protein Z7 can be carried out by eliminating the influence of temperature, weather, and human factors, it is more versatile than the measurement of NIBEM value, and It is possible to accurately determine the goodness of foam.

  Here, the “NIBEM value” means that the height of foam generated by pouring malt fermented beverage at 20 ° C. into a standard glass using carbon dioxide gas by a standard bubble pouring machine for NIBEM value measurement is reduced by 30 mm. Time (seconds).

The determination method preferably determines that the malt fermented beverage having a smaller numerical value obtained by multiplying the concentration of protein Z7 (M _Z7 ) by a negative coefficient is a malt fermented beverage having good foaming. More preferably, the numerical value is calculated as a−b × M _Z7 (where a is a positive number, a negative number, or 0, and b is a positive number). Further, it is more preferable if a and b are single regression coefficients of a single regression equation for predicting the NIBEM value of a malt fermented beverage when _MZ7 is used as a variable.

The single regression coefficient of the single regression equation for predicting the NIBEM value of the malt fermented beverage is a raw material before fermentation of barley seeds, malt, wort, malt fermented beverage or malt fermented beverage for a plurality of malt fermented beverages having measured NIBEM values. Measure the concentration of protein Z7 (M _Z7 ) in the liquid or raw material during fermentation, and use the NIBEM value of each malt fermented beverage as the objective variable and perform the single regression analysis using the concentration of protein Z7 (M _Z7 ) as the explanatory variable Can be obtained.

  The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

(Example 1) Correlation between the concentration of protein Z7 in the seeds of barley as a raw material for beer and the NIBEM value Seven barley varieties (variety A: Haruna Nijo, variety B: CDC Kendall, variety C: Amagi Nijo, Variety D: CDC Copeland, Variety E: Hokuiku No. 39, Variety F: Ryofu, Variety G: Lofty Nijo) each produces malt, and the same raw materials are used for the raw materials other than malt necessary for beer brewing 7 types of beer were brewed using The concentration of protein Z7 in the seeds of the seven barley varieties used as raw materials for beer and the NIBEM values of the seven types of beer were measured, respectively, and the correlation between the concentration of protein Z7 in the seeds of each barley variety and the NIBEM value. Investigated about.

  The concentration of protein Z7 contained in the seeds of seven barley varieties was quantified as follows. First, pulverized seeds of each barley variety (50 mg) are taken into a 2 mL tube with a screw cap, and 1 mL of PBS (Phosphate Buffer Saline) containing 0.28% dithiothreitol (Wako Co., Ltd.) is added and suspended. Protein Z7 was extracted by shaking overnight at 4 ° C. Thereafter, the suspension was centrifuged at 15,000 rpm for 5 minutes at 4 ° C., and the resulting supernatant was appropriately diluted, and the protein Z7 concentration was quantified with an anti-protein Z7 antibody. The anti-protein Z7 antibody was purchased from Dr. Evans of the University of Tasmania, Australia, and the quantification of protein Z7 using the anti-protein Z7 antibody was performed by Dr. Evans et al. (Amer. Soc. Brew. Chem., 2003). 61, p.55-62).

  Moreover, about the beer brewing which used the seed of each barley variety independently, it performed by the following method. First, seven kinds of barley varieties (variety A to G) were soaked in water in a soaking tank to a water content of about 43%, germinated in a germination room for 6 days, and then dried with hot air. Each barley variety of malt was produced.

  After that, corn starch, corn grits, rice and water, which are auxiliary materials, are added to each malt and subjected to saccharification treatment at 50 to 60 ° C. for 20 minutes, 65 ° C. for 40 minutes and 73 ° C. for 3 minutes (saccharification step). Hops were added to the resulting wort, boiled for 90 minutes, cooled to 10 ° C., yeast was added, and alcohol fermentation was performed at 10 to 15 ° C. for 7 to 10 days (fermentation step). After completion of fermentation, the mixture was aged at -1 to 2 ° C for 2 to 3 weeks, and the obtained fermented product was used as beer for measurement of NIBEM values.

  The NIBEM value was measured using a NIBEM-T apparatus manufactured by HAFFMANS, INPACK2000, and a standard glass for NIBEM value measurement. Specifically, each of the above malt fermented beverages is brought to a state of 20 ° C., poured into a standard glass using a carbon dioxide gas by a foam pouring machine, and the resulting drop in the height of the foam is tracked with a NIBEM-T apparatus. The time (seconds) required for the bubble height to drop by 30 mm was measured as the NIBEM value.

  Table 1 shows the concentration of protein Z7 contained in the seeds of 7 types of barley varieties (variety AG) and the NIBEM values of 7 types of beer brewed from the seeds of 7 types of barley varieties, respectively. It is a table.

  FIG. 1 is a graph obtained by performing a single regression analysis using the NIBEM value as an objective variable and the concentration of protein Z7 in barley seeds as an explanatory variable. * In the figure indicates that the risk rate is significant at 5%.

  As a result, a statistically significant correlation was observed between the NIBEM value and the concentration of protein Z7 in the barley seeds. The single regression equation was NIBEM value = 273.31 + (− 0.126 × protein Z7 concentration) (r = 0.848).

  From the above results, the concentration of protein Z7 in the barley seeds can be used as a negative factor for the foam retention of malt fermented beverages made from the barley seeds as a raw material, and can be used to determine the goodness of the malt fermented beverages. It was revealed that barley that improves the foam retention of malt fermented beverages can be selected by measuring the concentration of protein Z7 in the seeds.

(Example 2) Correlation between the concentration of protein Z7 in Congress wort and NIBEM value Seven barley varieties (variety A: Haruna Nijo, variety B: CDC Kendall, variety C: Amagi Nijo, variety D: CDC Copeland , Variety E: Kitaiku 39, Variety F: Ryofu, Variety G: Lofty Nijo), wort is produced, and the same raw materials are used for raw materials other than malt necessary for beer brewing. Seven types of beer were brewed. The concentration of protein Z7 and the NIBEM value of each of the seven types of beer produced from the seeds of the seven types of barley varieties used as raw materials for beer were measured. The correlation with the value was investigated.

  The production of the congress wort was performed by the following method. The malt of each barley variety produced according to the procedure described in Example 1 was pulverized, 200 mL of water was added to the pulverized malt (50 g), held at 45 ° C. for 30 minutes, and then 70 ° C. at a rate of 1 ° C. per minute. The temperature was raised to 70 ° C., and when it reached 70 ° C., 100 mL of water was added and held for 1 hour. Thereafter, water was added thereto so that the total amount became 450 g, and the wort obtained by filtration was designated as the congress wort.

  The brewing of beer and the measurement of NIBEM values were performed in the same procedure as the method described in Example 1. The concentration of protein Z7 in the conger wort was determined by appropriately diluting each solution and performing ELISA using an anti-protein Z7 antibody.

  Table 2 shows the concentration of protein Z7 contained in the conger wort produced from the seeds of seven types of barley varieties (variety A to G) and the NIBEM values of seven types of beer brewed using seven types of barley seeds as raw materials. It is the table | surface which showed.

  FIG. 2 is a graph obtained by performing a single regression analysis using the NIBEM value as an objective variable and the concentration of protein Z7 in the congress wort as an explanatory variable. * In the figure indicates that the risk rate is significant at 5%.

  As a result, a statistically significant correlation was observed between the NIBEM value and the concentration of protein Z7 in the conger wort. The single regression equation was NIBEM value = 273.05 + (− 0.9006 × protein Z7 concentration) (r = 0.852).

  From the above results, the concentration of protein Z7 in the conger wort can be used as a negative factor for the foam retention of the malt fermented beverage made from the wort as a raw material, and can be used to determine the goodness of the foam of the malt fermented beverage. By measuring the concentration of protein Z7 in the barley, it became clear that barley that improves the foam retention of malt fermented beverages can be selected.

(Example 3) Correlation between concentration of protein Z7 in beer and NIBEM value Seven barley varieties (variety A: Haruna Nijo, variety B: CDC Kendall, variety C: Amagi Nijo, variety D: CDC Copeland, variety E: Kitaiku 39, Variety F: Ryofu, Variety G: Lofty Nijo) each produces malt, and the same raw materials are used for the ingredients other than malt necessary for beer brewing. Brewed. The concentration of protein Z7 contained in 7 types of beer and the NIBEM value of 7 types of beer were measured, and the correlation between the concentration of protein Z7 in beer and the NIBEM value was examined.

  The brewing of beer and the measurement of NIBEM values were performed in the same procedure as the method described in Example 1. The concentration of protein Z7 in beer was determined by appropriately diluting each solution and quantifying it by ELISA using an anti-protein Z7 antibody.

  Table 3 is a table showing the concentration of protein Z7 contained in seven types of beer brewed using seeds of seven types of barley varieties (variety A to G) and NIBEM values of the seven types of beer.

  FIG. 3 is a graph obtained by performing a single regression analysis using the NIBEM value as an objective variable and the concentration of protein Z7 in beer as an explanatory variable. ** in the figure indicates that the risk rate is significant at 1%.

  As a result, a statistically significant correlation was observed between the NIBEM value and the concentration of protein Z7 in beer. The single regression equation was NIBEM value = 266.50 + (− 2.8089 × protein Z7 concentration) (r = 0.920).

  From the above results, the concentration of protein Z7 in beer can be used as a negative factor for the foam retention of the beer to determine the foaming goodness of malt fermented beverages, and by measuring the concentration of protein Z7 in beer It was revealed that barley that improves foam retention of malt fermented beverages can be selected.

It is the graph which performed the single regression analysis by making NIBEM value into an objective variable, and the density | concentration of protein Z7 in the barley seed as an explanatory variable. It is the graph which performed the single regression analysis by making NIBEM value into an objective variable and using the density | concentration of protein Z7 in Congress wort as an explanatory variable. It is the graph which performed the single regression analysis by making NIBEM value into an objective variable and using the density | concentration of protein Z7 in beer as an explanatory variable.

Claims (3)

A method for selecting barley that uses protein Z7 as a selection marker to improve the foam retention of a malt fermented beverage,
Included in the barley seeds, the malt obtained from the seeds, the wort obtained from the malt, the malt fermented beverage obtained by fermenting the wort, or the pre-fermentation raw material solution or the raw material solution during fermentation of the malt fermented beverage Measuring step of measuring the concentration of protein Z7,
When the concentration is low, the barley is determined to be barley that improves the foam retention of the malt fermented beverage; and
A selection method comprising: The measurement step includes the barley seeds, the malt obtained from the seeds, the wort obtained from the malt, the malt fermented drink obtained by fermenting the wort, or the raw material solution before fermentation of the malt fermented drink or during fermentation An extraction step for extracting protein from the raw material liquid;
A quantification step of quantifying the protein Z7 contained in the protein using an anti-protein Z7 antibody;
The selection method according to claim 1, further comprising: A method for selecting barley that uses protein Z7 as a selection marker to improve the foam retention of a malt fermented beverage,
An extraction step of extracting RNA from the barley seeds or malt obtained from the seeds;
A quantification step of quantifying protein Z7 mRNA expressed in the barley seeds or malt;
When the expression level of the protein Z7 mRNA is low, the determination step that the barley is barley that improves the foam retention of the malt fermented beverage;
A selection method comprising:

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