WO2022004713A1 - Non-alcoholic beer-flavored beverage - Google Patents

Non-alcoholic beer-flavored beverage Download PDF

Info

Publication number
WO2022004713A1
WO2022004713A1 PCT/JP2021/024537 JP2021024537W WO2022004713A1 WO 2022004713 A1 WO2022004713 A1 WO 2022004713A1 JP 2021024537 W JP2021024537 W JP 2021024537W WO 2022004713 A1 WO2022004713 A1 WO 2022004713A1
Authority
WO
WIPO (PCT)
Prior art keywords
ppm
alcoholic beer
beer
taste beverage
taste
Prior art date
Application number
PCT/JP2021/024537
Other languages
French (fr)
Japanese (ja)
Inventor
理沙 高木
菜子 首藤
恵子 岩佐
正晃 小沢
直人 神田
嘉英 松尾
Original Assignee
サントリーホールディングス株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by サントリーホールディングス株式会社 filed Critical サントリーホールディングス株式会社
Priority to JP2022534035A priority Critical patent/JPWO2022004713A1/ja
Publication of WO2022004713A1 publication Critical patent/WO2022004713A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients

Definitions

  • the present invention relates to a non-alcoholic beer-taste beverage.
  • Patent Document 1 discloses that a peptide having a specific molecular weight is contained in order to improve the flavor of a beer-taste beverage.
  • indexes such as beer-like taste, smooth taste flow, and roughness remaining in the mouth are used as evaluation indexes for beverages, and by containing a peptide of 10-20 kDa at a predetermined concentration, these indexes can be used. It is stated that a beverage with a high sensory evaluation score based on it can be obtained.
  • the taste that can be felt immediately after drinking a beer-taste beverage the strength, spread, thickness, and taste of the five basic tastes, that is, the tastes that cannot be expressed by sweetness, saltiness, acidity, bitterness, and umami, are maintained.
  • the characteristic that the strength of taste is well-balanced may be preferred.
  • Such a feature is referred to as "bulge" in the present specification.
  • the beverage described in Patent Document 1 is a beverage that has room for further improvement from the viewpoint of swelling, and a method for enhancing swelling has been desired.
  • An object of the present invention is to provide a beverage having an enhanced bulge.
  • Non-alcoholic beer-taste beverages containing malt as a raw material tend to have poor swelling, and therefore it is particularly desired to enhance swelling.
  • the present invention relates to the following non-alcoholic beer-taste beverages.
  • a non-alcoholic beer-taste beverage containing malt as a raw material which contains at least one diketopiperazine selected from the group consisting of cycloleucylproline, cyclovalylproline and cycloisoleuylproline, and contains the above-mentioned diketopiperazine.
  • the non-alcoholic beer-taste beverage of the present invention contains malt as a raw material.
  • the raw material here means a grain raw material other than water and hops and sugar.
  • rice, corn, millet, potato, starch, and wheat other than malt may be contained as a raw material.
  • Ingredients that can be added in trace amounts, such as acidulants, sweeteners, bitterness agents, seasonings, and flavors, are not included in the raw materials.
  • the weight of the extract content of the non-alcoholic beer-taste beverage of the present invention is not particularly limited, but is preferably 0.01 to 20.0% by weight.
  • the non-alcoholic beer-taste beverage is a beer-taste beverage having an alcohol content of less than 1%, and preferably contains substantially no alcohol. Further, the alcohol content may be 0%.
  • a beer-taste beverage is a beer-flavored carbonated beverage.
  • the beverages having a substantially no alcohol content do not exclude beverages containing an undetectable trace amount of alcohol. Beverages whose alcohol content is rounded to 0.0%, particularly those whose alcohol content is rounded to 0.00%, are included in non-alcoholic beer-taste beverages.
  • Types of non-alcoholic beer-taste beverages of the present invention include, for example, non-alcoholic beer-taste beverages, beer-taste soft drinks, and the like.
  • the "alcohol content (alcohol content)" here means the content of ethanol, and does not include the content of aliphatic alcohols other than ethanol.
  • the alcohol content of the non-alcoholic beer-taste beverage according to the present invention means the alcohol content (v / v%) in the beverage, and can be measured by any known method, for example, a vibration type. It can be measured by a densitometer. Specifically, a sample from which carbon dioxide gas has been removed from the beverage by filtration or ultrasonic waves is prepared, and the sample is directly distilled to measure the density of the obtained distillate at 15 ° C. Converted using the "Table 2 Alcohol Content and Density (15 ° C) and Specific Weight (15/15 ° C) Conversion Table" attached to (Heisei 19 National Tax Agency Royal Instruction No. 6, revised on June 22, 2007). Can be asked. When the alcohol content is less than 1.0%, a commercially available alcohol measuring device or gas chromatography may be used.
  • the non-alcoholic beer-taste beverage of the present invention contains at least one diketopiperazine selected from the group consisting of cycloleucylproline, cyclovalylproline and cycloisoroycilproline.
  • Diketopiperazine is a cyclic dipeptide in which two amino acids are linked.
  • Cycloleucylproline is a combination of leucine and proline, and is sometimes referred to as Cyclo (Leu-Pro) in the present specification.
  • Cyclovalylproline is a combination of valine and proline, and is sometimes referred to as Cyclo (Val-Pro) in the present specification.
  • Cycloisoleucine proline is a combination of isoleucine and proline, and is sometimes referred to as Cyclo (Ile-Pro) in the present specification.
  • the concentration of diketopiperazine contained most in the above-mentioned diketopiperazine is 0.4 ppm or more.
  • the concentration of these diketopiperazines is 0.4 ppm or more, the swelling in the non-alcoholic beer-taste beverage can be enhanced. It has not been known so far that the content of a specific diketopiperazine in a predetermined concentration or more is related to the swelling in a non-alcoholic beer-taste beverage, and it is a finding found by the present inventors.
  • the concentration of diketopiperazine, which is contained most in the diketopiperazine is preferably 10 ppm or less.
  • non-alcoholic beer-taste beverage of the present invention further contains a protein having a molecular weight of 35 to 50 kDa, and the concentration of the protein is preferably 5 ppm or more.
  • a protein having a molecular weight of 35 to 50 kDa is a protein found in the region having a molecular weight of 35 to 50 kDa when a non-alcoholic beer-taste beverage is subjected to electrophoresis by SDS-PAGE. Before subjecting the non-alcoholic beer-taste beverage to electrophoresis by SDS-PAGE, for example, the non-alcoholic beer-taste beverage may be subjected to ultrafiltration using a 30 kDa cut-off membrane as a pretreatment.
  • the protein is preferably a protein having a molecular weight of 35 to 45 kDa, more preferably a protein having a molecular weight of about 40 kDa. In the present specification, a protein having a molecular weight of 35 to 50 kDa is also referred to as a 40 kDa protein.
  • the 40 kDa protein is preferably a cereal-derived protein.
  • the cereal is preferably at least one selected from the group consisting of barley, wheat, corn, rice and soybean.
  • the cereal is wheat, it can contain a known protein derived from wheat used in the production of non-alcoholic beer-taste beverages. Examples of such wheat include barley, wheat, rye, oats, oats, and barley, and barley is preferable. Further, either germinated wheat or ungerminated wheat may be used, but germinated malt is preferable. These may be contained alone or in combination of two or more kinds.
  • Serpin Z4 also known as BSZ4, HorvuZ4, Major endosperm albumin or Protein Z
  • Serpin Z7 also known as BSZ7 or HorvZ
  • a protein having an amino acid sequence in which a part of the amino acids in the amino acid sequence is deleted, substituted, inserted and / or added may be used.
  • the swelling in non-alcoholic beer-taste beverages can be further enhanced.
  • concentration of the 40 kDa protein is preferably 30 ppm or less.
  • the swelling of the non-alcoholic beer-taste beverage can be effectively enhanced by the synergistic effect of diketopiperazine and 40 kDa protein.
  • the manufacturing process of a general non-alcoholic beer-taste beverage is shown below.
  • a non-alcoholic beer-taste beverage can be easily produced.
  • a non-alcoholic beer-taste beverage produced using malt as a raw material first, in addition to wheat such as malt, other grains, starch, saccharides, bitterness agents, coloring agents, etc., as necessary, etc. If necessary, an enzyme such as amylase is added to the mixture containing the raw material and water to gelatinize and saccharify, and the mixture is filtered to obtain a saccharified solution. If necessary, add hops and bitterness to the saccharified solution and boil, and remove solids such as coagulated protein in a clarification tank. As an alternative to this saccharified solution, hops may be added to a malt extract mixed with warm water and boiled. Hops may be mixed at any stage from the start of boiling to the end of boiling.
  • the boiling step As the conditions in the saccharification step, the boiling step, the solid content removing step and the like, known conditions may be used. After boiling, the obtained wort is filtered, and carbon dioxide gas is added to the obtained filtrate. After that, it is filled in a container and sterilized to obtain a desired non-alcoholic beer-taste beverage.
  • Aliphatic alcohol may be added to the non-alcoholic beer-taste beverage according to the present invention from the viewpoint of imparting a feeling of alcohol.
  • the aliphatic alcohol is not particularly limited as long as it is known, but an aliphatic alcohol having 4 to 5 carbon atoms is preferable.
  • preferred aliphatic alcohols include 2-methyl-1-propanol and 1-butanol as those having 4 carbon atoms and 3-methyl-1-butanol and 1-pen as those having 5 carbon atoms. Examples thereof include tanol and 2-pentanol. These can be used in one type or a combination of two or more types.
  • the content of the aliphatic alcohol having 4 to 5 carbon atoms is preferably 0.0002 to 0.0007% by weight, more preferably 0.0003 to 0.0006% by weight.
  • the content of the aliphatic alcohol can be measured by using a headspace gas chromatograph method.
  • the non-alcoholic beer-taste beverage according to the present invention is preferably low in calories in accordance with recent tastes for low calories. Therefore, the calorie content of the non-alcoholic beer-taste beverage according to the present invention is preferably less than 5 kcal / 100 mL, more preferably less than 4 kcal / 100 mL, still more preferably less than 3 kcal / 100 mL.
  • the number of calories contained in the non-alcoholic beer-taste beverage according to the present invention is basically calculated in accordance with "Analysis method of nutritional components, etc. in nutrition labeling standards" published in connection with the Health Promotion Law. That is, as a general rule, the amount of various nutritional components quantified is combined with the energy conversion coefficient of each component (protein: 4 kcal / g, lipid: 9 kcal / g, carbohydrate: 4 kcal / g, dietary fiber: 2 kcal / g, alcohol: It can be calculated as the sum of the products multiplied by 7 kcal / g and organic acid: 3 kcal / g). For details, refer to "Analysis method of nutritional components, etc. in nutrition labeling standards".
  • the specific method for measuring the amount of each nutritional component contained in the non-alcoholic beer-taste beverage according to the present invention is as follows according to the various analytical methods described in the health promotion method "Analysis method for nutritional components, etc. in nutrition labeling standards". good. Alternatively, if you ask the Japan Food Research Laboratories, you can know the amount of heat and / or the amount of each nutritional component.
  • the sugar contained in the non-alcoholic beer-taste beverage according to the present invention means a sugar based on the nutrition labeling standard for foods (Ministry of Health, Labor and Welfare Notification No. 176, 2003).
  • carbohydrate refers to food obtained by removing proteins, lipids, dietary fiber, ash, alcohol and water.
  • the amount of sugar in a food is calculated by subtracting the amount of protein, lipid, dietary fiber, ash and water from the weight of the food. In this case, the amounts of protein, lipid, dietary fiber, ash and water are measured by the methods listed in the nutrition labeling standards.
  • the amount of protein is measured by the nitrogen quantitative conversion method
  • the amount of lipid is measured by the ether extraction method, the chloroform / methanol mixed solution extraction method, the Gerbel method, the acid decomposition method or the Reesegotleave method
  • the amount of dietary fiber is measured. Is measured by high-speed liquid chromatograph method or Proski method
  • the amount of ash is measured by magnesium acetate-added ashing method, direct ashing method or sulfuric acid-added ashing method
  • the amount of water is measured by Karl Fisher method, drying aid. Measure by the method, vacuum overheating drying method, normal pressure heating drying method or plastic film method.
  • the non-alcoholic beer-taste beverage according to the present invention may be low-carbohydrate in accordance with the recent taste for low-carbohydrate. Therefore, the sugar content of the non-alcoholic beer-taste beverage according to the present invention may be less than 2.5 g / 100 mL or less than 0.5 g / 100 mL. Although the lower limit is not particularly set, it is usually about 0.1 g / 100 mL, and may be, for example, 0.15 g / 100 mL or more or 0.2 g / 100 mL or more.
  • the non-alcoholic beer-taste beverage according to the present invention may contain an acidulant.
  • the acidulant it is preferable to use one or more acids selected from the group consisting of citric acid, lactic acid, phosphoric acid, and malic acid.
  • succinic acid, tartaric acid, fumaric acid, glacial acetic acid and the like can also be used as acids other than the above acids. These can be used without limitation as long as they are approved to be added to foods.
  • the content of the acidulant in the non-alcoholic beer-taste beverage according to the present invention is preferably 200 ppm or more, more preferably 550 ppm or more, still more preferably 700 ppm or more, and further preferably 700 ppm or more, in terms of citric acid, from the viewpoint of imparting a beer-like feeling. From the viewpoint of acidity, 15,000 ppm or less is preferable, 5500 ppm or less is more preferable, and 2000 ppm or less is further preferable.
  • the content of the acidulant may be in a suitable range of 200 ppm to 15000 ppm, preferably 550 ppm to 5500 ppm, more preferably 700 ppm to 1500 ppm in terms of citric acid.
  • the citric acid conversion amount is an amount converted from the acidity of each acidulant based on the acidity of citric acid, and for example, the citric acid conversion amount corresponding to 100 ppm of lactic acid is used.
  • the citric acid equivalent amount corresponding to 120 ppm and 100 ppm of phosphoric acid is converted as 200 ppm
  • the citric acid equivalent amount corresponding to 100 ppm of apple acid is converted as 125 ppm.
  • the content of the acidulant in the non-alcoholic beer-taste beverage refers to the one calculated by analysis by high performance liquid chromatography (HPLC) or the like.
  • hops can be used as a part of the raw material.
  • ordinary pellet hops, powdered hops, and hop extracts used in the production of beer and the like can be appropriately selected and used according to the desired flavor.
  • processed hop products such as isometric hops and reduced hops may be used. These are included in the hops used in the non-alcoholic beer-taste beverage according to the present invention.
  • the amount of hops added is not particularly limited, but is typically about 0.0001 to 1% by weight based on the total amount of the beverage.
  • the non-alcoholic beer-taste beverage according to the present invention may use other raw materials as needed, as long as the effects of the present invention are not impaired.
  • sweeteners including high-sweetness sweeteners
  • bitterness agents including high-sweetness sweeteners
  • flavors yeast extracts
  • colorants such as caramel pigments
  • plant-extracted saponin-based substances such as soybean saponin and kiraya saponin
  • plant proteins such as corn and soybean
  • protein-based substances such as bovine serum albumin
  • seasonings such as dietary fiber and amino acids
  • antioxidants such as ascorbic acid
  • the non-alcoholic beer-taste beverage according to the present invention can be packed in a container.
  • the form of the container is not limited in any way, and it can be filled in a sealed container such as a bottle, a can, a barrel, or a PET bottle to make a beverage in a container.
  • the method for producing a non-alcoholic beer-taste beverage of the present invention is not particularly limited, and an example thereof is a method in which a predetermined amount of diketopiperazine is added to a non-alcoholic beer-taste beverage containing malt as a raw material. Further, it is preferable to add 40 kDa protein to a non-alcoholic beer-taste beverage containing malt as a raw material.
  • the diketopiperazine and 40 kDa protein to be added can be prepared, for example, by the procedure described in Examples described later. Further, regarding diketopiperazine and 40 kDa protein, the content thereof may be increased by adjusting various conditions in the manufacturing process of the non-alcoholic beer-taste beverage.
  • Diketopiperazine was purified according to the following. (1) Fractionation of beer by HP20 60 L of beer was fractionated using 10 L of Diaion (registered trademark) HP20 (manufactured by Mitsubishi Chemical Corporation). HP20 was washed 3 times with ethanol and then 3 times with 50% ethanol before use. The washed HP20 was filled in a mass fractionation column and replaced with water. The same amount of distilled water was mixed with 60 L of degassed beer and flowed to the HP20 column using a medium pressure pump. A solution that passed through the HP20 column was obtained as a passing fraction.
  • 40 L of distilled water was flowed using a medium pressure pump to obtain an eluate as a water-eluting fraction.
  • 40 L of hydrous ethanol (10% ethanol, 30% ethanol, and 70% ethanol) was poured, and the eluate was used as a 10% ethanol elution fraction, a 30% ethanol elution fraction, and a 70% ethanol elution fraction, respectively. Obtained.
  • Each eluted fraction was refrigerated as a dried product using an evaporator and a lyophilizer.
  • Protein analysis Protein identification was performed under the following conditions.
  • Search software Proteome Discoverer 2.2.0.3888 (manufactured by Thermo Fisher) Species: Barley (Hordeum vulgare), Hops (Humulus), Yeast (Saccharomyces cerevisiae) Search condition: Digestive enzyme: Chymotrypsin Precursor ion mass error range: Monoisotopic, ⁇ 10 ppm Product ion mass error range: ⁇ 0.02 Da Maximum number of missed cribes: 5 Confidence level (Percolator): High (the most probable level of the three levels of certainty) Database: SwissProt
  • the 40 kDa protein was Serpin Z4 derived from barley (sequence coverage: 77.2%) and Serpin Z7 derived from barley (sequence coverage: 72.8%).
  • the non-alcoholic beer-taste beverage is a non-alcoholic beer-taste beverage containing malt as a raw material.
  • the raw materials are malt, hops, carbonic acid, flavor, acidulant, caramel color, vitamin C, bitterness, and sweetener.
  • As nutritional components alcohol content is 0%, protein is 0 g, sugar is 0 g, and dietary fiber is 0 to 0. Contains 1 g and about 0 mg of purine.
  • the reference points for sensory evaluation are as follows. Five expert panels scored in increments of 0.05 points according to the following criteria, and the score values were averaged. The bulge strength is based on the following criteria. 0 points: Not felt at all 1 point: Slightly felt 2 points: Clearly felt 3 points: As a very felt reference point, a commercially available beer-taste alcoholic beverage different from the above-mentioned commercially available non-alcoholic beer-taste beverage to be evaluated As a standard beer-taste alcoholic beverage (I), the swelling was set to 0.7 points. Further, another commercially available beer-taste alcoholic beverage was used as the standard beer-taste alcoholic beverage (II), and its bulge was set to 1.5 points as the reference point.
  • the standard beer-taste alcoholic beverage (I) is a beer-taste alcoholic beverage in which the proportion of malt in the raw material exceeds 0% by weight and is less than 50% by weight.
  • the raw materials are low-malt beer, malt, hops, sugars, dietary fiber, and spirits (wheat).
  • alcohol content is 4%
  • protein is 0 to 0.2 g
  • sugar is 0.5 to 0.8 g
  • purine is used. Contains about 2.0 mg.
  • the standard beer-taste alcoholic beverage (II) is a beer-taste alcoholic beverage in which the ratio of malt in the raw material is 50% by weight or more.
  • the raw materials are malt and hops, and the nutritional components include 5.5% alcohol, 0.4 to 0.6 g of protein, 3.6 g of sugar, and about 12.5 mg of purines per 100 ml.
  • the procedure for sensory evaluation is as follows. (1) Dispense non-alcoholic beer-taste beverage into 1/10 volume (v / v) vial of final volume (2) Weigh and add diketopiperazine to any weight (3) Sonic for 30 seconds (4) Let stand at room temperature for 30 minutes (5) Fill up the non-alcoholic beer-taste beverage to the final volume (6) Dispense and evaluate by swallowing
  • Example 1 Evaluation by addition of Cyclo (Leu-Pro)
  • concentration of Cyclo (Leu-Pro) contained in the commercially available non-alcoholic beer-taste beverage was 0.008 ppm.
  • sensory evaluation was performed by adding Cyclo (Leu-Pro) so that the concentration of Cyclo (Leu-Pro) was 0.4 ppm, 1 ppm, 3 ppm, and 10 ppm, respectively. The results of the sensory evaluation are shown in Table 1.
  • Example 2 Evaluation of synergistic effect between Cyclo (Leu-Pro) and 40 kDa protein
  • Cyclo (Leu-Pro) by adding 40 kDa protein to a commercially available non-alcoholic beer-taste beverage based on a beverage with a Cyclo (Leu-Pro) concentration of 0.4 ppm.
  • the concentration of 40 kDa protein was 5 ppm.
  • Cyclo (Leu-Pro) is added by adding 40 kDa protein to a commercially available non-alcoholic beer-taste beverage based on a beverage in which the concentration of Cyclo (Leu-Pro) is 1 ppm.
  • the concentration of the 40 kDa protein was 5 ppm, 10 ppm, and 25 ppm.
  • the 40 kDa protein used was the one purified above. For comparison, only 40 kDa protein was added to a commercially available non-alcoholic beer-taste beverage, and the concentration of 40 kDa protein was evaluated as 5 ppm (contrast sample). The results of the sensory evaluation are shown in Table 2.
  • the increase value (0.11) from the control of the sensory evaluation in the sample 5 is larger than the additive effect (0.05)
  • the combination of diketopiperazine and the 40 kDa protein causes an unpredictable synergistic effect. It can be said that the effect is exhibited.
  • diketopiperazine and 40 kDa protein which are obtained by the sum of the increase value (0.12) from the sensory evaluation control in the sample 2 and the increase value (0.03) from the sensory evaluation control in the contrast sample, are used in combination. Since the increase value (0.20) from the control of the sensory evaluation in the sample 6 is larger than the additive effect (0.15) when the diketopiperazine is used in combination, an unpredictable synergistic effect is obtained. It can be said that the effect is exhibited.
  • Example 3 Evaluation by addition of Cyclo (Val-Pro)
  • concentration of Cyclo (Val-Pro) contained in the commercially available non-alcoholic beer-taste beverage was 0.012 ppm.
  • sensory evaluation was performed by adding Cyclo (Val-Pro) so that the Cyclo (Val-Pro) concentration was 0.4 ppm and 0.8 ppm, respectively. Further, the evaluation was performed with the concentration of 40 kDa protein set to 5 ppm. The results of the sensory evaluation are shown in Table 3.
  • the swelling can be enhanced by adding Cyclo (Val-Pro) to a commercially available non-alcoholic beer-taste beverage. Further, it can be seen that the swelling can be further enhanced by further adding 40 kDa protein. From the results of the comparison sample (see Table 2), it can be seen that the swelling can be enhanced only by adding the 40 kDa protein. However, with diketopiperazine, which is obtained by the sum of the increase value (0.02) from the sensory evaluation control in the sample 9 and the increase value (0.03) from the sensory evaluation control in the comparison sample shown in Table 2.
  • the increase value (0.09) from the control of the sensory evaluation in sample 10 is larger than the additive effect (0.05) when the 40 kDa protein is used in combination, the combination of diketopiperazine and the 40 kDa protein is used. It can be said that an unexpected synergistic effect is exhibited.
  • diketopiperazine which is obtained by the sum of the increase value (0.08) from the sensory evaluation control in the sample 11 and the increase value (0.03) from the sensory evaluation control in the comparison sample shown in Table 2. Since the increase value (0.20) from the control of the sensory evaluation in sample 12 is larger than the additive effect (0.11) when the 40 kDa protein is used in combination, the combination of diketopiperazine and the 40 kDa protein is used. It can be said that an unexpected synergistic effect is exhibited.
  • Example 4 Evaluation by addition of Cyclo (Ile-Pro)
  • concentration of Cyclo (Ile-Pro) contained in the commercially available non-alcoholic beer-taste beverage was 0 ppm.
  • sensory evaluation was performed by adding Cyclo (Ile-Pro) so that the Cyclo (Ile-Pro) concentration was 0.4 ppm and 0.8 ppm, respectively. Further, the evaluation was performed with the concentration of 40 kDa protein set to 5 ppm. The results of the sensory evaluation are shown in Table 4.
  • the swelling can be enhanced by adding Cyclo (Ile-Pro) to a commercially available non-alcoholic beer-taste beverage. Further, it can be seen that the swelling can be further enhanced by further adding 40 kDa protein. From the results of the comparison sample (see Table 2), it can be seen that the swelling can be enhanced only by adding the 40 kDa protein. However, with diketopiperazine, which is obtained by the sum of the increase value (0.04) from the sensory evaluation control in the sample 13 and the increase value (0.03) from the sensory evaluation control in the comparison sample shown in Table 2.
  • the increase value (0.11) from the control of the sensory evaluation in sample 14 is larger than the additive effect (0.07) when the 40 kDa protein is used in combination, the combination of diketopiperazine and the 40 kDa protein is used. It can be said that an unexpected synergistic effect is exhibited.
  • diketopiperazine which is obtained by the sum of the increase value (0.09) from the sensory evaluation control in the sample 15 and the increase value (0.03) from the sensory evaluation control in the comparison sample shown in Table 2. Since the increase value (0.21) from the control of the sensory evaluation in sample 16 is larger than the additive effect (0.12) when the 40 kDa protein is used in combination, the combination of diketopiperazine and the 40 kDa protein is used. It can be said that an unexpected synergistic effect is exhibited.

Landscapes

  • Health & Medical Sciences (AREA)
  • Nutrition Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Non-Alcoholic Beverages (AREA)
  • Alcoholic Beverages (AREA)

Abstract

A non-alcohol beer-flavored beverage containing malt as a raw material, the beverage comprising one or more diketopiperazines selected from the group consisting of cycloleucyl proline, cyclovalyl proline, and cycloisoleucyl proline, wherein the concentration of the most abundant one of the diketopiperazines is at least 0.4 ppm.

Description

ノンアルコールビールテイスト飲料Non-alcoholic beer taste beverage
本発明は、ノンアルコールビールテイスト飲料に関する。 The present invention relates to a non-alcoholic beer-taste beverage.
近年の消費者の嗜好の多様化にともなって、様々な香味特徴をもつノンアルコールビールテイスト飲料の開発が望まれている。 With the diversification of consumer tastes in recent years, it is desired to develop non-alcoholic beer-taste beverages having various flavor characteristics.
特許文献１には、ビールテイスト飲料の香味を改善するために、特定の分子量のペプチドを含有させることが開示されている。 Patent Document 1 discloses that a peptide having a specific molecular weight is contained in order to improve the flavor of a beer-taste beverage.
特開２０１６－１４９９７５号公報Japanese Unexamined Patent Publication No. 2016-149975
特許文献１では、飲料の評価指標として、ビールらしい味わい、スムーズな味の流れ、口内に残るざらつき、といった指標を用いており、１０－２０ｋＤａのペプチドを所定濃度含有することにより、これらの指標に基づく官能評価スコアが高い飲料が得られることが記載されている。 In Patent Document 1, indexes such as beer-like taste, smooth taste flow, and roughness remaining in the mouth are used as evaluation indexes for beverages, and by containing a peptide of 10-20 kDa at a predetermined concentration, these indexes can be used. It is stated that a beverage with a high sensory evaluation score based on it can be obtained.
ここで、ビールテイスト飲料を飲んだ直後から感じられる味覚として、５基本味、すなわち、甘味、塩味、酸味、苦味及びうま味では表せない味覚で、味の強さ、広がり、厚み、味が持続する又は味の強さのバランスがとれているという特徴が好まれることがある。このような特徴を本明細書中では「ふくらみ」という。
特許文献１に記載された飲料は、ふくらみの観点からはさらに改良の余地がある飲料であるといえ、ふくらみを増強する方法が望まれていた。 Here, as the taste that can be felt immediately after drinking a beer-taste beverage, the strength, spread, thickness, and taste of the five basic tastes, that is, the tastes that cannot be expressed by sweetness, saltiness, acidity, bitterness, and umami, are maintained. Alternatively, the characteristic that the strength of taste is well-balanced may be preferred. Such a feature is referred to as "bulge" in the present specification.
It can be said that the beverage described in Patent Document 1 is a beverage that has room for further improvement from the viewpoint of swelling, and a method for enhancing swelling has been desired.
本発明は、ふくらみが増強された飲料を提供することを目的とする。特に原料に麦芽を含むノンアルコールビールテイスト飲料において、ふくらみが増強された飲料を提供することを目的とする。原料に麦芽を含むノンアルコールビールテイスト飲料は、ふくらみが乏しい傾向があるために、ふくらみを増強することが特に望まれる飲料である。 An object of the present invention is to provide a beverage having an enhanced bulge. In particular, it is an object of the present invention to provide a beverage with enhanced swelling in a non-alcoholic beer-taste beverage containing malt as a raw material. Non-alcoholic beer-taste beverages containing malt as a raw material tend to have poor swelling, and therefore it is particularly desired to enhance swelling.
すなわち、本発明は、以下のノンアルコールビールテイスト飲料に関する。
〔１〕原料に麦芽を含むノンアルコールビールテイスト飲料であって、シクロロイシルプロリン、シクロバリルプロリン及びシクロイソロイシルプロリンからなる群から選択される少なくとも１種のジケトピペラジンを含み、上記ジケトピペラジンのうち最も多く含まれるジケトピペラジンの濃度が０．４ｐｐｍ以上であるノンアルコールビールテイスト飲料。
〔２〕上記ジケトピペラジンのうち最も多く含まれるジケトピペラジンの濃度が１０ｐｐｍ以下である上記〔１〕に記載のノンアルコールビールテイスト飲料。
〔３〕さらに、分子量３５～５０ｋＤａのタンパク質を含み、上記タンパク質の濃度が５ｐｐｍ以上である上記〔１〕又は〔２〕に記載のノンアルコールビールテイスト飲料。
〔４〕上記タンパク質の濃度が３０ｐｐｍ以下である上記〔３〕に記載のノンアルコールビールテイスト飲料。 That is, the present invention relates to the following non-alcoholic beer-taste beverages.
[1] A non-alcoholic beer-taste beverage containing malt as a raw material, which contains at least one diketopiperazine selected from the group consisting of cycloleucylproline, cyclovalylproline and cycloisoleuylproline, and contains the above-mentioned diketopiperazine. A non-alcoholic beer-taste beverage in which the concentration of diketopiperazine, which is the most contained in ketopiperazine, is 0.4 ppm or more.
[2] The non-alcoholic beer-taste beverage according to [1] above, wherein the concentration of diketopiperazine contained most in the above-mentioned diketopiperazine is 10 ppm or less.
[3] The non-alcoholic beer-taste beverage according to the above [1] or [2], which further contains a protein having a molecular weight of 35 to 50 kDa and has a protein concentration of 5 ppm or more.
[4] The non-alcoholic beer-taste beverage according to [3] above, wherein the concentration of the protein is 30 ppm or less.
本発明によれば、原料に麦芽を含むノンアルコールビールテイスト飲料において、ふくらみが増強された飲料を提供することができる。 According to the present invention, it is possible to provide a non-alcoholic beer-taste beverage containing malt as a raw material with enhanced swelling.
本発明のノンアルコールビールテイスト飲料は、原料に麦芽を含む。
ここでいう原料とは、水とホップ以外の穀物原料及び糖類を意味する。
原料として麦芽の他、米、トウモロコシ、コウリャン、バレイショ、デンプン、麦芽以外の麦を含んでいてもよい。酸味料、甘味料、苦味料、調味料、香料など、微量に添加され得る成分については原料には含めない。本発明のノンアルコールビールテイスト飲料のエキス分重量は特に限定されないが、好ましくは０．０１～２０．０重量％である。 The non-alcoholic beer-taste beverage of the present invention contains malt as a raw material.
The raw material here means a grain raw material other than water and hops and sugar.
In addition to malt, rice, corn, millet, potato, starch, and wheat other than malt may be contained as a raw material. Ingredients that can be added in trace amounts, such as acidulants, sweeteners, bitterness agents, seasonings, and flavors, are not included in the raw materials. The weight of the extract content of the non-alcoholic beer-taste beverage of the present invention is not particularly limited, but is preferably 0.01 to 20.0% by weight.
ノンアルコールビールテイスト飲料とは、アルコール度数が１％未満のビールテイスト飲料であり、好ましくは、アルコールを実質的に含まない。また、アルコール度数が０％であってもよい。
ビールテイスト飲料とは、ビール風味の炭酸飲料である。
ここで、アルコールを実質的に含まない態様の飲料は、検出できない程度の極微量のアルコールを含有する飲料を除くものではない。アルコール度数が四捨五入により０．０％となる飲料、中でも、アルコール度数が四捨五入により０．００％となる飲料は、ノンアルコールビールテイスト飲料に包含される。本発明のノンアルコールビールテイスト飲料の種類としては、例えば、ノンアルコールのビールテイスト飲料、ビールテイストの清涼飲料などが含まれる。なお、ここでの「アルコール度数（アルコール含有量）」はエタノールの含有量を意味し、エタノール以外の脂肪族アルコールの含有量は含まれない。 The non-alcoholic beer-taste beverage is a beer-taste beverage having an alcohol content of less than 1%, and preferably contains substantially no alcohol. Further, the alcohol content may be 0%.
A beer-taste beverage is a beer-flavored carbonated beverage.
Here, the beverages having a substantially no alcohol content do not exclude beverages containing an undetectable trace amount of alcohol. Beverages whose alcohol content is rounded to 0.0%, particularly those whose alcohol content is rounded to 0.00%, are included in non-alcoholic beer-taste beverages. Types of non-alcoholic beer-taste beverages of the present invention include, for example, non-alcoholic beer-taste beverages, beer-taste soft drinks, and the like. The "alcohol content (alcohol content)" here means the content of ethanol, and does not include the content of aliphatic alcohols other than ethanol.
本発明にかかるノンアルコールビールテイスト飲料のアルコール度数は、飲料中のアルコール分の含有量（ｖ／ｖ％）を意味し、公知のいずれの方法によっても測定することができるが、例えば、振動式密度計によって測定することができる。具体的には、飲料から濾過又は超音波によって炭酸ガスを抜いた試料を調製し、そして、その試料を直火蒸留し、得られた留液の１５℃における密度を測定し、国税庁所定分析法（平１９国税庁訓令第６号、平成１９年６月２２日改訂）の付表である「第２表アルコール分と密度（１５℃）及び比重（１５／１５℃）換算表」を用いて換算して求めることができる。アルコール度数が１．０％未満の場合は、市販のアルコール測定装置や、ガスクロマトグラフィーを用いても良い。 The alcohol content of the non-alcoholic beer-taste beverage according to the present invention means the alcohol content (v / v%) in the beverage, and can be measured by any known method, for example, a vibration type. It can be measured by a densitometer. Specifically, a sample from which carbon dioxide gas has been removed from the beverage by filtration or ultrasonic waves is prepared, and the sample is directly distilled to measure the density of the obtained distillate at 15 ° C. Converted using the "Table 2 Alcohol Content and Density (15 ° C) and Specific Weight (15/15 ° C) Conversion Table" attached to (Heisei 19 National Tax Agency Royal Instruction No. 6, revised on June 22, 2007). Can be asked. When the alcohol content is less than 1.0%, a commercially available alcohol measuring device or gas chromatography may be used.
本発明のノンアルコールビールテイスト飲料は、シクロロイシルプロリン、シクロバリルプロリン及びシクロイソロイシルプロリンからなる群から選択される少なくとも１種のジケトピペラジンを含む。
ジケトピペラジンは、アミノ酸が二つ結合した環状のジペプチドである。
シクロロイシルプロリンはロイシンとプロリンが結合したものであり、本明細書中でＣｙｃｌｏ（Ｌｅｕ－Ｐｒｏ）と表記することがある。
シクロバリルプロリンはバリンとプロリンが結合したものであり、本明細書中でＣｙｃｌｏ（Ｖａｌ－Ｐｒｏ）と表記することがある。
シクロイソロイシルプロリンはイソロイシンとプロリンが結合したものであり、本明細書中でＣｙｃｌｏ（Ｉｌｅ－Ｐｒｏ）と表記することがある。 The non-alcoholic beer-taste beverage of the present invention contains at least one diketopiperazine selected from the group consisting of cycloleucylproline, cyclovalylproline and cycloisoroycilproline.
Diketopiperazine is a cyclic dipeptide in which two amino acids are linked.
Cycloleucylproline is a combination of leucine and proline, and is sometimes referred to as Cyclo (Leu-Pro) in the present specification.
Cyclovalylproline is a combination of valine and proline, and is sometimes referred to as Cyclo (Val-Pro) in the present specification.
Cycloisoleucine proline is a combination of isoleucine and proline, and is sometimes referred to as Cyclo (Ile-Pro) in the present specification.
本発明のノンアルコールビールテイスト飲料では、上記ジケトピペラジンのうち最も多く含まれるジケトピペラジンの濃度が０．４ｐｐｍ以上である。
これらのジケトピペラジンの濃度が０．４ｐｐｍ以上であると、ノンアルコールビールテイスト飲料におけるふくらみを増強することができる。特定のジケトピペラジンを所定濃度以上含有することとノンアルコールビールテイスト飲料におけるふくらみが関連することは、これまで知られておらず、本発明者らが見出した知見である。 In the non-alcoholic beer-taste beverage of the present invention, the concentration of diketopiperazine contained most in the above-mentioned diketopiperazine is 0.4 ppm or more.
When the concentration of these diketopiperazines is 0.4 ppm or more, the swelling in the non-alcoholic beer-taste beverage can be enhanced. It has not been known so far that the content of a specific diketopiperazine in a predetermined concentration or more is related to the swelling in a non-alcoholic beer-taste beverage, and it is a finding found by the present inventors.
本発明のノンアルコールビールテイスト飲料では、ジケトピペラジンのうち最も多く含まれるジケトピペラジンの濃度が１０ｐｐｍ以下であることが好ましい。 In the non-alcoholic beer-taste beverage of the present invention, the concentration of diketopiperazine, which is contained most in the diketopiperazine, is preferably 10 ppm or less.
また、本発明のノンアルコールビールテイスト飲料では、さらに、分子量３５～５０ｋＤａのタンパク質を含み、上記タンパク質の濃度が５ｐｐｍ以上であることが好ましい。 Further, the non-alcoholic beer-taste beverage of the present invention further contains a protein having a molecular weight of 35 to 50 kDa, and the concentration of the protein is preferably 5 ppm or more.
分子量３５～５０ｋＤａのタンパク質は、ノンアルコールビールテイスト飲料をＳＤＳ－ＰＡＧＥによる電気泳動に供した場合に分子量が３５～５０ｋＤａの領域にみられるタンパク質である。ノンアルコールビールテイスト飲料をＳＤＳ－ＰＡＧＥによる電気泳動に供する前に、例えば、前処理としてノンアルコールビールテイスト飲料に対して３０ｋＤａのカットオフ膜を用いて限外濾過を行ってもよい。
上記タンパク質は、好ましくは分子量が３５～４５ｋＤａのタンパク質であり、より好ましくは約４０ｋＤａのタンパク質である。本明細書では分子量３５～５０ｋＤａのタンパク質を４０ｋＤａタンパク質ともいう。 A protein having a molecular weight of 35 to 50 kDa is a protein found in the region having a molecular weight of 35 to 50 kDa when a non-alcoholic beer-taste beverage is subjected to electrophoresis by SDS-PAGE. Before subjecting the non-alcoholic beer-taste beverage to electrophoresis by SDS-PAGE, for example, the non-alcoholic beer-taste beverage may be subjected to ultrafiltration using a 30 kDa cut-off membrane as a pretreatment.
The protein is preferably a protein having a molecular weight of 35 to 45 kDa, more preferably a protein having a molecular weight of about 40 kDa. In the present specification, a protein having a molecular weight of 35 to 50 kDa is also referred to as a 40 kDa protein.
４０ｋＤａタンパク質は、穀類由来タンパク質であることが好ましい。
上記穀類は、大麦、小麦、トウモロコシ、イネ、大豆からなる群より選択される少なくとも１種であることが好ましい。
また、穀類が麦である場合、ノンアルコールビールテイスト飲料の製造に使用される公知の麦に由来するタンパク質を含有することができる。このような麦としては、大麦、小麦、ライ麦、カラス麦、オート麦、エン麦などが挙げられ、好ましくは大麦である。また、発芽した麦、未発芽の麦のいずれでもよいが、好ましくは発芽した麦の麦芽である。これらは、単独で含有していてもよく、２種以上を組み合わせて含有していてもよい。 The 40 kDa protein is preferably a cereal-derived protein.
The cereal is preferably at least one selected from the group consisting of barley, wheat, corn, rice and soybean.
When the cereal is wheat, it can contain a known protein derived from wheat used in the production of non-alcoholic beer-taste beverages. Examples of such wheat include barley, wheat, rye, oats, oats, and barley, and barley is preferable. Further, either germinated wheat or ungerminated wheat may be used, but germinated malt is preferable. These may be contained alone or in combination of two or more kinds.
また、４０ｋＤａタンパク質として、大麦（学名：Ｈｏｒｄｅｕｍ　ｖｕｌｇａｒｅ）由来のＳｅｒｐｉｎ　Ｚ４（別名：ＢＳＺ４、ＨｏｒｖｕＺ４、Ｍａｊｏｒ　ｅｎｄｏｓｐｅｒｍ　ａｌｂｕｍｉｎ又はＰｒｏｔｅｉｎ　Ｚ）、大麦由来のＳｅｒｐｉｎ　Ｚ７（別名：ＢＳＺ７又はＨｏｒｖｕＺ７）が好ましい。また、上記タンパク質において、そのアミノ酸配列の一部のアミノ酸が欠失、置換、挿入及び／又は付加されたアミノ酸配列を有するタンパク質であってもよい。 Further, as the 40 kDa protein, Serpin Z4 (also known as BSZ4, HorvuZ4, Major endosperm albumin or Protein Z) derived from barley (scientific name: Hordeum bulgare) and Serpin Z7 (also known as BSZ7 or HorvZ) derived from barley are preferable. Further, in the above protein, a protein having an amino acid sequence in which a part of the amino acids in the amino acid sequence is deleted, substituted, inserted and / or added may be used.
ジケトピペラジンに加えて４０ｋＤａタンパク質をさらに含有させることによって、ノンアルコールビールテイスト飲料におけるふくらみをさらに増強させることができる。
また、４０ｋＤａタンパク質の濃度は３０ｐｐｍ以下であることが好ましい。 By further containing 40 kDa protein in addition to diketopiperazine, the swelling in non-alcoholic beer-taste beverages can be further enhanced.
The concentration of the 40 kDa protein is preferably 30 ppm or less.
また、ジケトピペラジンと４０ｋＤａタンパク質をともに含有する場合には、ジケトピペラジンと４０ｋＤａタンパク質の相乗効果によりノンアルコールビールテイスト飲料のふくらみを効果的に増強させることができる。 When both diketopiperazine and 40 kDa protein are contained, the swelling of the non-alcoholic beer-taste beverage can be effectively enhanced by the synergistic effect of diketopiperazine and 40 kDa protein.
以下に、一般的なノンアルコールビールテイスト飲料の製造工程を示す。
酵母による発酵工程を有さないことにより、ノンアルコールビールテイスト飲料を容易に製造することができる。 The manufacturing process of a general non-alcoholic beer-taste beverage is shown below.
By not having a fermentation step with yeast, a non-alcoholic beer-taste beverage can be easily produced.
麦芽を原料として使用して製造されるノンアルコールビールテイスト飲料を製造する場合には、まず、麦芽等の麦の他、必要に応じて他の穀物、でんぷん、糖類、苦味料、又は着色料などの原料及び水を含む混合物に、必要に応じてアミラーゼなどの酵素を添加し、糊化、糖化を行なわせ、ろ過し、糖化液とする。必要に応じてホップや苦味料などを糖化液に加えて煮沸し、清澄タンクにて凝固タンパク質などの固形分を取り除く。この糖化液の代替として、麦芽エキスに温水を加えたものにホップを加えて煮沸してもよい。ホップは煮沸開始から煮沸終了前のどの段階で混合してもよい。糖化工程、煮沸工程、固形分除去工程などにおける条件は、知られている条件を用いればよい。煮沸後、得られた麦汁を濾過し、得られた濾過液に炭酸ガスを加える。その後、容器に充填し殺菌工程を経て目的のノンアルコールビールテイスト飲料を得る。 When producing a non-alcoholic beer-taste beverage produced using malt as a raw material, first, in addition to wheat such as malt, other grains, starch, saccharides, bitterness agents, coloring agents, etc., as necessary, etc. If necessary, an enzyme such as amylase is added to the mixture containing the raw material and water to gelatinize and saccharify, and the mixture is filtered to obtain a saccharified solution. If necessary, add hops and bitterness to the saccharified solution and boil, and remove solids such as coagulated protein in a clarification tank. As an alternative to this saccharified solution, hops may be added to a malt extract mixed with warm water and boiled. Hops may be mixed at any stage from the start of boiling to the end of boiling. As the conditions in the saccharification step, the boiling step, the solid content removing step and the like, known conditions may be used. After boiling, the obtained wort is filtered, and carbon dioxide gas is added to the obtained filtrate. After that, it is filled in a container and sterilized to obtain a desired non-alcoholic beer-taste beverage.
本発明にかかるノンアルコールビールテイスト飲料に、酒感を付与する観点から、脂肪族アルコールを添加してもよい。脂肪族アルコールとしては、公知のものであれば特に制限されないが、炭素数４～５の脂肪族アルコールが好ましい。本発明において、好ましい脂肪族アルコールとしては、炭素数４のものとして、２－メチル－１－プロパノール、１－ブタノール等が、炭素数５のものとして、３－メチル－１－ブタノール、１－ペンタノール、２－ペンタノール等が挙げられる。これらは１種又は２種以上の組み合せで用いることができる。
炭素数４～５の脂肪族アルコールの含有量は好ましくは０．０００２～０．０００７重量％であり、より好ましくは０．０００３～０．０００６重量％である。本明細書において、脂肪族アルコールの含有量は、ヘッドスペースガスクロマトグラフ法を用いて測定することができる。 Aliphatic alcohol may be added to the non-alcoholic beer-taste beverage according to the present invention from the viewpoint of imparting a feeling of alcohol. The aliphatic alcohol is not particularly limited as long as it is known, but an aliphatic alcohol having 4 to 5 carbon atoms is preferable. In the present invention, preferred aliphatic alcohols include 2-methyl-1-propanol and 1-butanol as those having 4 carbon atoms and 3-methyl-1-butanol and 1-pen as those having 5 carbon atoms. Examples thereof include tanol and 2-pentanol. These can be used in one type or a combination of two or more types.
The content of the aliphatic alcohol having 4 to 5 carbon atoms is preferably 0.0002 to 0.0007% by weight, more preferably 0.0003 to 0.0006% by weight. In the present specification, the content of the aliphatic alcohol can be measured by using a headspace gas chromatograph method.
本発明にかかるノンアルコールビールテイスト飲料については、近年の低カロリー嗜好に合わせて、低カロリーであることが望ましい。従って、本発明にかかるノンアルコールビールテイスト飲料のカロリー数は、好ましくは５ｋｃａｌ／１００ｍＬ未満、より好ましくは４ｋｃａｌ／１００ｍＬ未満、更に好ましくは３ｋｃａｌ／１００ｍＬ未満である。 The non-alcoholic beer-taste beverage according to the present invention is preferably low in calories in accordance with recent tastes for low calories. Therefore, the calorie content of the non-alcoholic beer-taste beverage according to the present invention is preferably less than 5 kcal / 100 mL, more preferably less than 4 kcal / 100 mL, still more preferably less than 3 kcal / 100 mL.
本発明にかかるノンアルコールビールテイスト飲料に含まれるカロリー数は、基本的に健康増進法に関連して公表されている「栄養表示基準における栄養成分等の分析方法等について」に従って算出する。すなわち、原則として、定量した各種栄養成分の量に、それぞれの成分のエネルギー換算係数（タンパク質：４ｋｃａｌ／ｇ、脂質：９ｋｃａｌ／ｇ、糖質：４ｋｃａｌ／ｇ、食物繊維：２ｋｃａｌ／ｇ、アルコール：７ｋｃａｌ／ｇ、有機酸：３ｋｃａｌ／ｇ）を乗じたものの総和として算出することができる。詳細は、「栄養表示基準における栄養成分等の分析方法等について」を参照されたい。 The number of calories contained in the non-alcoholic beer-taste beverage according to the present invention is basically calculated in accordance with "Analysis method of nutritional components, etc. in nutrition labeling standards" published in connection with the Health Promotion Law. That is, as a general rule, the amount of various nutritional components quantified is combined with the energy conversion coefficient of each component (protein: 4 kcal / g, lipid: 9 kcal / g, carbohydrate: 4 kcal / g, dietary fiber: 2 kcal / g, alcohol: It can be calculated as the sum of the products multiplied by 7 kcal / g and organic acid: 3 kcal / g). For details, refer to "Analysis method of nutritional components, etc. in nutrition labeling standards".
本発明にかかるノンアルコールビールテイスト飲料に含まれる各栄養成分量の具体的な測定手法は、健康増進法「栄養表示基準における栄養成分等の分析方法等について」に記載の各種分析法に従えばよい。または、財団法人日本食品分析センターに依頼すれば、このような熱量及び／又は各栄養成分量を知ることができる。 The specific method for measuring the amount of each nutritional component contained in the non-alcoholic beer-taste beverage according to the present invention is as follows according to the various analytical methods described in the health promotion method "Analysis method for nutritional components, etc. in nutrition labeling standards". good. Alternatively, if you ask the Japan Food Research Laboratories, you can know the amount of heat and / or the amount of each nutritional component.
本発明に係るノンアルコールビールテイスト飲料に含まれる糖質とは、食品の栄養表示基準（平成１５年厚生労働省告示第１７６号）に基づく糖質をいう。具体的には、糖質は、食品から、タンパク質、脂質、食物繊維、灰分、アルコール分及び水分を除いたものをいう。また、食品中の糖質の量は、当該食品の重量から、タンパク質、脂質、食物繊維、灰分及び水分の量を控除することにより算定される。この場合に、タンパク質、脂質、食物繊維、灰分及び水分の量は、栄養表示基準に掲げる方法により測定する。具体的には、タンパク質の量は窒素定量換算法で測定し、脂質の量はエーテル抽出法、クロロホルム・メタノール混液抽出法、ゲルベル法、酸分解法またはレーゼゴットリーブ法で測定し、食物繊維の量は高速液体クロマトグラフ法またはプロスキー法で測定し、灰分の量は酢酸マグネシウム添加灰化法、直接灰化法または硫酸添加灰化法で測定し、水分の量はカールフィッシャー法、乾燥助剤法、減圧過熱乾燥法、常圧加熱乾燥法またはプラスチックフィルム法で測定する。 The sugar contained in the non-alcoholic beer-taste beverage according to the present invention means a sugar based on the nutrition labeling standard for foods (Ministry of Health, Labor and Welfare Notification No. 176, 2003). Specifically, carbohydrate refers to food obtained by removing proteins, lipids, dietary fiber, ash, alcohol and water. The amount of sugar in a food is calculated by subtracting the amount of protein, lipid, dietary fiber, ash and water from the weight of the food. In this case, the amounts of protein, lipid, dietary fiber, ash and water are measured by the methods listed in the nutrition labeling standards. Specifically, the amount of protein is measured by the nitrogen quantitative conversion method, the amount of lipid is measured by the ether extraction method, the chloroform / methanol mixed solution extraction method, the Gerbel method, the acid decomposition method or the Reesegotleave method, and the amount of dietary fiber is measured. Is measured by high-speed liquid chromatograph method or Proski method, the amount of ash is measured by magnesium acetate-added ashing method, direct ashing method or sulfuric acid-added ashing method, and the amount of water is measured by Karl Fisher method, drying aid. Measure by the method, vacuum overheating drying method, normal pressure heating drying method or plastic film method.
本発明に係るノンアルコールビールテイスト飲料は、近年の低糖質嗜好に合わせて、低糖質であってもよい。従って、本発明に係るノンアルコールビールテイスト飲料の糖質の含有量は、２．５ｇ／１００ｍＬ未満であってもよく、０．５ｇ／１００ｍＬ未満であってもよい。また、下限は特に設定されないが、通常、０．１ｇ／１００ｍＬ程度であり、例えば、０．１５ｇ／１００ｍＬ以上であっても、０．２ｇ／１００ｍＬ以上であってもよい。 The non-alcoholic beer-taste beverage according to the present invention may be low-carbohydrate in accordance with the recent taste for low-carbohydrate. Therefore, the sugar content of the non-alcoholic beer-taste beverage according to the present invention may be less than 2.5 g / 100 mL or less than 0.5 g / 100 mL. Although the lower limit is not particularly set, it is usually about 0.1 g / 100 mL, and may be, for example, 0.15 g / 100 mL or more or 0.2 g / 100 mL or more.
本発明にかかるノンアルコールビールテイスト飲料は、酸味料を含んでいてもよい。酸味料としては、クエン酸、乳酸、リン酸、及びリンゴ酸からなる群より選ばれる１種以上の酸を用いることが好ましい。また、本発明においては、上記酸以外の酸として、コハク酸、酒石酸、フマル酸および氷酢酸等も用いることができる。これらは食品に添加することが認められているものであれば制限なく用いることができる。本発明においては、まろやかな酸味を適切に付与する観点から乳酸と、やや刺激感のある酸味を適切に付与する観点からリン酸との組み合わせを用いることが好ましい。 The non-alcoholic beer-taste beverage according to the present invention may contain an acidulant. As the acidulant, it is preferable to use one or more acids selected from the group consisting of citric acid, lactic acid, phosphoric acid, and malic acid. Further, in the present invention, succinic acid, tartaric acid, fumaric acid, glacial acetic acid and the like can also be used as acids other than the above acids. These can be used without limitation as long as they are approved to be added to foods. In the present invention, it is preferable to use a combination of lactic acid from the viewpoint of appropriately imparting a mild acidity and phosphoric acid from the viewpoint of appropriately imparting a slightly irritating acidity.
酸味料の含有量は、本発明にかかるノンアルコールビールテイスト飲料中、クエン酸換算で、ビールテイスト感の付与の観点から、２００ｐｐｍ以上が好ましく、５５０ｐｐｍ以上がより好ましく、７００ｐｐｍ以上がさらに好ましく、また、酸味の観点から、１５０００ｐｐｍ以下が好ましく、５５００ｐｐｍ以下がより好ましく、２０００ｐｐｍ以下がさらに好ましい。従って、本発明において、酸味料の含有量は、クエン酸換算で、２００ｐｐｍ～１５０００ｐｐｍ、好ましくは５５０ｐｐｍ～５５００ｐｐｍ、より好ましくは７００ｐｐｍ～１５００ｐｐｍなどの好適範囲が挙げられる。なお、本明細書において、クエン酸換算量とは、クエン酸の酸味度を基準として各酸味料の酸味度から換算される量のことであり、例えば、乳酸１００ｐｐｍに相当するクエン酸換算量は１２０ｐｐｍ、リン酸１００ｐｐｍに相当するクエン酸換算量は２００ｐｐｍ、リンゴ酸１００ｐｐｍに相当するクエン酸換算量は１２５ｐｐｍとして換算する。 The content of the acidulant in the non-alcoholic beer-taste beverage according to the present invention is preferably 200 ppm or more, more preferably 550 ppm or more, still more preferably 700 ppm or more, and further preferably 700 ppm or more, in terms of citric acid, from the viewpoint of imparting a beer-like feeling. From the viewpoint of acidity, 15,000 ppm or less is preferable, 5500 ppm or less is more preferable, and 2000 ppm or less is further preferable. Therefore, in the present invention, the content of the acidulant may be in a suitable range of 200 ppm to 15000 ppm, preferably 550 ppm to 5500 ppm, more preferably 700 ppm to 1500 ppm in terms of citric acid. In the present specification, the citric acid conversion amount is an amount converted from the acidity of each acidulant based on the acidity of citric acid, and for example, the citric acid conversion amount corresponding to 100 ppm of lactic acid is used. The citric acid equivalent amount corresponding to 120 ppm and 100 ppm of phosphoric acid is converted as 200 ppm, and the citric acid equivalent amount corresponding to 100 ppm of apple acid is converted as 125 ppm.
ノンアルコールビールテイスト飲料中の酸味料の含有量については、高速液体クロマトグラフィー（ＨＰＬＣ）等により分析して算出されたものを指す。 The content of the acidulant in the non-alcoholic beer-taste beverage refers to the one calculated by analysis by high performance liquid chromatography (HPLC) or the like.
本発明に係るノンアルコールビールテイスト飲料においては、原料の一部にホップを用いることができる。
ホップを使用する際には、ビール等の製造に使用される通常のペレットホップ、粉末ホップ、ホップエキスを、所望の香味に応じて適宜選択して使用することができる。また、イソ化ホップ、還元ホップなどのホップ加工品を用いてもよい。本発明に係るノンアルコールビールテイスト飲料に使用されるホップには、これらのものが包含される。また、ホップの添加量は特に限定されないが、典型的には、飲料全量に対して０．０００１～１重量％程度である。 In the non-alcoholic beer-taste beverage according to the present invention, hops can be used as a part of the raw material.
When hops are used, ordinary pellet hops, powdered hops, and hop extracts used in the production of beer and the like can be appropriately selected and used according to the desired flavor. Further, processed hop products such as isometric hops and reduced hops may be used. These are included in the hops used in the non-alcoholic beer-taste beverage according to the present invention. The amount of hops added is not particularly limited, but is typically about 0.0001 to 1% by weight based on the total amount of the beverage.
本発明に係るノンアルコールビールテイスト飲料は、本発明の効果を妨げない範囲で、必要に応じて、その他の原料を用いてもよい。例えば、甘味料（高甘味度甘味料を含む）、苦味料、香料、酵母エキス、カラメル色素などの着色料、大豆サポニンやキラヤサポニン等の植物抽出サポニン系物質、コーンや大豆などの植物タンパク質およびペプチド含有物、ウシ血清アルブミン等のタンパク質系物質、食物繊維やアミノ酸などの調味料、アスコルビン酸等の酸化防止剤を、本発明の効果を妨げない範囲で必要に応じて用いることができる。 The non-alcoholic beer-taste beverage according to the present invention may use other raw materials as needed, as long as the effects of the present invention are not impaired. For example, sweeteners (including high-sweetness sweeteners), bitterness agents, flavors, yeast extracts, colorants such as caramel pigments, plant-extracted saponin-based substances such as soybean saponin and kiraya saponin, plant proteins such as corn and soybean, and Peptide-containing substances, protein-based substances such as bovine serum albumin, seasonings such as dietary fiber and amino acids, and antioxidants such as ascorbic acid can be used as needed as long as they do not interfere with the effects of the present invention.
本発明に係るノンアルコールビールテイスト飲料は、容器詰めとすることができる。容器の形態は何ら制限されず、ビン、缶、樽、またはペットボトル等の密封容器に充填して、容器入り飲料とすることができる。 The non-alcoholic beer-taste beverage according to the present invention can be packed in a container. The form of the container is not limited in any way, and it can be filled in a sealed container such as a bottle, a can, a barrel, or a PET bottle to make a beverage in a container.
本発明のノンアルコールビールテイスト飲料の製造方法は、特に限定されるものではないが、原料に麦芽を含むノンアルコールビールテイスト飲料に対して、ジケトピペラジンを所定量添加する方法が例示される。
また、原料に麦芽を含むノンアルコールビールテイスト飲料に対して、４０ｋＤａタンパク質を添加することが好ましい。
添加するジケトピペラジン及び４０ｋＤａタンパク質の調製は例えば後述する実施例に記載の手順で行うことができる。
また、ジケトピペラジン及び４０ｋＤａタンパク質については、ノンアルコールビールテイスト飲料の製造過程における諸条件を調整することによって、これらの含有量が増えるようにしてもよい。 The method for producing a non-alcoholic beer-taste beverage of the present invention is not particularly limited, and an example thereof is a method in which a predetermined amount of diketopiperazine is added to a non-alcoholic beer-taste beverage containing malt as a raw material.
Further, it is preferable to add 40 kDa protein to a non-alcoholic beer-taste beverage containing malt as a raw material.
The diketopiperazine and 40 kDa protein to be added can be prepared, for example, by the procedure described in Examples described later.
Further, regarding diketopiperazine and 40 kDa protein, the content thereof may be increased by adjusting various conditions in the manufacturing process of the non-alcoholic beer-taste beverage.
以下、実施例を示して本発明を具体的に説明するが、本発明は下記実施例に制限されるものではない。 Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to the following Examples.
（ジケトピペラジンの精製）
下記に従いジケトピペラジンの精製を行った。
（１）ビールのＨＰ２０による分画
６０Ｌのビールを、１０Ｌのダイヤイオン(登録商標)ＨＰ２０（三菱ケミカル株式会社製）を用いて分画した。ＨＰ２０は使用前に、エタノールにより３回洗浄し、次いで５０％エタノールにより３回洗浄した。洗浄したＨＰ２０を大量分画用カラムへ充填し、水により置換した。脱気させた６０Ｌのビールへ同量の蒸留水を混合し、中圧ポンプを用いてＨＰ２０カラムへ流した。ＨＰ２０カラムを素通りした溶液を素通り画分として得た。中圧ポンプを用いて４０Ｌの蒸留水を流し、溶出液を水溶出画分として得た。同様に、含水エタノール（１０％エタノール、３０％エタノール、および７０％エタノール）を４０Ｌずつ流し、溶出液をそれぞれ１０％エタノール溶出画分、３０％エタノール溶出画分、および７０％エタノール溶出画分として得た。それぞれの溶出画分は、エバポレーターおよび凍結乾燥機を用いて、乾燥体として冷蔵保存した。 (Purification of diketopiperazine)
Diketopiperazine was purified according to the following.
(1) Fractionation of beer by HP20 60 L of beer was fractionated using 10 L of Diaion (registered trademark) HP20 (manufactured by Mitsubishi Chemical Corporation). HP20 was washed 3 times with ethanol and then 3 times with 50% ethanol before use. The washed HP20 was filled in a mass fractionation column and replaced with water. The same amount of distilled water was mixed with 60 L of degassed beer and flowed to the HP20 column using a medium pressure pump. A solution that passed through the HP20 column was obtained as a passing fraction. 40 L of distilled water was flowed using a medium pressure pump to obtain an eluate as a water-eluting fraction. Similarly, 40 L of hydrous ethanol (10% ethanol, 30% ethanol, and 70% ethanol) was poured, and the eluate was used as a 10% ethanol elution fraction, a 30% ethanol elution fraction, and a 70% ethanol elution fraction, respectively. Obtained. Each eluted fraction was refrigerated as a dried product using an evaporator and a lyophilizer.
（２）３０％エタノール溶出画分のＬＨ－２０分画
ＨＰ２０分画物のうち、３０％エタノール溶出画分について、１．２ｋｇのＳｅｐｈａｄｅｘ(登録商標)ＬＨ－２０を用いて分画した。エタノールにより洗浄したＬＨ－２０を大量分画用カラムへ充填し、水により置換した。ＨＰ２０分画により得られた３０％エタノール溶出画分（８７．９ｇ）のうち、１７．６ｇを蒸留水に溶解させ、ＬＨ－２０カラムへアプライした。中圧ポンプを用いて１３．５Ｌの蒸留水を流し、水溶出画分－１～６を得た。次いで、含水エタノール（３５％エタノール、７０％エタノール、および１００％エタノール）を７Ｌずつ流し、溶出液をそれぞれ３５％エタノール溶出画分、７０％エタノール溶出画分、および１００％エタノール溶出画分として得た。それぞれの溶出画分は、エバポレーターおよび凍結乾燥機を用いて、乾燥体として冷蔵保存した。 (2) LH-20 Fraction of 30% Ethanol Elution Fraction Of the HP20 fractions, the 30% ethanol-eluting fraction was fractionated using 1.2 kg of Sephadex® LH-20. LH-20 washed with ethanol was filled in a mass fractionation column and replaced with water. Of the 30% ethanol-eluted fraction (87.9 g) obtained by the HP 20 fraction, 17.6 g was dissolved in distilled water and applied to the LH-20 column. 13.5 L of distilled water was flowed using a medium pressure pump to obtain water elution fractions -1 to 6. Then, 7 L of hydrous ethanol (35% ethanol, 70% ethanol, and 100% ethanol) was poured, and the eluate was obtained as a 35% ethanol elution fraction, a 70% ethanol elution fraction, and a 100% ethanol elution fraction, respectively. rice field. Each eluted fraction was refrigerated as a dried product using an evaporator and a lyophilizer.
（３）ジケトピペラジンの分離
ＬＨ－２０分画により得られた水溶出画分－３（１．０４ｇ）のうち８６．７ｍｇについて、ＨＰＬＣ（ＣＯＳＭＯＳＩＬ　５Ｃ１８－ＰＡＱ，２０×２５０ｍｍ）を用いて、１０％エタノールにより溶出させた。次いで、１５ｍｉｎから２０ｍｉｎの溶出液を濃縮し、ＨＰＬＣ（ＣＯＳＭＯＳＩＬ　５Ｃ１８－ＰＡＱ，２０×２５０ｍｍ）を用いて、エタノール－水（５：９５→２０：８０）の濃度勾配の混液により溶出させ、化合物１（６．７ｍｇ，ｔＲ＝２５ｍｉｎ）を得た。同様に、２７．５ｍｉｎから４０ｍｉｎの溶出液を濃縮し、ＨＰＬＣ（ＣＯＳＭＯＳＩＬ　５Ｃ１８－ＰＡＱ，２０×２５０ｍｍ）を用いて、エタノール－水（５：９５→２０：８０）の濃度勾配の混液により溶出させ、化合物２（２．２ｍｇ，ｔＲ＝４３ｍｉｎ）および化合物３（２．６ｍｇ，ｔＲ＝４６ｍｉｎ）を得た。
化合物１は、ＭＳ、ＮＭＲの物理学的データの解析および文献値によりＣｙｃｌｏ（Ｐｒｏ－Ｖａｌ）であると同定した。化合物２は、ＭＳ、ＮＭＲの物理学的データの解析および文献値によりＣｙｃｌｏ（Ｉｌｅ－Ｐｒｏ）であると同定した。化合物３は、ＭＳ、ＮＭＲの物理学的データの解析および文献値によりＣｙｃｌｏ（Ｌｅｕ－Ｐｒｏ）であると同定した。
参照した参考文献は、Ｊ．　Ａｇｒｉｃ．　Ｆｏｏｄ　Ｃｈｅｍ．　２００７，５５，７５－７９．である。
使用した分析機器は以下の通りである。
ＬＣ－ＭＳ；Ｑ　Ｅｘａｃｔｉｖｅ，Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製
ＮＭＲ；ＡＶＡＮＣＥ４００，Ｂｒｕｋｅｒ社製 (3) Separation of Diketopiperazine 86.7 mg of the water-eluted fraction-3 (1.04 g) obtained by the LH-20 fraction was used by HPLC (COSMOSIL 5C18-PAQ, 20 × 250 mm). It was eluted with 10% ethanol. Then, the eluate from 15 min to 20 min was concentrated and eluted with a mixed solution of ethanol-water (5:95 → 20:80) using HPLC (COSMOSIL 5C18-PAQ, 20 × 250 mm) to compound 1. (6.7 mg, tR = 25 min) was obtained. Similarly, the eluate from 27.5 min to 40 min is concentrated and eluted with a mixed solution of ethanol-water (5:95 → 20:80) using HPLC (COSMOSIL 5C18-PAQ, 20 × 250 mm). , Compound 2 (2.2 mg, tR = 43 min) and Compound 3 (2.6 mg, tR = 46 min) were obtained.
Compound 1 was identified as Cyclo (Pro-Val) by analysis of physical data of MS and NMR and literature values. Compound 2 was identified as Cyclo (Ile-Pro) by analysis of physical data of MS and NMR and literature values. Compound 3 was identified as Cyclo (Leu-Pro) by analysis of physical data of MS and NMR and literature values.
The references referred to are J. Mol. Agric. Food Chem. 2007,55,75-79. Is.
The analytical instruments used are as follows.
LC-MS; Q Active, Thermo Fisher Scientific NMR; AVANCE400, Bruker
（４０ｋＤａタンパク質の精製）
市販のビール１Ｌから下記に従い４０ｋＤａタンパク質の精製を行った。 (Purification of 40 kDa protein)
40 kDa protein was purified from 1 L of commercially available beer according to the following.
（１）陽イオン交換樹脂による分画
陽イオン交換樹脂ＳＰ　Ｓｅｐｈａｒｏｓｅ５０ｍＬを空きカラムに入れた。ビールを樹脂に吸着させた。その後、樹脂をカラムに移し替え、２０ｍＭ酢酸ナトリウム緩衝液（ｐＨ４．５）で洗浄した。次いで、２０ｍＭ酢酸ナトリウム（ｐＨ４．５）＋０．５Ｍ－ＮａＣｌで溶出し画分を集めた。得られた画分をＳＤＳ－ＰＡＧＥで評価し、４０ｋＤａのタンパク質が含まれる画分を集め陽イオン交換樹脂結合画分とした。 (1) Fractionation by cation exchange resin 50 mL of cation exchange resin SP Sepharose was placed in an empty column. Beer was adsorbed on the resin. Then, the resin was transferred to a column and washed with 20 mM sodium acetate buffer (pH 4.5). Then, it was eluted with 20 mM sodium acetate (pH 4.5) + 0.5M-NaCl and fractions were collected. The obtained fraction was evaluated by SDS-PAGE, and the fraction containing a protein of 40 kDa was collected and used as a cation exchange resin bound fraction.
（２）限外濾過（バッファー交換）
水洗浄した限外ろ過ユニット（Ｍｅｒｃｋ製　Ａｍｉｃｏｎ　Ｕｌｔｒａ－１５　３０Ｋ）に（１）で得た陽イオン交換樹脂結合画分を１０ｍＬずつ添加し、３５００ｒｐｍで遠心し限外ろ過し濃縮液を得た。 (2) Extrafiltration (buffer exchange)
To a water-washed ultrafiltration unit (Amicon Ultra-15 30K manufactured by Merck), 10 mL each of the cation exchange resin-bound fraction obtained in (1) was added, and the mixture was centrifuged at 3500 rpm for ultrafiltration to obtain a concentrated solution.
（３）硫安分画
２０ｍＭリン酸バッファー（ｐＨ７．０）＋２Ｍ硫酸アンモニウムをビーカーに入れ、（２）で得た濃縮液を滴下、撹拌した。次に懸濁液を遠心（２３３０ｇ、１０分間、室温）した。上清を別容器に集めた。集めた溶液は限外ろ過ユニットを用いて濃縮した。濃縮液を２０ｍＭ酢酸ナトリウム（ｐＨ４．５）に加えて遠心（２３３０ｇ、１０分間、室温）し、濃縮を行い、４０ｋＤａタンパク質精製品（Ｂｒａｄｆｏｒｄ定量（ウシ血清アルブミン（ＢＳＡ）換算）、２０．４ｍｇ／ｍＬ、２．２１ｍＬ）を得た。得られた４０ｋＤａタンパク質の純度はＳＤＳ－ＰＡＧＥで確認した。 (3) Ammonium sulfate fraction 20 mM phosphate buffer (pH 7.0) + 2M ammonium sulfate was placed in a beaker, and the concentrate obtained in (2) was added dropwise and stirred. The suspension was then centrifuged (2330 g, 10 minutes, room temperature). The supernatant was collected in a separate container. The collected solution was concentrated using an ultrafiltration unit. The concentrate is added to 20 mM sodium acetate (pH 4.5), centrifuged (2330 g, 10 minutes, room temperature), concentrated, and 40 kDa protein assay (Bradford quantification (bovine serum albumin (BSA) equivalent), 20.4 mg / mL, 2.21 mL) was obtained. The purity of the obtained 40 kDa protein was confirmed by SDS-PAGE.
４０ｋＤａタンパク質を酵素で消化後、ＬＣ－ＭＳ／ＭＳで分析することにより、タンパク質の同定を試みた。
ＳＤＳ－ＰＡＧＥで分離した４０ｋＤａ付近のバンドを切り出し、ジチオスレイトールによる還元（５６℃、１時間）、ヨードアセトアミドによるカルバミドメチル化（遮光下，室温，４５分間）を行った。次いで０．０１％ＰｒｏｔｅａｓｅＭａｘ含有１０ｎｇ／μＬキモトリプシン溶液（５ｍＭ塩化カルシウム，５０ｍＭ炭酸水素アンモニウム溶液）１５μＬ、５ｍＭ塩化カルシウム、５０ｍＭ炭酸水素アンモニウム溶液１５μＬを添加し一晩インキュベートした後、酵素消化液を回収した。回収した溶液を減圧乾固し、０．１％ギ酸溶液に再溶解した。
これをＬＣ－ＭＳ／ＭＳ分析に使用した。 An attempt was made to identify the protein by digesting the 40 kDa protein with an enzyme and then analyzing it by LC-MS / MS.
Bands around 40 kDa separated by SDS-PAGE were excised, reduced with dithiothreitol (56 ° C., 1 hour), and carbamid methylation with iodoacetamide (light-shielded, room temperature, 45 minutes). Then, 15 μL of 10 ng / μL chymotrypsin solution (5 mM calcium chloride, 50 mM ammonium hydrogencarbonate solution) containing 0.01% ProteinMax, 15 μL of 5 mM calcium chloride, and 15 μL of 50 mM ammonium hydrogencarbonate solution were added and incubated overnight, and then the enzyme digestive juice was recovered. .. The recovered solution was dried under reduced pressure and redissolved in a 0.1% formic acid solution.
This was used for LC-MS / MS analysis.
（ＬＣ－ＭＳ／ＭＳによる測定）
ＬＣ－ＭＳ／ＭＳの測定は下記の条件で行った。
使用装置：ダイレクトフローｎａｎｏＬＣシステムＥａｓｙ－ｎＬＣ　１０００ＴＭ　（Ｔｈｅｒｍｏ　Ｓｃｉｅｎｔｉｆｉｃ）
トラップカラム：Ａｃｃｌａｉｍ　ＰｅｐＭａｐ（登録商標）（Ｔｈｅｒｍｏ　Ｓｃｉｅｎｔｉｆｉｃ）
分析カラム：ＮＡＮＯ　ＨＰＬＣ　ＣＡＰＩＬＬＡＲＹ　ＣＯＬＵＭＮ（日京テクノス（株））
液体クロマトグラフ質量分析計　Ｑ　Ｅｘａｃｔｉｖｅ　Ｐｌｕｓ（Ｔｈｅｒｍｏ　Ｓｃｉｅｎｔｉｆｉｃ）
移動相：Ａ液：０．１％ギ酸／水、Ｂ液：０．１％ギ酸／アセトニトリル
流速：３００ｎＬ／ｍｉｎ
グラジエント：０－４０％Ｂ／０－３０ｍｉｎ、４０－６０％Ｂ／３０－３５ｍｉｎ、６０－９０％Ｂ／３５－３７ｍｉｎ、９０％Ｂ／３７－４５ｍｉｎ
注入量：１０μＬ
イオン化モード：ＥＳＩ　Ｐｏｓｉｔｉｖｅ
測定範囲：ＭＳ１（ｍ／ｚ　３５０－１７５０）
Ｄａｔａ　Ｄｅｐｅｎｄｅｎｔ　Ｓｃａｎモード (Measurement by LC-MS / MS)
The LC-MS / MS measurement was performed under the following conditions.
Equipment used: Direct flow nanoLC system Easy-nLC 1000TM (Thermo Scientific)
Trap column: Acclim PepMap® (Thermo Scientific)
Analytical column: NANO HPLC CAPILLARY COLUMN (Nikkyo Technos Co., Ltd.)
Liquid Chromatograph Mass Spectrometer Q Active Plus (Thermo Scientific)
Mobile phase: Liquid A: 0.1% formic acid / water, liquid B: 0.1% formic acid / acetonitrile Flow velocity: 300 nL / min
Radiant: 0-40% B / 0-30min, 40-60% B / 30-35min, 60-90% B / 35-37min, 90% B / 37-45min
Injection volume: 10 μL
Ionization mode: ESI Positive
Measurement range: MS1 (m / z 350-1750)
Data Dependent Scan mode
（４）タンパク質の解析
タンパク質同定は下記の条件で行った。
検索ソフト：Ｐｒｏｔｅｏｍｅ　Ｄｉｓｃｏｖｅｒｅｒ　２．２．０．３８８（ＴｈｅｒｍｏＦｉｓｈｅｒ社製）
生物種：大麦（Ｈｏｒｄｅｕｍ　ｖｕｌｇａｒｅ）、ホップ（Ｈｕｍｕｌｕｓ）、酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ）
検索条件：消化酵素：Ｃｈｙｍｏｔｒｙｐｓｉｎ
プリカーサーイオン質量誤差範囲：Ｍｏｎｏｉｓｏｔｏｐｉｃ、±１０ｐｐｍ
プロダクトイオン質量誤差範囲：±０．０２Ｄａ
最大ミスクリベージ数：５
コンフィデンスレベル（Ｐｅｒｃｏｌａｔｏｒ）：Ｈｉｇｈ（確からしさ３段階のうち最も確率が高いレベル）
データベース：ＳｗｉｓｓＰｒｏｔ (4) Protein analysis Protein identification was performed under the following conditions.
Search software: Proteome Discoverer 2.2.0.3888 (manufactured by Thermo Fisher)
Species: Barley (Hordeum vulgare), Hops (Humulus), Yeast (Saccharomyces cerevisiae)
Search condition: Digestive enzyme: Chymotrypsin
Precursor ion mass error range: Monoisotopic, ± 10 ppm
Product ion mass error range: ± 0.02 Da
Maximum number of missed cribes: 5
Confidence level (Percolator): High (the most probable level of the three levels of certainty)
Database: SwissProt
その結果、４０ｋＤａタンパク質は大麦由来のＳｅｒｐｉｎ　Ｚ４（配列カバー率：７７．２％）及び大麦由来のＳｅｒｐｉｎ　Ｚ７（配列カバー率：７２．８％）であることがわかった。 As a result, it was found that the 40 kDa protein was Serpin Z4 derived from barley (sequence coverage: 77.2%) and Serpin Z7 derived from barley (sequence coverage: 72.8%).
（市販のノンアルコールビールテイスト飲料にジケトピペラジンを添加した場合の官能評価）
市販のノンアルコールビールテイスト飲料に、ジケトピペラジンを添加し、ふくらみの官能評価を行った。
当該ノンアルコールビールテイスト飲料は、原料に麦芽を含むノンアルコールビールテイスト飲料である。
原材料は麦芽、ホップ、炭酸、香料、酸味料、カラメル色素、ビタミンＣ、苦味料、甘味料であり、栄養成分として１００ｍｌあたりアルコール分０％、タンパク質０ｇ、糖質０ｇ、食物繊維０～０．１ｇ、プリン体約０ｍｇを含む。 (Sensory evaluation when diketopiperazine is added to a commercially available non-alcoholic beer-taste beverage)
Diketopiperazine was added to a commercially available non-alcoholic beer-taste beverage, and the sensory evaluation of swelling was performed.
The non-alcoholic beer-taste beverage is a non-alcoholic beer-taste beverage containing malt as a raw material.
The raw materials are malt, hops, carbonic acid, flavor, acidulant, caramel color, vitamin C, bitterness, and sweetener. As nutritional components, alcohol content is 0%, protein is 0 g, sugar is 0 g, and dietary fiber is 0 to 0. Contains 1 g and about 0 mg of purine.
官能評価の基準点は以下の通りである。
専門パネル５名により０．０５点刻みで下記の基準によりスコア化、そのスコア値を平均化した。
ふくらみ強度は以下の基準である。
０点：全く感じられない
１点：やや感じられる
２点：明確に感じる
３点：非常に感じる
基準点として、評価対象とする上記市販のノンアルコールビールテイスト飲料とは異なる市販のビールテイストアルコール飲料を基準のビールテイストアルコール飲料（Ｉ）としてふくらみを０．７点とした。また、他の市販のビールテイストアルコール飲料を基準のビールテイストアルコール飲料（ＩＩ）としてそのふくらみを基準点の１．５点とした。また、上記の市販のビールテイストアルコール飲料（Ｉ）及び（ＩＩ）のふくらみを基準として、評価対象とする上記市販のノンアルコールビールテイスト飲料のふくらみを０．５点とした。
当該基準のビールテイストアルコール飲料（Ｉ）は原料中の麦芽の比率が０重量％を超え、５０重量％未満であるビールテイストアルコール飲料である。
原材料は発泡酒、麦芽、ホップ、糖類、食物繊維、スピリッツ（小麦）であり、栄養成分として１００ｍｌあたりアルコール分４％、タンパク質０～０．２ｇ、糖質０．５～０．８ｇ、プリン体約２．０ｍｇを含む。
当該基準のビールテイストアルコール飲料（ＩＩ）は原料中の麦芽の比率が５０重量％以上であるビールテイストアルコール飲料である。
原材料は麦芽、ホップであり、栄養成分として１００ｍｌあたりアルコール分５．５％、タンパク質０．４～０．６ｇ、糖質３．６ｇ、プリン体約１２．５ｍｇを含む。 The reference points for sensory evaluation are as follows.
Five expert panels scored in increments of 0.05 points according to the following criteria, and the score values were averaged.
The bulge strength is based on the following criteria.
0 points: Not felt at all 1 point: Slightly felt 2 points: Clearly felt 3 points: As a very felt reference point, a commercially available beer-taste alcoholic beverage different from the above-mentioned commercially available non-alcoholic beer-taste beverage to be evaluated As a standard beer-taste alcoholic beverage (I), the swelling was set to 0.7 points. Further, another commercially available beer-taste alcoholic beverage was used as the standard beer-taste alcoholic beverage (II), and its bulge was set to 1.5 points as the reference point. Further, based on the swelling of the above-mentioned commercially available beer-taste alcoholic beverages (I) and (II), the swelling of the above-mentioned commercially available non-alcoholic beer-taste beverage to be evaluated was set to 0.5 points.
The standard beer-taste alcoholic beverage (I) is a beer-taste alcoholic beverage in which the proportion of malt in the raw material exceeds 0% by weight and is less than 50% by weight.
The raw materials are low-malt beer, malt, hops, sugars, dietary fiber, and spirits (wheat). As nutritional components, alcohol content is 4%, protein is 0 to 0.2 g, sugar is 0.5 to 0.8 g, and purine is used. Contains about 2.0 mg.
The standard beer-taste alcoholic beverage (II) is a beer-taste alcoholic beverage in which the ratio of malt in the raw material is 50% by weight or more.
The raw materials are malt and hops, and the nutritional components include 5.5% alcohol, 0.4 to 0.6 g of protein, 3.6 g of sugar, and about 12.5 mg of purines per 100 ml.
官能評価の手順は以下の通りである。
（１）ノンアルコールビールテイスト飲料を最終容量の１／１０容量（ｖ／ｖ）バイアル瓶に分注する
（２）ジケトピペラジンを任意の重量で秤量して加える
（３）３０秒ソニケーションする
（４）３０分室温で静置する
（５）ノンアルコールビールテイスト飲料を最終容量にフィルアップする
（６）分注して飲み込み評価する The procedure for sensory evaluation is as follows.
(1) Dispense non-alcoholic beer-taste beverage into 1/10 volume (v / v) vial of final volume (2) Weigh and add diketopiperazine to any weight (3) Sonic for 30 seconds (4) Let stand at room temperature for 30 minutes (5) Fill up the non-alcoholic beer-taste beverage to the final volume (6) Dispense and evaluate by swallowing
（市販のノンアルコールビールテイスト飲料の分析）
官能評価に使用した市販のノンアルコールビールテイスト飲料に含まれるジケトピペラジンの濃度を以下の手順によりＬＣ－ＭＳで定量した。
（１）標品の調製及び検量線の作製
ジケトピペラジンについて、それぞれ下記の濃度となるように希釈し、０．２２μｍのフィルターに通してから測定に供した。
最終濃度：０．００１ｐｐｍ，０．０２５ｐｐｍ，０．０５０ｐｐｍ，０．１００ｐｐｍ，０．２００ｐｐｍ，０．３００ｐｐｍ，０．５００ｐｐｍ，０．７５０ｐｐｍ，１．０００ｐｐｍ
（１ｐｐｍ＝１μｇ／ｍＬである）
希釈液には、５％（ｖ／ｖ）のエタノール水溶液を用いた。
なお、標品の分析結果において、検量線の直線性が保たれる範囲（Ｒ２＞０．９９）に測定値が入るような希釈倍率の測定値を採用した。 (Analysis of commercially available non-alcoholic beer-taste beverages)
The concentration of diketopiperazine contained in the commercially available non-alcoholic beer-taste beverage used for the sensory evaluation was quantified by LC-MS by the following procedure.
(1) Preparation of standard and preparation of calibration curve Diketopiperazine was diluted to the following concentrations, passed through a 0.22 μm filter, and then subjected to measurement.
Final concentration: 0.001 ppm, 0.025 ppm, 0.050 ppm, 0.100 ppm, 0.200 ppm, 0.300 ppm, 0.500 ppm, 0.750 ppm, 1.000 ppm
(1 ppm = 1 μg / mL)
A 5% (v / v) aqueous ethanol solution was used as the diluent.
Note that in the analysis of a preparation was employed measurements of the dilution ratio as the measured value is in the range (R 2> 0.99) the linearity of the calibration curve is maintained.
ＬＣ－ＭＳの測定条件は以下の通りである。
ＬＣ－ＭＳ：エービー・サイエックス社製Ｘ５００Ｒ
分離カラム：Ｗａｔｅｒｓ社製　ＨＳＳＴ３　１．８μｍ，２．１ｘ１５０ｍｍ
溶離液：
Ａ液：０．１％ギ酸／水、Ｂ液：０．１％ギ酸／アセトニトリル
グラジエント：Ａ液：Ｂ液＝９８：２→２：９８（２７ｍｉｎ）
注入量：５μＬ
流速：０．２ｍＬ／ｍｉｎ
カラムオーブン：４０℃
（ＭＳ）
イオン化モード：ＥＳＩ　Ｐｏｓｉｔｉｖｅ
測定範囲：ＭＳ１（ｍ／ｚ　１００－１０００）
Ｄａｔａ　Ｉｎｄｅｐｅｎｄｅｎｔ　Ｓｃａｎモード
イオン源温度：３５０℃ The measurement conditions for LC-MS are as follows.
LC-MS: X500R manufactured by AB SIX Co., Ltd.
Separation column: Waters HSST3 1.8 μm, 2.1x150 mm
Eluent:
Liquid A: 0.1% formic acid / water, liquid B: 0.1% formic acid / acetonitrile gradient: liquid A: liquid B = 98: 2 → 2:98 (27 min)
Injection volume: 5 μL
Flow rate: 0.2 mL / min
Column oven: 40 ° C
(MS)
Ionization mode: ESI Positive
Measurement range: MS1 (m / z 100-1000)
Data Independent Scan mode Ion source temperature: 350 ° C
（２）市販のノンアルコールビールテイスト飲料からの測定用試料の調製
市販のノンアルコールビールテイスト飲料をソニケーションにより脱気し、気泡が落ち着いてから適宜希釈し、０．２２μｍのフィルターに通してから測定に供した。
希釈液には、５％（ｖ／ｖ）のエタノール水溶液を用いた。
市販のノンアルコールビールテイスト飲料に含まれる各ジケトピペラジンの濃度をコントロールとした。 (2) Preparation of measurement sample from commercially available non-alcoholic beer-taste beverages The commercially available non-alcoholic beer-taste beverages are degassed by sonication, diluted appropriately after the bubbles have settled, and passed through a 0.22 μm filter. It was used for measurement.
A 5% (v / v) aqueous ethanol solution was used as the diluent.
The concentration of each diketopiperazine contained in a commercially available non-alcoholic beer-taste beverage was controlled.
（実施例１：Ｃｙｃｌｏ（Ｌｅｕ－Ｐｒｏ）添加による評価）
市販のノンアルコールビールテイスト飲料に含まれるＣｙｃｌｏ（Ｌｅｕ－Ｐｒｏ）の濃度は０．００８ｐｐｍであった。
これに対してＣｙｃｌｏ（Ｌｅｕ－Ｐｒｏ）濃度が０．４ｐｐｍ、１ｐｐｍ、３ｐｐｍ、１０ｐｐｍにそれぞれなるようにＣｙｃｌｏ（Ｌｅｕ－Ｐｒｏ）を加えて官能評価を行った。
官能評価の結果を表１に示した。 (Example 1: Evaluation by addition of Cyclo (Leu-Pro))
The concentration of Cyclo (Leu-Pro) contained in the commercially available non-alcoholic beer-taste beverage was 0.008 ppm.
On the other hand, sensory evaluation was performed by adding Cyclo (Leu-Pro) so that the concentration of Cyclo (Leu-Pro) was 0.4 ppm, 1 ppm, 3 ppm, and 10 ppm, respectively.
The results of the sensory evaluation are shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
表１に示す結果から、原料に麦芽を含むノンアルコールビールテイスト飲料につき、Ｃｙｃｌｏ（Ｌｅｕ－Ｐｒｏ）濃度が０．４ｐｐｍ以上であるとふくらみが増強されることが分かる。 From the results shown in Table 1, it can be seen that the swelling of the non-alcoholic beer-taste beverage containing malt as a raw material is enhanced when the Cyclo (Leu-Pro) concentration is 0.4 ppm or more.
（実施例２：Ｃｙｃｌｏ（Ｌｅｕ－Ｐｒｏ）と４０ｋＤａタンパク質の相乗効果の評価）
市販のノンアルコールビールテイスト飲料に、Ｃｙｃｌｏ（Ｌｅｕ－Ｐｒｏ）を加えてＣｙｃｌｏ（Ｌｅｕ－Ｐｒｏ）の濃度を０．４ｐｐｍとした飲料を基準として、さらに４０ｋＤａタンパク質を加えることによりＣｙｃｌｏ（Ｌｅｕ－Ｐｒｏ）と４０ｋＤａタンパク質の相乗効果を評価した。４０ｋＤａタンパク質の濃度は５ｐｐｍとした。
また、市販のノンアルコールビールテイスト飲料に、Ｃｙｃｌｏ（Ｌｅｕ－Ｐｒｏ）を加えてＣｙｃｌｏ（Ｌｅｕ－Ｐｒｏ）の濃度を１ｐｐｍとした飲料を基準として、さらに４０ｋＤａタンパク質を加えることによりＣｙｃｌｏ（Ｌｅｕ－Ｐｒｏ）と４０ｋＤａタンパク質の相乗効果を評価した。４０ｋＤａタンパク質の濃度は５ｐｐｍ、１０ｐｐｍ、２５ｐｐｍとした。
４０ｋＤａタンパク質には、上記で精製したものを使用した。
また、対比のために市販のノンアルコールビールテイスト飲料に４０ｋＤａタンパク質だけを添加して４０ｋＤａタンパク質の濃度を５ｐｐｍとしての評価も行った（対比試料）。
官能評価の結果を表２に示した。 (Example 2: Evaluation of synergistic effect between Cyclo (Leu-Pro) and 40 kDa protein)
Cyclo (Leu-Pro) by adding 40 kDa protein to a commercially available non-alcoholic beer-taste beverage based on a beverage with a Cyclo (Leu-Pro) concentration of 0.4 ppm. And the synergistic effect of 40 kDa protein was evaluated. The concentration of 40 kDa protein was 5 ppm.
In addition, Cyclo (Leu-Pro) is added by adding 40 kDa protein to a commercially available non-alcoholic beer-taste beverage based on a beverage in which the concentration of Cyclo (Leu-Pro) is 1 ppm. And the synergistic effect of 40 kDa protein was evaluated. The concentration of the 40 kDa protein was 5 ppm, 10 ppm, and 25 ppm.
The 40 kDa protein used was the one purified above.
For comparison, only 40 kDa protein was added to a commercially available non-alcoholic beer-taste beverage, and the concentration of 40 kDa protein was evaluated as 5 ppm (contrast sample).
The results of the sensory evaluation are shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
表２に示す結果から、市販のノンアルコールビールテイスト飲料に、Ｃｙｃｌｏ（Ｌｅｕ－Ｐｒｏ）を加えてさらに４０ｋＤａタンパク質を加えることにより、ふくらみをより増強できることが分かる。
対比試料の結果から、４０ｋＤａタンパク質を添加するだけでもふくらみを増強することができることがわかる。
しかしながら、上記試料１における官能評価のコントロールからの増加値（０．０２）と対比試料における官能評価のコントロールからの増加値（０．０３）の和で求められる、ジケトピペラジンと４０ｋＤａタンパク質を併用したときの相加効果（０．０５）よりも、試料５における官能評価のコントロールからの増加値（０．１１）が大きいことから、ジケトピペラジンと４０ｋＤａタンパク質を併用することにより、予想できない相乗効果が発揮されているといえる。
また、上記試料２における官能評価のコントロールからの増加値（０．１２）と対比試料における官能評価のコントロールからの増加値（０．０３）の和で求められる、ジケトピペラジンと４０ｋＤａタンパク質を併用したときの相加効果（０．１５）よりも、試料６における官能評価のコントロールからの増加値（０．２０）が大きいことから、ジケトピペラジンと４０ｋＤａタンパク質を併用することにより、予想できない相乗効果が発揮されているといえる。 From the results shown in Table 2, it can be seen that the swelling can be further enhanced by adding Cyclo (Leu-Pro) to a commercially available non-alcoholic beer-taste beverage and further adding 40 kDa protein.
From the results of the comparison sample, it can be seen that the swelling can be enhanced only by adding the 40 kDa protein.
However, diketopiperazine and 40 kDa protein, which are obtained by the sum of the increase value (0.02) from the sensory evaluation control in the sample 1 and the increase value (0.03) from the sensory evaluation control in the contrast sample, are used in combination. Since the increase value (0.11) from the control of the sensory evaluation in the sample 5 is larger than the additive effect (0.05), the combination of diketopiperazine and the 40 kDa protein causes an unpredictable synergistic effect. It can be said that the effect is exhibited.
In addition, diketopiperazine and 40 kDa protein, which are obtained by the sum of the increase value (0.12) from the sensory evaluation control in the sample 2 and the increase value (0.03) from the sensory evaluation control in the contrast sample, are used in combination. Since the increase value (0.20) from the control of the sensory evaluation in the sample 6 is larger than the additive effect (0.15) when the diketopiperazine is used in combination, an unpredictable synergistic effect is obtained. It can be said that the effect is exhibited.
（実施例３：Ｃｙｃｌｏ（Ｖａｌ－Ｐｒｏ）添加による評価）
市販のノンアルコールビールテイスト飲料に含まれるＣｙｃｌｏ（Ｖａｌ－Ｐｒｏ）の濃度は０．０１２ｐｐｍであった。
これに対してＣｙｃｌｏ（Ｖａｌ－Ｐｒｏ）濃度が０．４ｐｐｍ、０．８ｐｐｍにそれぞれなるようにＣｙｃｌｏ（Ｖａｌ－Ｐｒｏ）を加えて官能評価を行った。また、さらに４０ｋＤａタンパク質の濃度を５ｐｐｍにしての評価も行った。
官能評価の結果を表３に示した。 (Example 3: Evaluation by addition of Cyclo (Val-Pro))
The concentration of Cyclo (Val-Pro) contained in the commercially available non-alcoholic beer-taste beverage was 0.012 ppm.
On the other hand, sensory evaluation was performed by adding Cyclo (Val-Pro) so that the Cyclo (Val-Pro) concentration was 0.4 ppm and 0.8 ppm, respectively. Further, the evaluation was performed with the concentration of 40 kDa protein set to 5 ppm.
The results of the sensory evaluation are shown in Table 3.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
表３に示す結果から、市販のノンアルコールビールテイスト飲料に、Ｃｙｃｌｏ（Ｖａｌ－Ｐｒｏ）を加えることによりふくらみを増強できることが分かる。
また、さらに４０ｋＤａタンパク質を加えることにより、ふくらみをより増強できることが分かる。
対比試料の結果（表２参照）から、４０ｋＤａタンパク質を添加するだけでもふくらみを増強することができることがわかる。
しかしながら、上記試料９における官能評価のコントロールからの増加値（０．０２）と表２に示す対比試料における官能評価のコントロールからの増加値（０．０３）の和で求められる、ジケトピペラジンと４０ｋＤａタンパク質を併用したときの相加効果（０．０５）よりも、試料１０における官能評価のコントロールからの増加値（０．０９）が大きいことから、ジケトピペラジンと４０ｋＤａタンパク質を併用することにより、予想できない相乗効果が発揮されているといえる。
また、上記試料１１における官能評価のコントロールからの増加値（０．０８）と表２に示す対比試料における官能評価のコントロールからの増加値（０．０３）の和で求められる、ジケトピペラジンと４０ｋＤａタンパク質を併用したときの相加効果（０．１１）よりも、試料１２における官能評価のコントロールからの増加値（０．２０）が大きいことから、ジケトピペラジンと４０ｋＤａタンパク質を併用することにより、予想できない相乗効果が発揮されているといえる。 From the results shown in Table 3, it can be seen that the swelling can be enhanced by adding Cyclo (Val-Pro) to a commercially available non-alcoholic beer-taste beverage.
Further, it can be seen that the swelling can be further enhanced by further adding 40 kDa protein.
From the results of the comparison sample (see Table 2), it can be seen that the swelling can be enhanced only by adding the 40 kDa protein.
However, with diketopiperazine, which is obtained by the sum of the increase value (0.02) from the sensory evaluation control in the sample 9 and the increase value (0.03) from the sensory evaluation control in the comparison sample shown in Table 2. Since the increase value (0.09) from the control of the sensory evaluation in sample 10 is larger than the additive effect (0.05) when the 40 kDa protein is used in combination, the combination of diketopiperazine and the 40 kDa protein is used. It can be said that an unexpected synergistic effect is exhibited.
Further, with diketopiperazine, which is obtained by the sum of the increase value (0.08) from the sensory evaluation control in the sample 11 and the increase value (0.03) from the sensory evaluation control in the comparison sample shown in Table 2. Since the increase value (0.20) from the control of the sensory evaluation in sample 12 is larger than the additive effect (0.11) when the 40 kDa protein is used in combination, the combination of diketopiperazine and the 40 kDa protein is used. It can be said that an unexpected synergistic effect is exhibited.
（実施例４：Ｃｙｃｌｏ（Ｉｌｅ－Ｐｒｏ）添加による評価）
市販のノンアルコールビールテイスト飲料に含まれるＣｙｃｌｏ（Ｉｌｅ－Ｐｒｏ）の濃度は０ｐｐｍであった。
これに対してＣｙｃｌｏ（Ｉｌｅ－Ｐｒｏ）濃度が０．４ｐｐｍ、０．８ｐｐｍにそれぞれなるようにＣｙｃｌｏ（Ｉｌｅ－Ｐｒｏ）を加えて官能評価を行った。また、さらに４０ｋＤａタンパク質の濃度を５ｐｐｍにしての評価も行った。
官能評価の結果を表４に示した。 (Example 4: Evaluation by addition of Cyclo (Ile-Pro))
The concentration of Cyclo (Ile-Pro) contained in the commercially available non-alcoholic beer-taste beverage was 0 ppm.
On the other hand, sensory evaluation was performed by adding Cyclo (Ile-Pro) so that the Cyclo (Ile-Pro) concentration was 0.4 ppm and 0.8 ppm, respectively. Further, the evaluation was performed with the concentration of 40 kDa protein set to 5 ppm.
The results of the sensory evaluation are shown in Table 4.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
表４に示す結果から、市販のノンアルコールビールテイスト飲料に、Ｃｙｃｌｏ（Ｉｌｅ－Ｐｒｏ）を加えることによりふくらみを増強できることが分かる。
また、さらに４０ｋＤａタンパク質を加えることにより、ふくらみをより増強できることが分かる。
対比試料の結果（表２参照）から、４０ｋＤａタンパク質を添加するだけでもふくらみを増強することができることがわかる。
しかしながら、上記試料１３における官能評価のコントロールからの増加値（０．０４）と表２に示す対比試料における官能評価のコントロールからの増加値（０．０３）の和で求められる、ジケトピペラジンと４０ｋＤａタンパク質を併用したときの相加効果（０．０７）よりも、試料１４における官能評価のコントロールからの増加値（０．１１）が大きいことから、ジケトピペラジンと４０ｋＤａタンパク質を併用することにより、予想できない相乗効果が発揮されているといえる。
また、上記試料１５における官能評価のコントロールからの増加値（０．０９）と表２に示す対比試料における官能評価のコントロールからの増加値（０．０３）の和で求められる、ジケトピペラジンと４０ｋＤａタンパク質を併用したときの相加効果（０．１２）よりも、試料１６における官能評価のコントロールからの増加値（０．２１）が大きいことから、ジケトピペラジンと４０ｋＤａタンパク質を併用することにより、予想できない相乗効果が発揮されているといえる。 From the results shown in Table 4, it can be seen that the swelling can be enhanced by adding Cyclo (Ile-Pro) to a commercially available non-alcoholic beer-taste beverage.
Further, it can be seen that the swelling can be further enhanced by further adding 40 kDa protein.
From the results of the comparison sample (see Table 2), it can be seen that the swelling can be enhanced only by adding the 40 kDa protein.
However, with diketopiperazine, which is obtained by the sum of the increase value (0.04) from the sensory evaluation control in the sample 13 and the increase value (0.03) from the sensory evaluation control in the comparison sample shown in Table 2. Since the increase value (0.11) from the control of the sensory evaluation in sample 14 is larger than the additive effect (0.07) when the 40 kDa protein is used in combination, the combination of diketopiperazine and the 40 kDa protein is used. It can be said that an unexpected synergistic effect is exhibited.
Further, with diketopiperazine, which is obtained by the sum of the increase value (0.09) from the sensory evaluation control in the sample 15 and the increase value (0.03) from the sensory evaluation control in the comparison sample shown in Table 2. Since the increase value (0.21) from the control of the sensory evaluation in sample 16 is larger than the additive effect (0.12) when the 40 kDa protein is used in combination, the combination of diketopiperazine and the 40 kDa protein is used. It can be said that an unexpected synergistic effect is exhibited.
本発明によれば、原料に麦芽を含むノンアルコールビールテイスト飲料において、ふくらみが増強された飲料を提供することができる。 According to the present invention, it is possible to provide a non-alcoholic beer-taste beverage containing malt as a raw material with enhanced swelling.

Claims (4)

  1. 原料に麦芽を含むノンアルコールビールテイスト飲料であって、シクロロイシルプロリン、シクロバリルプロリン及びシクロイソロイシルプロリンからなる群から選択される少なくとも１種のジケトピペラジンを含み、前記ジケトピペラジンのうち最も多く含まれるジケトピペラジンの濃度が０．４ｐｐｍ以上であるノンアルコールビールテイスト飲料。 A non-alcoholic beer-taste beverage containing malt as a raw material, which contains at least one diketopiperazine selected from the group consisting of cycloleuicylproline, cyclovalylproline and cycloisoleuicylproline, and contains the diketopiperazine. A non-alcoholic beer-taste beverage with the highest concentration of diketopiperazine of 0.4 ppm or more.
  2. 前記ジケトピペラジンのうち最も多く含まれるジケトピペラジンの濃度が１０ｐｐｍ以下である請求項１に記載のノンアルコールビールテイスト飲料。 The non-alcoholic beer-taste beverage according to claim 1, wherein the concentration of diketopiperazine contained most in the diketopiperazine is 10 ppm or less.
  3. さらに、分子量３５～５０ｋＤａのタンパク質を含み、
    前記タンパク質の濃度が５ｐｐｍ以上である請求項１又は２に記載のノンアルコールビールテイスト飲料。     In addition, it contains a protein with a molecular weight of 35-50 kDa,
    The non-alcoholic beer-taste beverage according to claim 1 or 2, wherein the protein concentration is 5 ppm or more.
  4. 前記タンパク質の濃度が３０ｐｐｍ以下である請求項３に記載のノンアルコールビールテイスト飲料。

          The non-alcoholic beer-taste beverage according to claim 3, wherein the protein concentration is 30 ppm or less.
PCT/JP2021/024537 2020-07-01 2021-06-29 Non-alcoholic beer-flavored beverage WO2022004713A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2022534035A JPWO2022004713A1 (en) 2020-07-01 2021-06-29

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2020-114421 2020-07-01
JP2020114421 2020-07-01

Publications (1)

Publication Number Publication Date
WO2022004713A1 true WO2022004713A1 (en) 2022-01-06

Family

ID=79316270

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2021/024537 WO2022004713A1 (en) 2020-07-01 2021-06-29 Non-alcoholic beer-flavored beverage

Country Status (3)

Country Link
JP (1) JPWO2022004713A1 (en)
TW (1) TW202214115A (en)
WO (1) WO2022004713A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4083056A4 (en) * 2019-12-27 2024-01-24 Suntory Holdings Ltd Non-alcoholic beer-flavored beverage, rich taste enhancing agent and sourness reducing agent for non-alcoholic beer-flavored beverages, and rich taste enhancing method and sourness reducing method for non-alcoholic beer-flavored beverages

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014079220A (en) * 2012-10-18 2014-05-08 Asahi Breweries Ltd Manufacturing method of fermented malt beverage inducing enhanced feeling of stimulation
WO2014200000A1 (en) * 2013-06-10 2014-12-18 サントリーホールディングス株式会社 Plant extract containing diketopiperazine and method for producing same
WO2017168718A1 (en) * 2016-03-31 2017-10-05 サントリーホールディングス株式会社 Packaged beverage

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014079220A (en) * 2012-10-18 2014-05-08 Asahi Breweries Ltd Manufacturing method of fermented malt beverage inducing enhanced feeling of stimulation
WO2014200000A1 (en) * 2013-06-10 2014-12-18 サントリーホールディングス株式会社 Plant extract containing diketopiperazine and method for producing same
WO2017168718A1 (en) * 2016-03-31 2017-10-05 サントリーホールディングス株式会社 Packaged beverage

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SAKAMURA SADAO, FURUKAWA KATSUHISA, KASAI TAKANORI: "Bitter Diketopiperazines in Roasted Malts for Beer Brewing", AGRICULTURAL AND BIOLOGICAL CHEMISTRY, AGRICULTURAL CHEMICAL SOCIETY OF JAPAN, JP, vol. 42, no. 3, 1 March 1978 (1978-03-01), JP , pages 607 - 612, XP055895898, ISSN: 0002-1369, DOI: 10.1080/00021369.1978.10863026 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4083056A4 (en) * 2019-12-27 2024-01-24 Suntory Holdings Ltd Non-alcoholic beer-flavored beverage, rich taste enhancing agent and sourness reducing agent for non-alcoholic beer-flavored beverages, and rich taste enhancing method and sourness reducing method for non-alcoholic beer-flavored beverages

Also Published As

Publication number Publication date
TW202214115A (en) 2022-04-16
JPWO2022004713A1 (en) 2022-01-06

Similar Documents

Publication Publication Date Title
JP7257144B2 (en) beer-taste beverages
WO2021131924A1 (en) Non-alcoholic beer-flavored beverage, rich taste enhancing agent and sourness reducing agent for non-alcoholic beer-flavored beverages, and rich taste enhancing method and sourness reducing method for non-alcoholic beer-flavored beverages
JP2020103208A (en) Beer taste beverage
JP2023126640A (en) Beer taste beverage
WO2022004713A1 (en) Non-alcoholic beer-flavored beverage
WO2022004711A1 (en) Non-alcoholic beer-like beverage
WO2022004725A1 (en) Non-alcoholic beer-flavored beverage
WO2022004715A1 (en) Beer-taste alcoholic beverage
WO2022004724A1 (en) Beer-flavored alcoholic beverage
JP7313278B2 (en) Beer-taste alcoholic beverage, body-taste enhancer for beer-taste-alcoholic beverage, and method for enhancing body-taste of beer-taste-alcoholic beverage
WO2020157890A1 (en) Beer-taste beverage
JP7411513B2 (en) beer taste alcoholic beverage
JP6605033B2 (en) Beer-taste beverage
WO2022004719A1 (en) Non-alcoholic beer-taste beverage
WO2022004717A1 (en) Beer-taste alcoholic beverage
WO2022004722A1 (en) Non-alcoholic beer taste beverage
JP7313277B2 (en) Beer-taste alcoholic beverage, body-taste enhancer for beer-taste-alcoholic beverage, and method for enhancing body-taste of beer-taste-alcoholic beverage
WO2022004720A1 (en) Beer-taste alcoholic beverage
WO2022004721A1 (en) Beer-taste alcoholic beverage
EP4268603A1 (en) Non-alcoholic beer-flavored beverage, method for producing non-alcoholic beer-flavored beverage, and method for improving quality of bitterness of non-alcoholic beer-flavored beverage
JP2021106540A (en) Beer taste alcoholic beverage

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21833135

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022534035

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21833135

Country of ref document: EP

Kind code of ref document: A1