JP6429749B2 - Beverage containing enzyme-treated isoquercitrin - Google Patents

Description

  The present invention relates to a beverage containing enzyme-treated isoquercitrin.

  Quercetin (3,3 ', 4', 5,7-pentahydroxyflavone, also called quercetin) is a polyphenol component that is abundant in vegetables and fruits, as it is or in the form of glycosides, citrus fruits, It is contained in various plants such as onion, buckwheat and enju. Quercetin is known to have various physiological functions such as platelet aggregation inhibition and adhesion inhibition action, vasodilatory action, and anticancer action in addition to strong antioxidant activity (Non-patent Document 1) (Non-patent Document 1). Reference 2).

  Isoquercitrin, one of the quercetin glycosides, is a flavonol glycoside in which one glucose is β-bonded at the 3-position of quercetin, and has a strong antioxidant activity and can prevent dye fading. It is a known material (Patent Document 1), and is expected to have an effect on the living body such as anti-arteriosclerosis and blood flow improvement. Recently, it has been found that isoquercitrin has an effect of reducing body fat of obese people, and it has been reported that it is effective when ingested in the form of a beverage (Non-patent Document 3).

  However, since isoquercitrin is hardly soluble in water, there is a problem that its use as an aqueous composition such as a beverage is limited. Therefore, a method for preparing α-glucosylisoquercitrin (referred to as “enzyme-treated isoquercitrin” in this specification) by transferring glucose to the glucose residue site of isoquercitrin using glycosyltransferase has been proposed. (Patent Document 2). “Enzyme-treated isoquercitrin” is known to be a readily water-soluble substance with improved solubility in water while maintaining the action of isoquercitrin.

JP 2008-131888 A JP-A-1-213293

Middlton EJ. Et al., Pharmacol Rev., 52, 673-751, 2000 Pharmacology and treatment, p123-131, vol.37, No.2, 2009 Pharmacology and treatment, p919-930, vol.36, No.10, 2008

  Thus, since enzyme-treated isoquercitrin is superior in solubility to isoquercitrin, it is possible to produce a beverage containing enzyme-treated isoquercitrin at a high concentration. It is difficult to keep the color of the beverage stable over a long period of time. An object of the present invention is to provide an enzyme-treated isoquercitrin-containing beverage with little color change even after long-term storage.

As a result of intensive studies to solve the above problems, the present inventors have included quinic acid at a concentration of more than 10 mg / kg and less than 4000 mg / kg with respect to a beverage containing enzyme-treated isoquercitrin. The inventors have found that color change during long-term storage can be suppressed, and have completed the present invention. The present invention is not limited to this, but relates to the following.
(1) the following components (A) and (B);
(A) Enzyme-treated isoquercitrin: 200 mg / kg or more (B) Quinic acid: greater than 10 mg / kg and less than 4000 mg / kg,
The drink whose mass ratio [(B) / (A)] of an ingredient (A) and an ingredient (B) is 0.05 <[(B) / (A)] <20.

(2) The beverage according to (1), wherein the concentration of quinic acid is 3500 mg / kg or less.
(3) The beverage according to (1) or (2), wherein the concentration of the enzyme-treated isoquercitrin is less than 4000 mg / kg.
(4) A method for suppressing color change over time of a beverage containing enzyme-treated isoquercitrin at a concentration of 200 mg / kg or more,
Adjusting the concentration of quinic acid to greater than 10 mg / kg and less than 4000 mg / kg; and
Adjusting the mass ratio of quinic acid to enzyme treated isoquercitrin to be greater than 0.05 and less than 20.

  According to the present invention, it is possible to obtain a beverage with little color change even when stored for a long period of time while containing enzyme-treated isoquercitrin at a high concentration. Furthermore, the present inventors have also surprisingly found that the quinic acid reduces the taste caused by a high concentration of enzyme-treated isoquercitrin. Since quinic acid is known as a component that contributes to bitterness and sourness, it was an unexpected result that the savory taste of enzyme-treated isoquercitrin was reduced by the addition of quinic acid. These effects were not seen when other acids such as citric acid or malic acid were used instead of quinic acid.

(Enzyme-treated isoquercitrin)
The enzyme-treated isoquercitrin referred to in the present invention is an isoquercitrin in which the number of glucose residues (n) is 0 in the following formula 1, and an integer having a glucose residue number (n) of 1 or more (preferably 1 to 1). 15 is an integer of 15 and more preferably an integer of 1 to 7). In the present specification, QG1 containing 1 glucose in quercetin (n = 0 in the formula 1) and 1 QG2 (n = 1 in the formula 1) (Q = 1) In Formula 1, n = 2) may be expressed as QG3 (hereinafter, every time glucose increases, QG4, QG5, QG6,...).

  As the enzyme-treated isoquercitrin, a commercially available one may be used, or one prepared by enzyme treatment of isoquercitrin or rutin may be used. Isoquercitrin can be produced, for example, by the method described in WO2005 / 030975, that is, a method in which rutin is treated with naringinase in the presence of a specific edible component. Furthermore, as described in WO2005 / 030975, α-glycosylisoquercitrin can be obtained by treating isoquercitrin with a glycosyltransferase.

  The beverage of the present invention contains enzyme-treated isoquercitrin at a concentration sufficient to exert a physiological effect. Specifically, it contains enzyme-treated isoquercitrin at a concentration of 200 mg / kg or more. If the concentration of the enzyme-treated isoquercitrin is high, precipitation may occur during refrigerated storage. Therefore, the concentration of the enzyme-treated isoquercitrin is preferably 200 mg / kg or more and less than 4000 mg / kg, more preferably 200 mg / kg. It is 3000 mg / kg or less, more preferably 200 mg / kg or more and 2000 mg / kg or less. The concentration of the enzyme-treated isoquercitrin in the beverage when used for drinking without dilution is desirably 200 mg / kg or more and 800 mg / kg or less.

  In the present invention, when referring to the concentration of enzyme-treated isoquercitrin in the beverage, unless otherwise specified, the total concentration of enzyme-treated isoquercitrin is converted to QG1, and quercetin produced by hydrolysis of QG1 The concentration of The concentration of quercetin generated by hydrolysis of QG1 can be determined by (molecular concentration 302 of quercetin and molecular weight 464 of QG1) (concentration of enzyme-treated isoquercitrin ÷ 464) × 302. In addition, when referring to the concentration or amount of a beverage component in the present invention, it refers to the concentration or amount in the final product, unless otherwise specified.

  The concentration of the enzyme-treated isoquercitrin can be measured by a conventional method well known to those skilled in the art. The concentration of enzyme-treated isoquercitrin may be determined by the following method using QG1 to QG7 as the components involved unless otherwise specified: That is, using Quercetin 3-0-glucoside (QG1) as a standard substance, Using HPLC, create a calibration curve based on the area at an ultraviolet absorbance of 350 nm and the standard substance concentration. Since enzyme-treated isoquercitrin is hydrolyzed to quercetin in the small intestine, QG1 to QG7 are considered to be physiologically equivalent, and the 3-position glycoside of quercetin is independent of the sugar chain length. , All have a maximum absorption at 350 nm, and its absorbance depends on quercetin, which is an aglycon moiety. Therefore, although molecular weights are different, QG1 to QG7 are considered to be equal in terms of molar absorbance, and the components involved are quantified in terms of QG1. Specifically, the analysis sample is subjected to HPLC under the same conditions as the standard substance, and the peak that matches the elution retention time of the standard substance is specified in the obtained chart. Then, the peak of enzyme-treated isoquercitrin QG2 to QG7 detected before the peak of QG1 is specified (if any), and a calibration curve created using a standard substance is used from the total of each peak area. The concentration of the enzyme-treated isoquercitrin in the analysis sample is calculated.

(Quinic acid)
Quinic acid (molecular formula C ₇ H ₁₂ O ₆ , IUPAC name (1S, 3R, 4S, 5R) -1,3,4,5-tetrahydroxycyclohexanecarboxylic acid) was discovered from the skin of Kina, a tree of Rubiaceae Component. It is known to have antibacterial action and is known to be contained in cranberry fruits, coffee beans, grapefruit, kiwifruit, apples, peaches, and the like.

  The quinic acid in the present invention may be in the form of a quinic acid lactone. Quinic acid lactone is produced from quinic acid by intramolecular dehydration, and returns to quinic acid by hydrolysis of the intramolecular ester. In the present invention, the concentration of quinic acid in the beverage can be adjusted by converting quinic acid lactone as the amount of quinic acid before preparing the beverage. Further, when referring to the concentration of quinic acid in the prepared beverage, the amount of quinic acid lactone in the beverage can be converted to the amount of quinic acid and added to the concentration of quinic acid in the beverage.

  The beverage of the present invention contains quinic acid at a concentration greater than 10 mg / kg and less than 4000 mg / kg. The mass ratio of quinic acid to enzyme-treated isoquercitrin (quinic acid / enzyme-treated isoquercitrin) is set to be larger than 0.05 and less than 20. Enzymatically treated isoquercitrin-containing beverage by adjusting the amount of quinic acid and the mass ratio of quinic acid to enzymatically treated isoquercitrin within the above range with respect to beverage containing 200 mg / kg or more of enzyme-treated isoquercitrin The change in color over time can be suppressed.

  The concentration of quinic acid is preferably more than 10 mg / kg and not more than 3500 mg / kg, more preferably not less than 200 mg / kg and not more than 3000 mg / kg, from the viewpoint of the color change inhibiting effect.

  The mass ratio of quinic acid to enzyme-treated isoquercitrin is preferably 0.1 to 15, and more preferably 0.2 to 10. Furthermore, it is more preferable that the mass ratio is 0.5 to 10 because the change in color is particularly low, and 3 to 4 is more preferable.

  In another aspect, the present invention provides a beverage containing 200 mg / kg or more of enzyme-treated isoquercitrin, wherein the concentration of quinic acid in the beverage is greater than 10 mg / kg and less than 4000 mg / kg, and the enzyme treatment for quinic acid is performed. The present invention relates to a method for suppressing a change in color of a beverage over time by adjusting the mass ratio of isoquercitrin (quinic acid / enzyme-treated isoquercitrin) to be greater than 0.05 and less than 20.

  In addition to the above-described effect of suppressing color change, the present inventors have added an enzyme treatment by adding quinic acid in the above-mentioned concentration range and mass ratio to a high-concentration enzyme-treated isoquercitrin-containing beverage. It has been found that the taste of isoquercitrin can be reduced. Since quinic acid is known as a component that contributes to bitterness and sourness, the addition of quinic acid has improved the egg taste of enzyme-treated isoquercitrin, and improved the ease of drinking beverages. It was an unexpected effect.

(Color change of beverage)
According to the present invention, it is possible to suppress a change in color over time when a beverage containing an enzyme-treated isoquercitrin having a high concentration of 200 mg / kg or more is stored. The change in beverage color can be expressed as ΔE based on the following formula in the L ^* a ^* b ^* color system:
ΔE = ((L ^* −L ^* ′) ² + (a ^* −a ^* ′) ² + (b ^* −b ^* ′) ² ) ^1/2
In the formula, L ^* , a ^* , b ^* , and L ^* ′, a ^* ′, b ^* ′ represent values before and after storage of beverages in the L ^* a ^* b ^* color system, respectively. Is. The L ^* , a ^* , and b ^* of the beverage can be measured, for example, by the method described in Examples described later.

(Beverage)
The type of beverage in the beverage of the present invention is not particularly limited, and carbonated beverages, non-carbonated beverages, alcoholic beverages, non-alcoholic beverages, coffee beverages, fruit beverages, tea beverages, dairy beverages, vegetable beverages, sports beverages, and cocoa beverages. It may be any of a nutrient drink, a functional drink, a near water drink, and the like.

  In addition to enzyme-treated isoquercitrin and quinic acid, the beverage of the present invention may contain various additives depending on the type of beverage. Examples of the various additives include sweeteners, acidulants, fragrances, vitamins, pigments, antioxidants, emulsifiers, preservatives, extracts, dietary fiber, pH adjusters, quality stabilizers, and the like.

  The drink of this invention is good also as a container-packed drink through processes, such as sterilization, as needed. For example, a sterilized container-packed beverage can be produced by a method of performing heat sterilization after filling a beverage into a container, or a method of sterilizing a beverage and then filling the container in an aseptic environment.

  The kind of container is not particularly limited, and examples thereof include PET bottles, cans, bottles, paper packs, and the like. In particular, colorless and transparent PET bottles and bottles are suitable for suppressing the color change of a beverage over time using the present invention because the color of the beverage in the container is easily visible from the outside.

  In addition, the beverage of the present invention can be provided in the form of a so-called BIB (bag-in-box), and can be provided as it is just before drinking, or can be appropriately diluted with water, carbonated water or the like. In particular, when provided in a diluted form, the content of the enzyme-treated isoquercitrin contained in the beverage in the BIB becomes high, so the effect of the present invention can be expected more.

  Hereinafter, the present invention will be specifically described using experimental examples, but the present invention is not limited thereto.

(Measurement method of enzyme-treated isoquercitrin concentration in beverages)
1. Reagent / Acetonitrile: High-performance liquid chromatograph purity 99.8% (manufactured by Nacalai Tesque)
・ Water: Impurity for high performance liquid chromatograph 0.001% or less (manufactured by Nacalai Tesque)
・ Trifluoroacetic acid: 99% purity (manufactured by Nacalai Tesque)
・ Isoquercitrin (Quercetin 3-O-glucoside: QG1): SSX1327S, purity 93.8% (Funakoshi Co., Ltd.)
・ Ethanol: 99.8% purity for high performance liquid chromatography (manufactured by Nacalai Tesque)
Dimethyl sulfoxide (hereinafter referred to as DMSO): purity 99.0% (manufactured by Nacalai Tesque).
2. Analytical instrument high performance liquid chromatograph (hereinafter referred to as HPLC)
Pump: LC-10ADvp
Detector: SPD-M10Avp detector analysis software: Class LC solution (Shimadzu Corporation)
3. Preparation of analytical sample The beverage stock solution is diluted 5-fold with 20% ethanol / water and filtered through a 0.45 μm filter (Mirex LH-4: manufactured by Millipore) for HPLC.
4). Preparation of calibration curve 1.0 mg of isoquercitrin (purity 93.8%), the standard substance, was accurately weighed and dissolved in 0.5 ml dimethyl sulfoxide (purity 99.0%) in a 5 ml volumetric flask, and 20% ethanol (purity 99.8%). %) / Water to 5 ml. This 200 μg / ml solution is diluted sequentially with 20% ethanol / water to make 10, 25, 50, and 100 μg / ml solutions. 10 μl of each concentration solution is subjected to HPLC. The elution retention time of the peak detected at this time is about 14.5 minutes. At this time, a calibration curve is prepared based on the area and concentration at an ultraviolet absorbance of 350 nm.

Calculate an approximate straight line passing through the origin, calculate the concentration from QG1 to QG7 using this, and calculate the amount of quercetin glycoside by multiplying the sum by the purity of the standard substance (93.8%).
5. Test procedure and qualitative test: Analyze the analysis sample under the same conditions as the standard, and set the peak that matches the elution retention time of the QG1 standard to QG1. QG1 is a quercetin glycoside in which one glucose is bound to quercetin.
Quantitative test: The six peaks detected before the QG1 peak are glycosides of quercetin further glucose-bound to QG1. In HPLC analysis, QG1 and a compound in which 1 to 6 glucoses were further bound to QG1 were detectable, and these (QG1 to QG7) were set as the components involved. In addition, since quercetin glycoside is hydrolyzed to quercetin in the small intestine, QG1 to QG7 are considered to be physiologically equivalent and are the main constituents of quercetin glycoside, and standard products are available QG1 is set as the index component, and the amount in terms of QG1 is calculated. The peak areas of the seven elution peaks of quercetin glycoside are measured, and the concentration of quercetin glycoside in the analysis sample is calculated from a calibration curve created based on the peak area of the QG1 standard product.

  Isoquercitrin (QG1) is a compound in which one molecule of glucose is β-bonded to the 3-position of quercetin. QG2 to QG7 are a group of compounds in which 1 to 6 glucoses are further α-1,4 linked to QG1, and 7 components QG1 and QG2 to QG7 are involved components.

  Regardless of the length of the sugar chain, quercetin 3-position glycosides all have a maximum absorption at 350 nm, and the absorbance is contributed by quercetin, which is an aglycon moiety. Therefore, although the molecular weight is different, QG1 to QG7 are considered to be equal in terms of molar absorbance, and the components involved are determined in terms of QG1. The obtained quercetin glycoside (enzyme-treated isoquercitrin) concentration in terms of QG1 was further converted into the concentration of quercetin.

(Measurement of quinic acid and quinic acid lactone concentrations in beverages)
The concentration of quinic acid and quinic acid lactone in the beverage can be measured by a known method, for example, the Japan Food Analysis Center method by HPLC (column: manufactured by Tosoh Corporation, TSKGEL OApak, φ7.8 mm × 300 mm, Column temperature: 40 ° C., mobile phase: 0.75 mM sulfuric acid, reaction solution: 15 mM disodium hydrogenphosphate solution containing 0.2 mM bromthymol blue, flow rate: mobile phase 0.8 mL / min, reaction solution 0.8 mL / min, (Detection wavelength: 445 nm).

(Measurement method of beverage color change (ΔE))
Using a colorimetric color difference meter ZE-2000 (manufactured by JEOL Ltd.), the values of L ^* , a ^* , b ^* are measured for the beverage just prepared and the beverage stored at 55 ° C. for 4 days, and the following formula Is used to calculate the color difference ΔE:
ΔE = ((L ^* −L ^* ′) ² + (a ^* −a ^* ′) ² + (b ^* −b ^* ′) ² ) ^1/2
In the formula, L ^* , a ^* , and b ^* represent L ^* , a ^* , and b ^* values of the beverage immediately after preparation, and L ^* ′, a ^* ′, and b ^* ′ represent the same beverage at 4 ° C. at 55 ° C. L ^* , a ^* , b ^* values after storage for days are expressed. It can be said that the greater the ΔE, the greater the change in beverage color due to storage.

(Example 1)
As the enzyme-treated isoquercitrin (QG), Sanemik P15 (San-Eigen FFI Co., Ltd.) was used. Each beverage was prepared by dissolving quinic acid and enzyme-treated isoquercitrin in water so as to have the amounts shown in Table 1 below. The L ^* , a ^* , and b ^* values immediately after preparation of each beverage were measured by the above method. Thereafter, each beverage was filled in a colorless and transparent PET container, sterilized by heating, and stored at 55 ° C. for 4 days. L ^* , a ^* , b ^* values of each beverage after storage at 55 ° C. for 4 days were measured, and ΔE was calculated by the above formula. Separately from this, each beverage was allowed to stand at 5 ° C., and it was confirmed whether or not a precipitate was formed. The results are shown in Table 1.

  From the results of Table 1, the enzyme-treated isoquercitrin is contained at a concentration of 200 mg / kg or more, quinic acid is contained at a concentration of more than 10 mg / kg and less than 4000 mg / kg, and the mass ratio of quinic acid to the enzyme-treated isoquercitrin is from 0.05 It can be seen that a beverage having a large value of less than 20 has a small ΔE when stored at 55 ° C. for 4 days (that is, there is little color change). In addition, since beverages containing enzyme-treated isoquercitrin at a concentration of 4000 mg / kg cause precipitation at 5 ° C., it can be seen that the concentration of enzyme-treated isoquercitrin is preferably less than 4000 mg / kg.

(Example 2)
As the enzyme-treated isoquercitrin (QG), Sanemik P15 (San-Eigen FFI Co., Ltd.) was used. Each beverage was prepared by dissolving malic acid, citric acid, and enzyme-treated isoquercitrin in water so as to have the amounts shown in Table 2 below. The taste of these beverages and the taste of Comparative product 4 and Invention product 5 prepared in Example 1 were subjected to sensory evaluation in five stages by three panelists. In the evaluation, “5” indicates that the taste is extremely strong, “4” indicates that the taste is considerably felt, “3” indicates that the taste is slightly felt, “2” indicates that the taste is slightly felt, and “1” indicates that the taste is hardly felt. Table 2 shows the average scores of the three panelists.

  From the results of Table 2, it can be seen that the taste of enzyme-treated isoquercitrin can be reduced by adding a certain amount and mass ratio of quinic acid in a beverage containing a high concentration of enzyme-treated isoquercitrin. . This effect was not observed with other organic acids such as citric acid and malic acid, but it was found that the taste was rather enhanced with citric acid and malic acid. It has not been known so far that quinic acid has an effect of improving the taste of enzyme-treated isoquercitrin, which was an unexpected result.

Claims (4)

The following components (A) and (B);
(A) Enzyme-treated isoquercitrin: 200 mg / kg or more (B) Quinic acid: greater than 10 mg / kg and less than 4000 mg / kg,
The drink whose mass ratio [(B) / (A)] of an ingredient (A) and an ingredient (B) is 0.05 <[(B) / (A)] <20.   The beverage according to claim 1, wherein the concentration of quinic acid is 3500 mg / kg or less.   The beverage according to claim 1 or 2, wherein the concentration of the enzyme-treated isoquercitrin is less than 4000 mg / kg. A method for suppressing a change in color over time of a beverage containing enzyme-treated isoquercitrin at a concentration of 200 mg / kg or more,
Adjusting the concentration of quinic acid to greater than 10 mg / kg and less than 4000 mg / kg; and
Adjusting the mass ratio of quinic acid to enzyme treated isoquercitrin to be greater than 0.05 and less than 20.