WO2022224642A1 - Lactobacillus beverage having antioxidant effect - Google Patents

Lactobacillus beverage having antioxidant effect Download PDF

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Publication number
WO2022224642A1
WO2022224642A1 PCT/JP2022/011850 JP2022011850W WO2022224642A1 WO 2022224642 A1 WO2022224642 A1 WO 2022224642A1 JP 2022011850 W JP2022011850 W JP 2022011850W WO 2022224642 A1 WO2022224642 A1 WO 2022224642A1
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Prior art keywords
lactic acid
acid bacteria
ddmp
antioxidant
milk
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PCT/JP2022/011850
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French (fr)
Japanese (ja)
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修一 瀬川
圭介 田川
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日清ヨーク株式会社
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Priority to KR1020237033053A priority Critical patent/KR20230172466A/en
Priority to CN202280025876.4A priority patent/CN117098457A/en
Publication of WO2022224642A1 publication Critical patent/WO2022224642A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • 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
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • 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
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/302—Foods, ingredients or supplements having a functional effect on health having a modulating effect on age
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/60—Sugars, e.g. mono-, di-, tri-, tetra-saccharides
    • A23V2250/61—Glucose, Dextrose

Definitions

  • the present invention relates to lactic acid beverages.
  • it relates to a lactic acid bacteria drink having an antioxidant effect.
  • Lactic acid beverages are widely used and have been supported by people of all ages for many years.
  • Pilkul registered trademark: Nisshin York Co., Ltd.
  • Yakult registered trademark: Yakult Honsha Co., Ltd.
  • the lactic acid bacteria drink is widely known to have the function of improving the intestinal environment.
  • There are also known types that exhibit excellent effects such as alleviating stress, lowering blood sugar, and recovering from fatigue.
  • this lactic acid bacteria drink is expected to exhibit useful functionality and effects in addition to the above.
  • antioxidant properties it has not been known in the past that lactic acid bacteria beverages have antioxidant properties, nor have there been prior patent documents regarding antioxidant substances.
  • Patent Document 1 describes antioxidants for lactic acid bacteria beverages.
  • the patent document merely describes that the main purpose of the lactic acid bacteria drink is to contain docosahexaenoic acid (DHA), and an antioxidant is added as an incidental component.
  • DHA docosahexaenoic acid
  • the present inventors set a task to examine the antioxidant properties of lactic acid bacteria beverages. Moreover, it was made into the subject to discover about the active ingredient.
  • the present invention was completed by confirming that the lactic acid bacteria drink has an antioxidant effect. That is, the first invention of the present application is "A lactic acid bacteria drink containing an ingredient having an antioxidant action.”
  • the component with antioxidant action is DDMP (2,3-dihydro-3,5-dihydroxy-6- methyl-4H-pyran-4-one). That is, the second invention of the present application is "The lactic acid bacteria beverage according to claim 1, wherein said ingredient is DDMP (2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one)."
  • the Applicant also contemplates an antioxidant lactic acid bacteria beverage containing DDMP as an active ingredient. That is, the third invention of the present application is "An antioxidant lactic acid bacteria drink containing DDMP as an active ingredient.”
  • the DDMP is preferably contained in the lactic acid bacteria beverage at 150 ⁇ M ( ⁇ mol/L) or more. That is, the fourth invention of the present application is "The lactic acid bacteria beverage according to claim 2 or 3, which contains 150 ⁇ M or more of DDMP.”
  • the lactic acid bacteria beverage preferably includes a step of using skim milk and sugars as raw materials and sterilizing the milk containing the raw materials with heat. That is, the fifth invention of the present application is ⁇ The lactic acid beverage according to any one of claims 1 to 4, wherein the lactic acid beverage is produced by a production process including a step of heat sterilizing charged milk containing skim milk and sugar as raw materials.''
  • the saccharides preferably contain glucose. That is, the sixth invention of the present application is "The lactic acid bacteria beverage according to Claim 5, wherein said sugar contains glucose.”
  • the present applicant also intends a lactic acid bacterium drink labeled on the product package or the like to the effect that it has an antioxidant effect. That is, the seventh invention of the present application is "A lactic acid bacteria drink labeled as having an antioxidant effect.”
  • the lactic acid bacteria beverage of the present invention contains DDMP as an antioxidant component and has an antioxidant effect. As a result, various useful effects can be obtained.
  • FIG. 1 is a diagram comparing the DPPH radical scavenging activities of the lactic acid bacteria beverage of the first embodiment of the present invention and other commercially available lactic acid bacteria beverages in Test Example 1.
  • FIG. FIG. 4 is a diagram comparing the DPPH radical scavenging activity (antioxidant power) of the lactic acid bacteria beverage of the first embodiment of the present invention and Trolox using the slope of the regression line.
  • FIG. 2 is a flow diagram showing a fractionation scheme of a fermentation broth in Test Example 2;
  • FIG. 4 is a diagram showing the antioxidant activity of each fraction separated in FIG. 3.
  • FIG. FIG. 4 is a diagram showing a column-separated chromatogram of the XAD4-25% ethanol-eluted fraction in FIG.
  • FIG. 6 is a chromatographic chart obtained by further purifying fraction 7 in FIG. 5 using an ODS column.
  • 7 is a chart diagram showing an absorption spectrum of a peak portion in FIG. 6.
  • FIG. 7 is a chart showing the results of LC/MS analysis of the peak portion in FIG. 6.
  • FIG. 7 is a chart showing the results of identification by GC/MS of the peak portion in FIG. 6;
  • FIG. 10 is an LC chart showing measurement of DDMP contained in the first embodiment of the present application and other lactic acid bacteria beverages on the market in Test Example 3;
  • FIG. 2 is a diagram comparing the antioxidant power of the lactic acid bacteria drink of the first embodiment of the present invention using ascorbic acid and the slope of the regression line.
  • FIG. 11 is a diagram comparing the antioxidant power of other lactic acid bacteria beverages of Company A using the slope of the regression line, as in the case of FIG. 11 .
  • FIG. 10 is an LC chart showing changes in the amount of DDMP depending on the heating time of charged milk, etc. in Test Example 4.
  • FIG. 10 is a diagram comparing amounts of DDMP produced when sugar raw materials are changed in Test Example 5.
  • FIG. 10 is a diagram showing the effect of adding lysine in Test Example 6.
  • FIG. FIG. 10 is a diagram showing the effect of adding various proteases in Test Example 7.
  • the lactic acid bacteria beverage referred to in the present invention is prepared by the following steps. That is, first, as a raw material mixing step, skim milk, water and milk as raw materials are mixed to prepare milk (milk medium), and then as a sterilization and cooling step, the raw milk is heat-sterilized at a high temperature and fermented. cool to the required temperature. Next, in the lactic acid bacterium inoculation step, a separately prepared seed bacterium (lactic acid bacterium cultured (preculture)) is added to the heated milk. Next, as a fermentation process, the tank is kept at a constant temperature and fermented.
  • syrup, fruit juice, or the like is added to the fermented liquid after cultivation, and the mixture is adjusted with dilution water as necessary.
  • the homogenized product is filled in a container to complete the lactic acid beverage.
  • lactic acid bacteria beverages There are two types of lactic acid bacteria beverages: “dairy lactic acid beverages” and “lactic acid bacteria beverages.”
  • non-fat milk solids components of milk from which milk fat and water have been removed
  • lactic acid bacteria drink refers to a beverage with a non-fat milk solids content of less than 3.0% and a lactic acid bacteria or yeast count of 1,000,000/ml or more.
  • the lactic acid bacteria drink in the present invention includes both the above-mentioned "lactic acid bacteria drink” and "lactic acid bacteria drink”.
  • ⁇ Preferred method for producing lactic acid bacteria beverage in the present invention Although the general method for producing a lactic acid bacteria beverage is as described above, it is particularly preferable to generally produce the lactic acid bacteria beverage of the present invention in the following manner. However, the present invention is not limited to the manufacturing method described below.
  • a milk (milk medium) is prepared so that the milk raw material, mainly skimmed milk, is 5-30% by weight and the sugar is 2-20% by weight. Also preferably, the milk raw material is 15 to 20% by weight and the saccharide is 10 to 15% by weight.
  • the saccharides in the prepared milk contain glucose. Specifically, it is preferable to use glucose or high-fructose liquid sugar. Moreover, it is preferable to add lysine as an amino acid.
  • the milk is heated at 80°C to 100°C for about 30 minutes to 180 minutes.
  • the heating time is preferably 60 to 160 minutes. Further, it is more preferably 90 minutes to 140 minutes. Most preferably, it is 100 to 120 minutes.
  • Fermentation liquid pre-culture
  • starter solution lactic acid bacteria
  • a fermented liquid can be obtained by culturing until the lactic acid acidity reaches
  • Syrup solution A syrup solution base containing sugar, glucose, fructose, etc. is prepared, heat sterilized and cooled to obtain a syrup solution.
  • the fermented liquid and the syrup liquid are mixed at a weight ratio of about 1:1 or 1:5 to 5:1 and homogenized to complete the lactic acid beverage.
  • a preferred method for producing a lactic acid bacteria beverage after heating the milk containing skim milk powder and sugars, the milk after heating and the culture solution of lactic acid bacteria are mixed and fermented for a predetermined time.
  • a manufacturing method is adopted in which syrup is mixed with the fermented liquid after the fermentation. but, In this step, it is preferable to set the heating time of the skimmed milk powder and the saccharide-containing raw milk as described above.
  • Antioxidant properties are said to have various functions in living organisms. For example, they are said to be able to suppress excessively active enzymes that cause fatigue (and aging). That is, it has been reported that free radicals including active oxygen are the cause of aging, cancer, and lifestyle-related diseases.
  • free radical theory of aging proposed by Harman, aging is caused by the oxidation of DNA, proteins, and lipids by free radicals produced mainly in mitochondria as a by-product of energy metabolism ((1) D Harman, Free radical involvement in aging. Pathophysiology and therapeutic implications. , Drugs Aging 1993 3(1) 60-80 (2) Wulf Droge, Free radicals in the physiological control of cell function. Physiol Rev 2002 82(1)47-951).
  • Reactive oxygen has extremely high reactivity (Nakamura Shigeo Chemistry of Reactive Oxygen and Antioxidants Nippon Medical School Medical Journal 2013 9 164-169). Active oxygen reacts with lipids to produce lipid peroxides, which are reported to cause arteriosclerosis and myocardial infarction (J L Witztum, D Steinberg, Role of oxidized low density lipoprotein in atherogenesis. Clin Invest. 1991 88(6) 1785-92).
  • a living body has a mechanism for removing active oxygen generated in the living body.
  • Hydrogen peroxide removal enzymes such as superoxide dismutase (SOD) and catalase detoxify active oxygen generated in the body.
  • SOD superoxide dismutase
  • catalase detoxify active oxygen generated in the body.
  • low-molecular-weight compounds having antioxidant activity, and the body protects itself from oxidative damage caused by active oxygen by biosynthesizing these compounds or taking them in from food.
  • antioxidants include polyphenols such as ascorbic acid (vitamin C), ⁇ -tocopherol (vitamin E), catechins contained in green tea, and resveratrol contained in red wine (Nakamura Shigeo Active Oxygen and Antioxidant Chemistry of Substances Nippon Medical School Medical Journal 2013 9 164-169).
  • yogurt and lactic acid bacteria beverages contain lactic acid bacteria and have an intestinal regulation effect (Iva Hojsak, Probiotics in Functional Gastrointestinal Disorders. Adv Exp Med Biol 2019 1125 121-137.), an immunomodulatory effect (Yueh-Ting Tsai, Po-Ching Cheng). , Tzu-Ming Pan, The immunomodulatory effects of lactic acid bacteria for improving immune functions and benefits. Paula Wroblewska, Piotr Adamczuk, Wojciech Silny, Probiotic lacticacid bacteria and their potential in the prevention and treatment of allergic diseases.
  • DDMP the antioxidant capacity of the lactic acid bacteria beverage was evaluated by the DPPH radical scavenging method, and the antioxidant substance was isolated and purified to be DDMP.
  • DDMP is known as a substance produced by the Maillard reaction (Tetrahedron Letters No. 15, pp. 1243-1246, 1970). is preferred.
  • a lactic acid bacteria beverage was prepared as follows.
  • the lactic acid bacteria drink was tested.
  • ⁇ Method for producing lactic acid bacteria beverage according to the first embodiment of the present invention A milk medium containing 8% by weight of skim milk powder and 5% by weight of high-fructose corn syrup is prepared and sterilized by heating at 100° C. for about 120 minutes. It was inoculated and cultured at 37° C. until a predetermined lactic acid acidity was reached to obtain a fermented liquid.
  • a syrup base containing sugar and high-fructose liquid sugar was prepared, heat-sterilized and cooled to obtain a syrup.
  • 500 ml of the above syrup solution was mixed with 500 ml of the above fermented liquid together with a small amount of flavoring and homogenized to obtain 1000 ml of a lactic acid bacteria drink containing 3 ⁇ 10 8 /ml or more of viable lactic acid bacteria.
  • the DPPH radical scavenging activity evaluation method was performed as follows. First, the following reagents were used. (1) 200 mM MES (2-morpholinoethanesulphonic acid) buffer (pH 6.0) (2) 400 ⁇ M DPPH (1,1-diphenyl-2-picrylhydrazyl) ethanol solution After adding ethanol (100 mL) to 15.76 mg of DPPH (manufactured by Tokyo Kasei Kogyo Co., Ltd., D4313), add a rotor and stir with a stirrer. Dissolved over 30 minutes to 1 hour in the dark. The DPPH solution was freshly prepared due to its low stability.
  • the sample was dissolved in 50% ethanol aqueous solution. After dispensing 100 ⁇ L of samples of various concentrations into a 96-well plate in which 50 ⁇ L of 200 mM MES buffer was dispensed, 50 ⁇ L of 400 ⁇ M DPPH solution was added to initiate the reaction. After reacting for 20 minutes at room temperature in the dark, the absorbance at 520 nm or 495 nm was measured with a spectrophotometer or microplate reader. The Trolox solution with the above concentration was similarly reacted by adding the DPPH solution. The DPPH radical scavenging activity was determined according to the following formula as the amount of Trolox corresponding to the added amount of the analytical sample, using the slope of the regression line prepared with Trolox.
  • DPPH radical scavenging activity slope of analytical sample (A520 or A495/( ⁇ L or ⁇ g/assay))/slope of Trolox (A520 or A495/(nmol/assay)
  • the antioxidant activity of the sample of the lactic acid bacteria drink of the present invention was evaluated by the DPPH radical scavenging method.
  • the relative DPPH radical scavenging rate was calculated from the ratio of the absorbance at 520 nm when 40% of the sample solution was added to the measurement system and the absorbance at 520 nm when no sample was added, and the antioxidant activity was evaluated.
  • Each sample was centrifuged at 40,000 ⁇ g for 10 min at 4° C., and the supernatant from which lactic acid bacteria were removed was used for measurement. The results are shown in FIG.
  • the lactic acid bacteria beverages and fermented milk from other commercial companies also have DPPH radical scavenging activity.
  • the lactic acid bacteria drink obtained by the production method of the first embodiment of the present invention exhibits higher antioxidant activity than the other commercially available lactic acid bacteria drinks and fermented milk in the antioxidant activity evaluation method by the DPPH method. rice field.
  • the antioxidant power was calculated as the amount equivalent to Trolox.
  • the antioxidant capacity of the supernatant of the lactic acid bacteria drink of the first embodiment was calculated to be 2.46 (nmol-Trolox equivalent/mg).
  • the 25% ethanol eluted fraction exhibited a relatively high DPPH radical scavenging activity of 56.68 (nmol-Trolox equivalent/mg), and the recovery amount was greater than the 50% ethanol eluted fraction. Therefore, the XAD4-25% ethanol elution fraction (XAD4-25% EtOH Fr.) was further separated and purified by HPLC. XAD4-25% EtOH Fr. was separated using a Shodex Asahipak GS320HQ column under the separation conditions described in the experimental method.
  • FIG. 5 shows the chromatogram and the DPPH elimination activity of each fraction.
  • DPPH scavenging activity in Figure 5 was calculated from the difference in absorbance at 492 nm between the control and the sample. Relatively high DPPH scavenging activity was observed in fraction 7 (GS Fr.7) collected at a retention time of 30 to 35 minutes. Therefore, GS Fr.7 was further purified using an ODS column.
  • the column separation conditions are as follows. ⁇ Column and Separation Conditions ⁇ Column: Inertsil ODS-2 5 ⁇ m (4.6 ⁇ 150mm) Mobile phase: acetonitrile/0.1% formic acid Gradient conditions: acetonitrile concentration 0min 0% ⁇ 10min 10% ⁇ 20min 95% ⁇ 22min 95% Flow rate: 1.0 mL/min, Detection wavelength: 295nm Column temperature: 40°C Injection volume: 50 ⁇ L
  • DPPH scavenging activity was observed in the peak around 8 min in the chromatographic chart shown in FIG. Next, when the absorption spectrum was measured for this peak, it showed an absorption maximum at 295 nm as shown in FIG. In addition, as a result of LC/MS analysis of this peak, m/z 145 was shown in positive ion mode (Fig. 8).
  • this peak was analyzed by GC/MS, and a library search was performed from the mass spectrum pattern of the obtained target peak, and as a result, it was identified as DDMP (CAS No. 28564-83-2) (Fig. 9).
  • DDMP CAS No. 28564-83-2
  • Test Example 3 Amount of DDMP contained in other products DDMP was identified as a substance having DPPH radical scavenging activity separated and purified in the production method of the present invention.
  • three types of products Company A-1, Company A-2, Company A-3) that can be purchased in the market of Company A were tested using the DDMP sample of the present invention, Company A-1.
  • DDMP concentration was measured.
  • FIG. 10 shows a chromatogram (for company A, only company A-1) separated using an ODS column for each sample.
  • Table 1 shows the DDMP concentration of each sample calculated from the DDMP calibration curve.
  • the lactic acid bacteria drink produced by the production method of the present invention exhibited a DDMP concentration that was about twice as high as that of another company's lactic acid drink (Company A).
  • the antioxidant power was 0.397 (mol-Trolox/mol). This antioxidant power was calculated to be about 35% for ascorbic acid 1.141 (mol-Trolox/mol) (Fig. 11).
  • the slope of this line is 1.
  • the slope ratios of the lactic acid bacteria drink of the first embodiment and the lactic acid bacteria drink of Company A to the DDMP standard were 1.533 and 1.161, respectively. From this result, the contribution of DDMP to the antioxidant activity of the lactic acid bacteria of the first embodiment of the present invention and the lactic acid drink of Company A is estimated to be about 65% (1/1.533) and about 86% (1/1.161). rice field.
  • the lactic acid bacteria beverage of the first embodiment is obtained by lactic acid fermentation of milk (milk culture medium) composed of skim milk powder and high-fructose corn syrup. In addition, heat treatment is performed for sterilization in the manufacturing process of the prepared milk.
  • the change in the amount of DDMP in the prepared milk obtained when the heat sterilization time of the prepared milk was set to 10 minutes and 120 minutes (corresponding to the first embodiment of the present invention) was investigated. . From the area ratio of the DDMP peak, the amount of DDMP in the lactic acid beverage after 120 minutes of heat sterilization was 41.5 times that after 10 minutes of heat sterilization (Fig. 13(A)). In the case of 120 minutes, the amount of DDMP slightly decreased in the step of inoculating the starter solution (culture solution) of lactic acid bacteria into the milk for 120 minutes and fermenting with the lactic acid bacteria (Fig. 13(B)). From these experimental results, it was found that DDMP affects the heating time of the charged milk. Since (A) and (B) of FIG. 13 are different experiments, the peak areas before heat sterilization 120 minutes (A) and heat sterilization 120 minutes (B) before fermentation are different.
  • DDMP concentration in the recovered supernatant was measured by HPLC.
  • the DDMP concentration in the milk after heat sterilization for 3 hours was high in the order of glucose (1909 ⁇ M), high fructose liquid sugar (1585 ⁇ M) and fructose (734 ⁇ M). As described above, in the present invention, it was found that it is preferable to use glucose as the sugar in the milk.
  • Test Example 6 Effect of adding lysine to milk The effect of adding lysine to milk was verified. Lysine was added to the experimental milk preparation shown in the glucose (test group 3) and the high-fructose corn syrup (test group 1) in Test Example 5, and the heating time was varied to investigate (Fig. 15). As a result, when lysine was added when glucose was used as the sugar source (test group 4) or when lysine was added when glucose-fructose liquid sugar was used as the sugar source (test group 5), the lysine concentration dependence DDMP increased significantly (Fig. 15) It was found that DDMP can be increased by adding lysine to the formula.

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Abstract

[Problem] The present invention addresses the problem of examining the antioxidant effect of a lactobacillus beverage. The present invention also addresses the problem of finding out an active ingredient thereof. [Solution] It was found that a lactobacillus beverage has an antioxidant effect. It was also found that an antioxidant substance as an active ingredient thereof is 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP).　The present invention pertains to a lactobacillus beverage containing an antioxidant component. The antioxidant component is DDMP.

Description

抗酸化作用を有する乳酸菌飲料Lactic acid drink with antioxidant effect
　本発明は乳酸菌飲料に関するものである。特に、抗酸化作用を有する乳酸菌飲料に関するものである。 The present invention relates to lactic acid beverages. In particular, it relates to a lactic acid bacteria drink having an antioxidant effect.
　乳酸菌飲料は広く利用されており、長年において幅広い年代に支持されている。例えば、ピルクル（登録商標：日清ヨーク株式会社）やヤクルト（登録商標：株式会社ヤクルト本社）等が知られている。当該乳酸菌飲料については、腸内環境を改善する機能を有する点が広く知られている。また、ストレス改善や、血糖低下、疲労回復等の優れた効果も奏するタイプも知られている。 Lactic acid beverages are widely used and have been supported by people of all ages for many years. For example, Pilkul (registered trademark: Nisshin York Co., Ltd.) and Yakult (registered trademark: Yakult Honsha Co., Ltd.) are known. The lactic acid bacteria drink is widely known to have the function of improving the intestinal environment. There are also known types that exhibit excellent effects such as alleviating stress, lowering blood sugar, and recovering from fatigue.
　一方、この乳酸菌飲料については上記の他にも有用な機能性や効果を奏することが期待される。例えば、抗酸化性については、従来まで乳酸菌飲料において抗酸化性を有する点、又その抗酸化性物質について先行する特許文献は知られていなかった。
　乳酸菌飲料の抗酸化性に関連する特許文献としては、特許文献１に乳酸菌飲料についての抗酸化剤の記載がある。しかし、当該特許文献は乳製品乳酸菌飲料にドコサヘキサエン酸（ＤＨＡ）を含ませることを主目的とし、その付随的成分として抗酸化剤を添加することが記載されているに過ぎない。 On the other hand, this lactic acid bacteria drink is expected to exhibit useful functionality and effects in addition to the above. For example, with regard to antioxidant properties, it has not been known in the past that lactic acid bacteria beverages have antioxidant properties, nor have there been prior patent documents regarding antioxidant substances.
As a patent document related to the antioxidant properties of lactic acid bacteria beverages, Patent Document 1 describes antioxidants for lactic acid bacteria beverages. However, the patent document merely describes that the main purpose of the lactic acid bacteria drink is to contain docosahexaenoic acid (DHA), and an antioxidant is added as an incidental component.
特開1995-327593号Japanese Patent Application Laid-Open No. 1995-327593
　そこで、本発明者らは乳酸菌飲料の抗酸化性について検討することを課題とした。また、その有効成分について見出すことを課題とした。 Therefore, the present inventors set a task to examine the antioxidant properties of lactic acid bacteria beverages. Moreover, it was made into the subject to discover about the active ingredient.
　本発明者らの鋭意研究の結果、乳酸菌飲料について、抗酸化作用を有する機能を確認し、本発明を完成させたのである。
　すなわち、本願第一の発明は、
“抗酸化作用を有する成分を含有する乳酸菌飲料。”、である。 As a result of diligent research by the present inventors, the present invention was completed by confirming that the lactic acid bacteria drink has an antioxidant effect.
That is, the first invention of the present application is
"A lactic acid bacteria drink containing an ingredient having an antioxidant action."
　次に、上記抗酸化作用を有する成分は、ＤＤＭＰ（2,3-dihydro-3,5-dihydroxy-6-
methyl-4H-pyran-4-one）が含まれていることを見出した。
　すなわち、本願第二の発明は、
“前記成分がＤＤＭＰ（2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one）である請求項１に記載の乳酸菌飲料。”、である。 Next, the component with antioxidant action is DDMP (2,3-dihydro-3,5-dihydroxy-6-
methyl-4H-pyran-4-one).
That is, the second invention of the present application is
"The lactic acid bacteria beverage according to claim 1, wherein said ingredient is DDMP (2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one)."
　次に本出願人は、ＤＤＭＰを有効成分とする抗酸化用乳酸菌飲料も意図している。すなわち、本願第三の発明は、
“ＤＤＭＰを有効成分とする抗酸化用乳酸菌飲料。”、である。 Next, the Applicant also contemplates an antioxidant lactic acid bacteria beverage containing DDMP as an active ingredient. That is, the third invention of the present application is
"An antioxidant lactic acid bacteria drink containing DDMP as an active ingredient."
　次に、前記ＤＤＭＰについては、当該乳酸菌飲料において１５０μM（μmol/L）以上含有することが好ましい。
　すなわち、本願第四の発明は、
“ＤＤＭＰを１５０μM以上含有する請求項２又は３に記載の乳酸菌飲料。”、である。 Next, the DDMP is preferably contained in the lactic acid bacteria beverage at 150 μM (μmol/L) or more.
That is, the fourth invention of the present application is
"The lactic acid bacteria beverage according to claim 2 or 3, which contains 150 µM or more of DDMP."
　次に、前記乳酸菌飲料は、原料として脱脂乳及び糖類を利用し、当該原料を含む仕込乳を熱で殺菌する工程を含むことが好ましい。
　すなわち、本願第五の発明は、
“前記乳酸菌飲料が、原料として脱脂乳及び糖類を含む仕込乳を熱で殺菌する工程を含む製造工程によって製造される請求項１～４のいずれかに記載の乳酸菌飲料。”、である。 Next, the lactic acid bacteria beverage preferably includes a step of using skim milk and sugars as raw materials and sterilizing the milk containing the raw materials with heat.
That is, the fifth invention of the present application is
``The lactic acid beverage according to any one of claims 1 to 4, wherein the lactic acid beverage is produced by a production process including a step of heat sterilizing charged milk containing skim milk and sugar as raw materials.''
　次に、前記本願第四の発明においては、糖類がぶどう糖を含有することが好ましい。
　すなわち、本願第六の発明は、
“前記糖類がぶどう糖を含有する請求項５に記載の乳酸菌飲料。”、である。 Next, in the fourth invention of the present application, the saccharides preferably contain glucose.
That is, the sixth invention of the present application is
"The lactic acid bacteria beverage according to Claim 5, wherein said sugar contains glucose."
　次に、本出願人は、抗酸化作用を有する旨を商品パッケージ等に表記した乳酸菌飲料も意図している。
　すなわち、本願第七の発明は、
“抗酸化作用を有する旨を表示した乳酸菌飲料。”、である。 Next, the present applicant also intends a lactic acid bacterium drink labeled on the product package or the like to the effect that it has an antioxidant effect.
That is, the seventh invention of the present application is
"A lactic acid bacteria drink labeled as having an antioxidant effect."
　本発明の乳酸菌飲料は抗酸化成分としてＤＤＭＰを含有し、抗酸化作用を有する。これによって種々の有用な効果を奏することができる。
The lactic acid bacteria beverage of the present invention contains DDMP as an antioxidant component and has an antioxidant effect. As a result, various useful effects can be obtained.
試験例１において本発明の第一の実施態様の乳酸菌飲料及び他の市販の乳酸菌飲料のＤＰＰＨラジカル消去活性を比較した図である。1 is a diagram comparing the DPPH radical scavenging activities of the lactic acid bacteria beverage of the first embodiment of the present invention and other commercially available lactic acid bacteria beverages in Test Example 1. FIG. 本発明の第一の実施態様の乳酸菌飲料及びTroloxのＤＰＰＨラジカル消去活性（抗酸化力）を回帰直線の傾きを用いて比較した図である。FIG. 4 is a diagram comparing the DPPH radical scavenging activity (antioxidant power) of the lactic acid bacteria beverage of the first embodiment of the present invention and Trolox using the slope of the regression line. 試験例２における発酵液の分画スキームを示したフロー図である。FIG. 2 is a flow diagram showing a fractionation scheme of a fermentation broth in Test Example 2; 図３における分画した各フラクションの抗酸化活性を示した図である。FIG. 4 is a diagram showing the antioxidant activity of each fraction separated in FIG. 3. FIG. 図３におけるXAD4-25%エタノール溶出フラクションについてカラム・分離したクロマトチャートと各フラクションのＤＰＰＨ消去活性を示した図である。FIG. 4 is a diagram showing a column-separated chromatogram of the XAD4-25% ethanol-eluted fraction in FIG. 3 and the DPPH elimination activity of each fraction. 図５におけるフラクション7をODSカラムを用いてさらに精製を行ったクロマトチャート図である。FIG. 6 is a chromatographic chart obtained by further purifying fraction 7 in FIG. 5 using an ODS column. 図６におけるピーク部分の吸収スペクトルを示したチャート図である。7 is a chart diagram showing an absorption spectrum of a peak portion in FIG. 6. FIG. 図６におけるピーク部分のLC/MSによる分析を実施した結果のチャート図である。FIG. 7 is a chart showing the results of LC/MS analysis of the peak portion in FIG. 6. FIG. 図６におけるピーク部分のGC/MSによる同定を示した結果のチャート図である。FIG. 7 is a chart showing the results of identification by GC/MS of the peak portion in FIG. 6; 試験例３における本願の第一実施態様及び市場の他の乳酸菌飲料に含まれるＤＤＭＰを測定したＬＣチャート図である。FIG. 10 is an LC chart showing measurement of DDMP contained in the first embodiment of the present application and other lactic acid bacteria beverages on the market in Test Example 3; 本発明の第一の実施態様の乳酸菌飲料の抗酸化力についてアスコルビン酸と回帰直線の傾きを利用して比較した図である。FIG. 2 is a diagram comparing the antioxidant power of the lactic acid bacteria drink of the first embodiment of the present invention using ascorbic acid and the slope of the regression line. 図１１の場合と同様に他のＡ社の乳酸菌飲料の抗酸化力について回帰直線の傾きを利用して比較した図である。FIG. 11 is a diagram comparing the antioxidant power of other lactic acid bacteria beverages of Company A using the slope of the regression line, as in the case of FIG. 11 . 試験例４における仕込乳の加熱時間等によるＤＤＭＰ量の変化を示したＬＣチャート図である。FIG. 10 is an LC chart showing changes in the amount of DDMP depending on the heating time of charged milk, etc. in Test Example 4. FIG. 試験例５における糖原料を変えた場合の生成するＤＤＭＰ量を比較した図である。FIG. 10 is a diagram comparing amounts of DDMP produced when sugar raw materials are changed in Test Example 5. FIG. 試験例６におけるリジンの添加による効果を示した図である。FIG. 10 is a diagram showing the effect of adding lysine in Test Example 6. FIG. 試験例７における各種プロテアーゼの添加効果を示した図である。FIG. 10 is a diagram showing the effect of adding various proteases in Test Example 7. FIG.
　以下の本発明の内容を説明する。
─乳酸菌飲料─
　本発明にいう乳酸菌飲料とは、以下のような工程で調製される。すなわち、まず、原料の混合工程として、原料となる脱脂乳、水や牛乳を混ぜ合わせ仕込乳（乳培地）を調製し、次に殺菌・冷却工程として、仕込乳を高温で加熱殺菌し、発酵に必要な温度にまで冷やす。
　次に、乳酸菌の接種工程として、別に調製しておいた種菌（乳酸菌を培養しておいたもの（前培養））を加熱後の仕込乳に加える。次に、発酵工程としてタンク内で一定の温度に保ち発酵を行う。次に、冷却後、混合・希釈工程として、培養後の発酵液にシロップや果汁等を加え、必要に応じて希釈水で調整する。これを均質化したものを容器に充填して乳酸菌飲料が完成する。 The contents of the present invention are described below.
─Lactic Acid Beverage─
The lactic acid bacteria beverage referred to in the present invention is prepared by the following steps. That is, first, as a raw material mixing step, skim milk, water and milk as raw materials are mixed to prepare milk (milk medium), and then as a sterilization and cooling step, the raw milk is heat-sterilized at a high temperature and fermented. cool to the required temperature.
Next, in the lactic acid bacterium inoculation step, a separately prepared seed bacterium (lactic acid bacterium cultured (preculture)) is added to the heated milk. Next, as a fermentation process, the tank is kept at a constant temperature and fermented. Next, after cooling, as a mixing/dilution step, syrup, fruit juice, or the like is added to the fermented liquid after cultivation, and the mixture is adjusted with dilution water as necessary. The homogenized product is filled in a container to complete the lactic acid beverage.
　尚、乳酸菌飲料の種類別としては、「乳製品乳酸菌飲料」と「乳酸菌飲料」がある。まず、「乳製品乳酸菌飲料」については、無脂乳固形分（牛乳から乳脂肪分と水分を除いた成分）を3.0％以上含み、乳酸菌数又は酵母数が1000万/ml以上のものをいい、生菌タイプと殺菌タイプがある。
　次に、「乳酸菌飲料」については、無脂乳固形分が3.0％未満で乳酸菌数又は酵母数が100万/ml以上のものをいう。
　本発明における乳酸菌飲料とは、上記の「乳製品乳酸菌飲料」及び「乳酸菌飲料」のいずれも含むものとする。 There are two types of lactic acid bacteria beverages: "dairy lactic acid beverages" and "lactic acid bacteria beverages." First, regarding "dairy lactic acid bacteria beverages", non-fat milk solids (components of milk from which milk fat and water have been removed) contain 3.0% or more, and the number of lactic acid bacteria or yeast is 10 million/ml or more. There are live bacteria type and sterilization type.
Next, "lactic acid bacteria drink" refers to a beverage with a non-fat milk solids content of less than 3.0% and a lactic acid bacteria or yeast count of 1,000,000/ml or more.
The lactic acid bacteria drink in the present invention includes both the above-mentioned "lactic acid bacteria drink" and "lactic acid bacteria drink".
─本発明における好ましい乳酸菌飲料の製造方法─
　一般的な乳酸菌飲料の製造方法は上述した通りであるが、特に、本発明の乳酸菌飲料は概ね以下のように製造することが好ましい。但し、本発明は以下の製造方法に限定されるものではない。 ─Preferred method for producing lactic acid bacteria beverage in the present invention─
Although the general method for producing a lactic acid bacteria beverage is as described above, it is particularly preferable to generally produce the lactic acid bacteria beverage of the present invention in the following manner. However, the present invention is not limited to the manufacturing method described below.
（１）仕込乳溶解（乳培地の調製）
　脱脂乳を中心とした乳原料を5～30重量％、糖類を2～20重量％となる程度に仕込乳（乳培地）を調製する。また、好ましくは、乳原料を15～20重量％、糖類を10～15重量％である。
　尚、仕込乳における糖類は、ぶどう糖を含んでいることが好ましい。具体的には、ぶどう糖やぶどう糖果糖液糖を利用することが好ましい。また、アミノ酸としてリジンを添加することが好ましい。 (1) Milk dissolution (preparation of milk medium)
A milk (milk medium) is prepared so that the milk raw material, mainly skimmed milk, is 5-30% by weight and the sugar is 2-20% by weight. Also preferably, the milk raw material is 15 to 20% by weight and the saccharide is 10 to 15% by weight.
In addition, it is preferable that the saccharides in the prepared milk contain glucose. Specifically, it is preferable to use glucose or high-fructose liquid sugar. Moreover, it is preferable to add lysine as an amino acid.
（２）加熱
　前記仕込乳を80℃～100℃で30分～180分程度加熱する。尚、加熱時間については好ましくは60分～160分である。また、さらに好ましくは、90分～140分である。最も好ましくは、100分～120分とする。 (2) Heating The milk is heated at 80°C to 100°C for about 30 minutes to 180 minutes. Incidentally, the heating time is preferably 60 to 160 minutes. Further, it is more preferably 90 minutes to 140 minutes. Most preferably, it is 100 to 120 minutes.
（３）発酵液（前培養）
　乳酸菌（ラクトバチルス、ラクトコッカス、ペディオコッカス、ロイコノストック、ストレプトコッカス、エンテロコッカス等）を培養した培養液（スターター液）を前記の加熱後の乳培地に添加して30℃～40℃程度で所定の乳酸酸度となるまで培養して発酵液を得ることができる。 (3) Fermentation liquid (pre-culture)
Add a culture solution (starter solution) in which lactic acid bacteria (lactobacillus, lactococcus, pediococcus, leuconostoc, streptococcus, enterococcus, etc.) are cultured to the above-mentioned milk medium after heating and heat at about 30 to 40 ° C. A fermented liquid can be obtained by culturing until the lactic acid acidity reaches
（４）シロップ液
　砂糖、ぶどう糖、果糖等を含むシロップ液ベースを調製し、加熱殺菌及び冷却を行ってシロップ液を得る。 (4) Syrup solution A syrup solution base containing sugar, glucose, fructose, etc. is prepared, heat sterilized and cooled to obtain a syrup solution.
（５）混合・均質化
　前記発酵液とシロップ液を１：１又は１：５～５：１程度の重量比率で混合、均質化して乳酸菌飲料を完成させる。 (5) Mixing and homogenization The fermented liquid and the syrup liquid are mixed at a weight ratio of about 1:1 or 1:5 to 5:1 and homogenized to complete the lactic acid beverage.
　このように、本発明においては乳酸菌飲料の好ましい製造方法として、脱脂粉乳及び糖類を含有する仕込乳を加熱後、当該加熱後の仕込乳と乳酸菌の培養液を混合して所定時間の発酵後、当該発酵後の発酵液にシロップ液を混合して調製する製造方法を採用する。が、
　本工程のうち、脱脂粉乳及び糖類を含有する仕込乳の加熱時間を上述のようにすることが好ましい。
Thus, in the present invention, as a preferred method for producing a lactic acid bacteria beverage, after heating the milk containing skim milk powder and sugars, the milk after heating and the culture solution of lactic acid bacteria are mixed and fermented for a predetermined time. A manufacturing method is adopted in which syrup is mixed with the fermented liquid after the fermentation. but,
In this step, it is preferable to set the heating time of the skimmed milk powder and the saccharide-containing raw milk as described above.
─抗酸化性─
　抗酸化性については、生体において種々の機能があると言われているが、例えば、疲労（と老化）の原因である過剰な活性酵素を抑えることができるとされている。
　すなわち、活性酸素をはじめとするフリーラジカルは老化やがん、生活習慣病の原因であることが報告されている。Harmanによって提唱された老化のフリーラジカル説では、エネルギー代謝の副産物として主としてミトコンドリアで産生されたフリーラジカルが、DNAやタンパク質、脂質などを酸化することが老化の原因であるとする（(1) D Harman, Free radical involvement in aging. Pathophysiology and therapeutic implications.
, Drugs Aging 1993 3(1) 60-80　（2) Wulf Droge, Free radicals in the physiological control of cell function. Physiol Rev 2002 82(1)47-951)。ヒトは呼吸によって酸素を体内に取り込み、ミトコンドリアに存在する電子伝達系によりATPを産生し、生命活動に必要なエネルギーを獲得している。この過程でさまざまな活性酸素種が発生する。活性酸素は非常に高い反応性を有する（中村成夫 活性酸素と抗酸化物質の化学 日本医科大学医学会雑誌2013 9 164―169)。活性酸素は脂質と反応することで脂質過酸化物が生成し、動脈硬化、心筋梗塞などの原因となることが報告されている（J L Witztum, D Steinberg, Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest.
1991 88(6) 1785-92)。 ─ Antioxidant ─
Antioxidant properties are said to have various functions in living organisms. For example, they are said to be able to suppress excessively active enzymes that cause fatigue (and aging).
That is, it has been reported that free radicals including active oxygen are the cause of aging, cancer, and lifestyle-related diseases. According to the free radical theory of aging proposed by Harman, aging is caused by the oxidation of DNA, proteins, and lipids by free radicals produced mainly in mitochondria as a by-product of energy metabolism ((1) D Harman, Free radical involvement in aging. Pathophysiology and therapeutic implications.
, Drugs Aging 1993 3(1) 60-80 (2) Wulf Droge, Free radicals in the physiological control of cell function. Physiol Rev 2002 82(1)47-951). Humans take oxygen into the body through respiration, produce ATP through the electron transport system present in mitochondria, and acquire the energy necessary for life activities. Various reactive oxygen species are generated in this process. Reactive oxygen has extremely high reactivity (Nakamura Shigeo Chemistry of Reactive Oxygen and Antioxidants Nippon Medical School Medical Journal 2013 9 164-169). Active oxygen reacts with lipids to produce lipid peroxides, which are reported to cause arteriosclerosis and myocardial infarction (J L Witztum, D Steinberg, Role of oxidized low density lipoprotein in atherogenesis. Clin Invest.
1991 88(6) 1785-92).
　また、活性酸素は核酸と反応するとDNA鎖切断や核酸塩基の酸化的修飾によるDNAの変異により発がんを引き起こす可能性がある（Miral Dizdaroglu, Pawel Jaruga, Mechanisms of free radical-induced damage to DNA. Free Radic Res 2012 46(4) 382-419)。
　生体において発生した活性酸素を除去する仕組みを生体は備えている。スーパーオキシドディスムターゼ(SOD)やカタラーゼといった過酸化水素除去酵素などにより、生体内で発生した活性酸素が無害化される。一方、天然には抗酸化作用を有する低分子化合物が数多く存在し、生体はこれらを生合成したり、食物から取り込むことによって、活性酸素による酸化傷害から生体を防御している。代表的な天然の抗酸化物質としてアスコルビン酸（ビタミンC）、α-トコフェロール（ビタミンE）、緑茶に含まれるカテキン、赤ワインに含まれるレスベラトロールなどのポリフェノールがある（中村成夫 活性酸素と抗酸化物質の化学 日本医科大学医学会雑誌2013 9 164―169)。 In addition, when reactive oxygen reacts with nucleic acids, it may cause carcinogenesis through DNA mutation due to DNA strand breakage and oxidative modification of nucleobases (Miral Dizdaroglu, Pawel Jaruga, Mechanisms of free radical-induced damage to DNA. Free Radic Res 2012 46(4) 382-419).
A living body has a mechanism for removing active oxygen generated in the living body. Hydrogen peroxide removal enzymes such as superoxide dismutase (SOD) and catalase detoxify active oxygen generated in the body. On the other hand, there are many naturally occurring low-molecular-weight compounds having antioxidant activity, and the body protects itself from oxidative damage caused by active oxygen by biosynthesizing these compounds or taking them in from food. Representative natural antioxidants include polyphenols such as ascorbic acid (vitamin C), α-tocopherol (vitamin E), catechins contained in green tea, and resveratrol contained in red wine (Nakamura Shigeo Active Oxygen and Antioxidant Chemistry of Substances Nippon Medical School Medical Journal 2013 9 164-169).
　慢性疲労の原因については現時点で不明な点が多いが、慢性疲労者においてはフリーラジカルの産生が多く、酸化ストレスが原因である可能性が報告されている(A C Logan, C Wong, Chronic fatigue syndrome: oxidative stress and dietary modifications. Altern
Med Rev 2001 6(5) 450-9)。抗酸化作用を有するイミダゾールジペプチドを400mg配合した飲料の継続摂取において、日常作業において疲労を自覚している健常者の疲労感を軽減することが報告されている(清水惠一郎, 福田 正博, 山本 晴章, イミダゾールジペプチド配合飲料の日常的な作業のなかで疲労を自覚している健常者に対する継続摂取による有用性 薬理と治療）vol.37 no.3 2009 37(3) 255-637)。このように抗酸化物質を含む食品の日常的な摂取は疲労感を軽減する効果が期待される。 Although the cause of chronic fatigue is still largely unknown at present, it has been reported that chronic fatigue sufferers produce more free radicals and that oxidative stress may be the cause (A C Logan, C Wong, Chronic fatigue syndrome : oxidative stress and dietary modifications.
Med Rev 2001 6(5) 450-9). It has been reported that continuous ingestion of a drink containing 400 mg of imidazole dipeptide, which has an antioxidant effect, reduces fatigue in healthy subjects who are aware of fatigue during daily work (Keiichiro Shimizu, Masahiro Fukuda, Haruaki Yamamoto) , Usefulness of continuous ingestion of imidazole dipeptide-containing beverages for healthy subjects who are aware of fatigue during daily work. Pharmacology and Treatment) vol.37 no. Thus, daily intake of foods containing antioxidants is expected to have the effect of reducing fatigue.
　ここで、ヨーグルトや乳酸菌飲料は乳酸菌を含み、整腸作用(Iva Hojsak, Probiotics in Functional Gastrointestinal Disorders. Adv Exp Med Biol 2019 1125 121-137.)、免疫調節作用(Yueh-Ting Tsai, Po-Ching Cheng, Tzu-Ming Pan, The immunomodulatory effects of lactic acid bacteria for improving immune functions and benefits. Appl Microbiol Biotechnol 2012 96(4)853-62)、花粉症、アトピー性皮膚炎といったアレルギーの予防(Wioletta Zukiewicz-Sobczak, Paula Wroblewska, Piotr Adamczuk, Wojciech Silny, Probiotic lacticacid bacteria and their potential in the prevention and treatment of allergic diseases. Cent Eur JImmunol 2014 39(1) 104-8)、潰瘍性大腸炎の改善(Maria Jose Saez-Lara, Carolina Gomez-Llorente, Julio Plaza-Diaz, Angel Gil, The role ofprobiotic lactic acid bacteria and bifidobacteria in the prevention and treatment of inflammatorybowel disease and other related diseases: a systematic review of randomized human clinical trials.Biomed Res Int 2015 2015:505878)といったさまざまな生理活性が報告されている。 Here, yogurt and lactic acid bacteria beverages contain lactic acid bacteria and have an intestinal regulation effect (Iva Hojsak, Probiotics in Functional Gastrointestinal Disorders. Adv Exp Med Biol 2019 1125 121-137.), an immunomodulatory effect (Yueh-Ting Tsai, Po-Ching Cheng). , Tzu-Ming Pan, The immunomodulatory effects of lactic acid bacteria for improving immune functions and benefits. Paula Wroblewska, Piotr Adamczuk, Wojciech Silny, Probiotic lacticacid bacteria and their potential in the prevention and treatment of allergic diseases. Carolina Gomez-Llorente, Julio Plaza-Diaz, Angel Gil, The role of probiotic lactic acid bacteria and bifidobacteria in the prevention and treatment of inflammatorybowel disease and other related diseases: a systematic review of randomized human clinical trials.Biomed Res Int 015085 2015 ) have been reported to have various physiological activities.
　一方、乳酸菌飲料の抗酸化活性についての報告は少なく、勿論、乳酸菌飲料に含まれる抗酸化活性物質については同定されていない。今回、本発明者らは乳製品乳酸菌飲料についてＤＰＰＨ ラジカル消去法により抗酸化活性を評価し、乳製品乳酸菌飲料に含まれる抗酸化物質を分離・精製し、DDMPを同定した。 On the other hand, there are few reports on the antioxidant activity of lactic acid bacteria drinks, and of course, the antioxidant substances contained in lactic acid bacteria drinks have not been identified. This time, the present inventors evaluated the antioxidant activity of a lactic acid bacteria milk drink by the DPPH radical scavenging method, separated and purified the antioxidant substances contained in the lactic acid bacteria milk drink, and identified DDMP.
─ＤＤＭＰ─
　本件発明においては、乳酸菌飲料における抗酸化能をＤＰＰＨラジカル消去法により評価するとともに、その抗酸化性物質を分離・精製し、ＤＤＭＰであることを見出した。ここでＤＤＭＰとは、メイラード反応によって生成する物質として知られている（Tetrahedron Letters No. 15, pp. 1243-1246, 1970.）特に、本発明の乳酸菌飲料においては、ＤＤＭＰについて１５０μM以上を含有することが好ましい。
─DDMP─
In the present invention, the antioxidant capacity of the lactic acid bacteria beverage was evaluated by the DPPH radical scavenging method, and the antioxidant substance was isolated and purified to be DDMP. Here, DDMP is known as a substance produced by the Maillard reaction (Tetrahedron Letters No. 15, pp. 1243-1246, 1970). is preferred.
以下に本発明の実施例を記載する Examples of the present invention are described below.
[試験例１]本発明の乳酸菌飲料及び他社の乳酸菌飲料のDPPHのラジカル消去活性の比較
　本発明の第一の実施態様として以下のように乳酸菌飲料を調製した。当該乳酸菌飲料について試験した。
─本発明の第一実施態様の乳酸菌飲料の製法─
　脱脂粉乳を８重量％、ぶどう糖果糖液糖を５重量％含む乳培地を調製し１００℃、約１２０分加熱殺菌後、ラクトバチルス・パラカゼイのスターター液（培養液）を前記加熱後の乳培地に接種し、３７℃で所定の乳酸酸度となるまで培養し、発酵液を得た。
　一方、砂糖、ぶどう糖果糖液糖を含むシロップ液ベースを調製し、加熱殺菌及び冷却を行ってシロップ液を得た。
　その後、上記シロップ液５００ｍｌを、少量の香料とともに上記発酵液５００ｍｌと混合・均質化処理することにより、乳酸菌生菌を３×１０８／ｍｌ以上含有する乳酸菌飲料１０００ｍｌを得た。 [Test Example 1] Comparison of radical scavenging activity of DPPH between lactic acid bacteria beverage of the present invention and lactic acid bacteria beverages of other companies As a first embodiment of the present invention, a lactic acid bacteria beverage was prepared as follows. The lactic acid bacteria drink was tested.
─Method for producing lactic acid bacteria beverage according to the first embodiment of the present invention─
A milk medium containing 8% by weight of skim milk powder and 5% by weight of high-fructose corn syrup is prepared and sterilized by heating at 100° C. for about 120 minutes. It was inoculated and cultured at 37° C. until a predetermined lactic acid acidity was reached to obtain a fermented liquid.
On the other hand, a syrup base containing sugar and high-fructose liquid sugar was prepared, heat-sterilized and cooled to obtain a syrup.
After that, 500 ml of the above syrup solution was mixed with 500 ml of the above fermented liquid together with a small amount of flavoring and homogenized to obtain 1000 ml of a lactic acid bacteria drink containing 3×10 8 /ml or more of viable lactic acid bacteria.
─ＤＰＰＨラジカル消去活性評価法─
　ＤＰＰＨラジカル消去活性評価法については以下のように行った。まず、試薬としては以下を使用した。
(1)200 mM MES（2-morpholinoethanesulphonic acid）緩衝液（pH6.0）
(2)400 μM ＤＰＰＨ（1,1-diphenyl-2-picrylhydrazyl）エタノール溶液
　ＤＰＰＨ（東京化成工業社製，D4313）15.76mg にエタノール（100mL）を添加後、回転子を入れ、スターラーで撹拌して遮光下で30 分～1 時間かけて溶解した。ＤＰＰＨ溶液は安定性が低いことから用時調製した。
(3)2.0mM Trolox stock solutionと検量線用Trolox溶液
　Trolox（Wako社製，209-18881）12.51mgを50%エタノール水溶液で溶解し、25mLに定容した。Trolox stock solutionは200μLずつ分注し、-40°Cで保管した。2.0mM Trolox stock solution（200μL）に50%エタノール水溶液（3.9mL）を添加し、100μM Trolox溶液を使用直前に調製した。100μM Trolox 溶液を50%エタノール水溶液で希釈し、80，60，40,20mM Trolox溶液を調製した。 ─DPPH radical scavenging activity evaluation method─
The DPPH radical scavenging activity evaluation method was performed as follows. First, the following reagents were used.
(1) 200 mM MES (2-morpholinoethanesulphonic acid) buffer (pH 6.0)
(2) 400 μM DPPH (1,1-diphenyl-2-picrylhydrazyl) ethanol solution After adding ethanol (100 mL) to 15.76 mg of DPPH (manufactured by Tokyo Kasei Kogyo Co., Ltd., D4313), add a rotor and stir with a stirrer. Dissolved over 30 minutes to 1 hour in the dark. The DPPH solution was freshly prepared due to its low stability.
(3) 2.0 mM Trolox stock solution and Trolox solution for calibration curve Trolox (manufactured by Wako, 209-18881) (12.51 mg) was dissolved in 50% ethanol aqueous solution, and the volume was adjusted to 25 mL. 200 μL of Trolox stock solution was dispensed and stored at -40°C. 50% ethanol aqueous solution (3.9 mL) was added to 2.0 mM Trolox stock solution (200 μL) to prepare 100 μM Trolox solution immediately before use. A 100 μM Trolox solution was diluted with a 50% ethanol aqueous solution to prepare 80, 60, 40 and 20 mM Trolox solutions.
　サンプルは50%エタノール水溶液に溶解した。200mM MES緩衝液を50μL分注した96ウェルプレートに各種濃度のサンプルを100μL分注したのち、400μM ＤＰＰＨ溶液を50μL添加し、反応を開始させた。室温、遮光下で20分間反応後、分光光度計またはマイクロプレートリーダーで520nmまたは495nmの吸光度を測定した。上記濃度のTrolox溶液も同様にＤＰＰＨ溶液を加えて反応を行った。ＤＰＰＨラジカル消去活性はTroloxで作成した回帰直線の傾きを用いて、分析試料の添加量に相当するTrolox量として以下の計算式に従って求めた。
　ＤＰＰＨラジカル消去活性（nmol-Trolox 相当量/mol or mg）＝分析試料の傾き（A520またはA495/（μL or μg/assay））／Troloxの傾き（A520またはA495/（nmol/assay） The sample was dissolved in 50% ethanol aqueous solution. After dispensing 100 μL of samples of various concentrations into a 96-well plate in which 50 μL of 200 mM MES buffer was dispensed, 50 μL of 400 μM DPPH solution was added to initiate the reaction. After reacting for 20 minutes at room temperature in the dark, the absorbance at 520 nm or 495 nm was measured with a spectrophotometer or microplate reader. The Trolox solution with the above concentration was similarly reacted by adding the DPPH solution. The DPPH radical scavenging activity was determined according to the following formula as the amount of Trolox corresponding to the added amount of the analytical sample, using the slope of the regression line prepared with Trolox.
DPPH radical scavenging activity (nmol-Trolox equivalent/mol or mg) = slope of analytical sample (A520 or A495/(µL or µg/assay))/slope of Trolox (A520 or A495/(nmol/assay)
─試験法─
　本発明の乳酸菌飲料についてＤＰＰＨラジカル消去法によりサンプルの抗酸化活性を評価した。測定系にサンプル液を40%添加した際の520nmの吸光度とサンプル無添加の520nmの吸光度との比から相対的ＤＰＰＨラジカル消去率を算出し、抗酸化活性を評価した。各サンプルは40,000×g、10min、4℃の条件で遠心分離を行い、乳酸菌菌体を除去した上清を測定に用いた。結果を図１に示す。 ─Test method─
The antioxidant activity of the sample of the lactic acid bacteria drink of the present invention was evaluated by the DPPH radical scavenging method. The relative DPPH radical scavenging rate was calculated from the ratio of the absorbance at 520 nm when 40% of the sample solution was added to the measurement system and the absorbance at 520 nm when no sample was added, and the antioxidant activity was evaluated. Each sample was centrifuged at 40,000×g for 10 min at 4° C., and the supernatant from which lactic acid bacteria were removed was used for measurement. The results are shown in FIG.
─結果─
　本発明の第一の実施態様の方法で調製した乳酸菌飲料についてＤＰＰＨラジカル消去活性を有することを確認した。 ─ Results ─
It was confirmed that the lactic acid bacteria beverage prepared by the method of the first embodiment of the present invention has DPPH radical scavenging activity.
─他の市場の乳酸菌飲料についてのＤＰＰＨラジカル消去活性評価─
　本発明の第一の実施態様の乳酸菌飲料と比較するために他の市販の各社の乳酸菌飲料や発酵乳（Ａ社、Ｂ社-1、Ｂ社-2、Ｂ社-3、Ｃ社、Ｄ社）を市場で購入した。
　当該各社の乳酸菌飲料、発酵乳について上述のＤＰＰＨラジカル消去活性評価法を実施した。測定の結果を図１に示す。 ─Evaluation of DPPH radical scavenging activity of lactic acid bacteria drinks in other markets─
In order to compare with the lactic acid beverage of the first embodiment of the present invention, other commercially available lactic acid beverages and fermented milk (Company A, Company B-1, Company B-2, Company B-3, Company C, D company) was purchased on the market.
The DPPH radical scavenging activity evaluation method described above was performed on the lactic acid bacteria beverages and fermented milk of each company. The results of the measurements are shown in FIG.
　他の市販の各社の乳製品乳酸菌飲料、発酵乳についてもＤＰＰＨラジカル消去活性を有することを確認した。一方、本発明の第一の実施態様の製法により得られた乳酸菌飲料はＤＰＰＨ法による抗酸化活性評価法において他の市販の各社の乳製品乳酸菌飲料、発酵乳に比べて高い抗酸化活性を示した。 It was confirmed that the lactic acid bacteria beverages and fermented milk from other commercial companies also have DPPH radical scavenging activity. On the other hand, the lactic acid bacteria drink obtained by the production method of the first embodiment of the present invention exhibits higher antioxidant activity than the other commercially available lactic acid bacteria drinks and fermented milk in the antioxidant activity evaluation method by the DPPH method. rice field.
─Trolox相当量の算出─
　本発明の第一の実施態様の製造方法によって得られた乳酸菌飲料のＤＰＰＨラジカル消去活性はTroloxで作成した回帰直線の傾きを用いて、分析試料の添加量に相当するTrolox量として以下の計算式に従って求めた。
　ＤＰＰＨラジカル消去活性（nmol-Trolox相当量/mol or mg）＝分析試料の傾き（A520またはA495/（μL or μg/assay））／Trolox の傾き（A520またはA495 /（nmol/assay）とした。当該結果を図２に示す。 ─Calculation of Trolox Equivalent Amount─
The DPPH radical scavenging activity of the lactic acid bacteria beverage obtained by the production method of the first embodiment of the present invention is calculated using the following formula as the amount of Trolox corresponding to the added amount of the analysis sample using the slope of the regression line created with Trolox. sought according to
DPPH radical scavenging activity (nmol-Trolox equivalent/mol or mg)=slope of analytical sample (A520 or A495/(μL or μg/assay))/slope of Trolox (A520 or A495/(nmol/assay)). The results are shown in FIG.
　第一実施態様の乳酸菌飲料の上清添加量と520nmの吸光度で評価したＤＰＰＨラジカル消去量との関係から得られた直線の傾きからTrolox相当量として抗酸化力を算出した。その結果、第一実施態様の乳酸菌飲料の上清の抗酸化力は2.46(nmol-Trolox相当量/mg)と計算された。 From the slope of the straight line obtained from the relationship between the amount of supernatant added to the lactic acid bacteria beverage of the first embodiment and the amount of DPPH radical scavenging evaluated by absorbance at 520 nm, the antioxidant power was calculated as the amount equivalent to Trolox. As a result, the antioxidant capacity of the supernatant of the lactic acid bacteria drink of the first embodiment was calculated to be 2.46 (nmol-Trolox equivalent/mg).
[試験例２]　乳酸菌飲料からの抗酸化性物質の分離・同定
　本発明の製造方法によって製造した乳酸菌飲料について抗酸化性物質の分離・同定を試みた。
─抗酸化性成分の検索─
　本発明の第一実施態様の乳酸菌飲料の製法により調製した発酵液を利用して以下の実験を行った。当該発酵液を遠心分離によって乳酸菌を除去した上清を吸着樹脂XAD4に通液し、蒸留水、25%、50%、100%エタノールで順次溶出し、各フラクションを回収した。回収した各フラクションはロータリーエバポレーターもしくは凍結乾燥により溶媒を除去し乾固させた。各サンプルを所定の濃度となるように蒸留水で溶解し、ＤＰＰＨラジカル消去活性を評価した。分画スキームと各フラクションの抗酸化活性を図3及び図４に示す。 [Test Example 2] Separation and Identification of Antioxidant Substances from Lactic Acid Beverage An attempt was made to isolate and identify antioxidant substances in the lactic acid bacterium beverage produced by the production method of the present invention.
─Search for Antioxidant Ingredients─
The following experiment was conducted using the fermented liquid prepared by the method for producing a lactic acid bacteria beverage according to the first embodiment of the present invention. The fermented liquid was centrifuged to remove the lactic acid bacteria, and the supernatant was passed through an adsorption resin XAD4, followed by elution with distilled water, 25%, 50%, and 100% ethanol in order to collect each fraction. Each collected fraction was dried by removing the solvent by a rotary evaporator or freeze-drying. Each sample was dissolved in distilled water to a predetermined concentration, and DPPH radical scavenging activity was evaluated. Figures 3 and 4 show the fractionation scheme and the antioxidant activity of each fraction.
　25%エタノール溶出フラクションは56.68(nmol-Trolox相当量/mg)と比較的高いＤＰＰＨラジカル消去活性を示し、50%エタノール溶出フラクションと比較して回収量が大きかった。このため、XAD4-25%エタノール溶出フラクション(XAD4-25% EtOH Fr.)をさらにHPLCにて分離・精製を行った。Shodex Asahipak GS320HQカラムを用いて実験方法に記載した分離条件でXAD4-25% EtOH Fr.の分離を行った。
─カラム・分離条件─
カラム：Shodex Asahipak GS320 HQ
移動相：150mM Sodium phosphate buffer (pH2.5)
流速：0.6 mL/min
検出波長：260nm
カラム温度：35℃
注入量：50μL The 25% ethanol eluted fraction exhibited a relatively high DPPH radical scavenging activity of 56.68 (nmol-Trolox equivalent/mg), and the recovery amount was greater than the 50% ethanol eluted fraction. Therefore, the XAD4-25% ethanol elution fraction (XAD4-25% EtOH Fr.) was further separated and purified by HPLC. XAD4-25% EtOH Fr. was separated using a Shodex Asahipak GS320HQ column under the separation conditions described in the experimental method.
─Column and Separation Conditions─
Column: Shodex Asahipak GS320 HQ
Mobile phase: 150mM Sodium phosphate buffer (pH2.5)
Flow rate: 0.6mL/min
Detection wavelength: 260nm
Column temperature: 35°C
Injection volume: 50 μL
　サンプルインジェクション後、5分間隔で60分後まで溶離液を回収し(計12フラクション)、ＤＰＰＨラジカル消去活性を評価した。クロマトチャートと各フラクションのＤＰＰＨ消去活性を図５に示す。 After sample injection, the eluent was collected at 5-minute intervals for up to 60 minutes (total of 12 fractions), and the DPPH radical scavenging activity was evaluated. FIG. 5 shows the chromatogram and the DPPH elimination activity of each fraction.
　図５においてＤＰＰＨ消去活性はコントロールとサンプルの492nmの吸光度の差から算出した。リテンションタイム30から35分に回収したフラクション7(GS Fr.7)に比較的高いＤＰＰＨ消去活性が認められた。このため、GS Fr.7についてODSカラムを用いてさらに精製を行った。 　DPPH scavenging activity in Figure 5 was calculated from the difference in absorbance at 492 nm between the control and the sample. Relatively high DPPH scavenging activity was observed in fraction 7 (GS Fr.7) collected at a retention time of 30 to 35 minutes. Therefore, GS Fr.7 was further purified using an ODS column.
カラムの分離条件を以下の通りである。
─カラム・分離条件─
カラム：Inertsil ODS-2 5μm (4.6×150mm)
移動相：アセトニトリル/0.1% ギ酸
グラジエント条件：アセトニトリル濃度 0min 0% → 10min 10% → 20min 95% → 22min 95%
流速：1.0 mL/min,
検出波長：295nm
カラム温度：40℃
注入量：50μL The column separation conditions are as follows.
─Column and Separation Conditions─
Column: Inertsil ODS-2 5μm (4.6×150mm)
Mobile phase: acetonitrile/0.1% formic acid Gradient conditions: acetonitrile concentration 0min 0% → 10min 10% → 20min 95% → 22min 95%
Flow rate: 1.0 mL/min,
Detection wavelength: 295nm
Column temperature: 40℃
Injection volume: 50 μL
　図６に示すクロマトチャートの8min付近のピークにＤＰＰＨ消去活性が認められた。
次に、当該ピークについて吸収スペクトルを測定したところ、図７に示すように295nmに吸収極大を示した。
　また、このピークについてLC/MS による分析を実施した結果、ポジティブイオンモードでm/z145を示した(図８)。 DPPH scavenging activity was observed in the peak around 8 min in the chromatographic chart shown in FIG.
Next, when the absorption spectrum was measured for this peak, it showed an absorption maximum at 295 nm as shown in FIG.
In addition, as a result of LC/MS analysis of this peak, m/z 145 was shown in positive ion mode (Fig. 8).
　さらにこのピークをGC/MSにて分析を行い、得られたターゲットピークの質量スペクトルパターンからライブラリサーチを行った結果、ＤＤＭＰ(CAS No.28564-83-2)と同定された(図９)。このように本発明の乳酸菌飲料に抗酸化物質としてＤＤＭＰを含んでいることを見出した。 Furthermore, this peak was analyzed by GC/MS, and a library search was performed from the mass spectrum pattern of the obtained target peak, and as a result, it was identified as DDMP (CAS No. 28564-83-2) (Fig. 9). Thus, it was found that the lactic acid bacteria drink of the present invention contains DDMP as an antioxidant.
[試験例３]他の製品中に含まれるＤＤＭＰの量
　本発明の製造方法において分離・精製したＤＰＰＨラジカル消去活性を有する物質としてＤＤＭＰが同定された。次に、他のＡ社の市場で購入可能な３種類の製品（Ａ社-1,Ａ社-2,Ａ社-3）について、ＤＤＭＰの標品を用いて本発明、Ａ社-1のＤＤＭＰ濃度を測定した。それぞれのサンプルのODSカラムを用いて分離したクロマトチャート(Ａ社についてはＡ社-1のみ)を図１０に示す。ＤＤＭＰ検量線から計算した各サンプルのＤＤＭＰ濃度を表1に示す。本発明の製造方法により製造された乳酸菌飲料は他社の乳酸菌飲料（Ａ社）に対して２倍程度の高いＤＤＭＰ濃度を示した。 [Test Example 3] Amount of DDMP contained in other products DDMP was identified as a substance having DPPH radical scavenging activity separated and purified in the production method of the present invention. Next, three types of products (Company A-1, Company A-2, Company A-3) that can be purchased in the market of Company A were tested using the DDMP sample of the present invention, Company A-1. DDMP concentration was measured. FIG. 10 shows a chromatogram (for company A, only company A-1) separated using an ODS column for each sample. Table 1 shows the DDMP concentration of each sample calculated from the DDMP calibration curve. The lactic acid bacteria drink produced by the production method of the present invention exhibited a DDMP concentration that was about twice as high as that of another company's lactic acid drink (Company A).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
─本発明の乳酸菌飲料のＤＰＰＨラジカル消去活性におけるＤＤＭＰの寄与度について─
　本発明の第一の実施態様の製造方法による乳酸菌飲料についてＤＤＭＰ標品を用いてＤＰＰＨラジカル消去活性を測定した結果、0.397(mol-Trolox/mol)の抗酸化力を示した。この抗酸化力はアスコルビン酸1.141(mol-Trolox/mol)に対して約35%と計算された(図１１)。 -Contribution of DDMP to the DPPH radical scavenging activity of the lactic acid bacteria drink of the present invention-
As a result of measuring the DPPH radical scavenging activity of the lactic acid bacteria beverage produced by the production method of the first embodiment of the present invention using a DDMP standard, the antioxidant power was 0.397 (mol-Trolox/mol). This antioxidant power was calculated to be about 35% for ascorbic acid 1.141 (mol-Trolox/mol) (Fig. 11).
　次に、第一実施態様の乳酸菌飲料、A社の乳酸菌飲料に含まれるＤＤＭＰ量からそれぞれのＤＰＰＨ消去活性におけるＤＤＭＰの寄与度を算出した。図11に示すように第一実施態様の乳酸菌飲料、A社の乳酸菌飲料についてＤＤＭＰ量に対するＤＰＰＨラジカル消去を示す492nmの吸光度をプロットした際に得られた直線の傾きをＤＤＭＰ標品の傾きとの比から寄与度を算出した（図１２）。 Next, from the amount of DDMP contained in the lactic acid bacteria drink of the first embodiment and the lactic acid bacteria drink of Company A, the contribution of DDMP to each DPPH scavenging activity was calculated. As shown in FIG. 11, the slope of the straight line obtained when plotting the absorbance at 492 nm indicating DPPH radical scavenging against the amount of DDMP for the lactic acid beverage of the first embodiment and the lactic acid beverage of Company A is compared with the slope of the DDMP standard. The contribution was calculated from the ratio (Fig. 12).
　製品の抗酸化活性がＤＤＭＰのみで説明されると仮定すると、この直線の傾きは1となる。第一実施態様の乳酸菌飲料、A社の乳酸菌飲料のＤＤＭＰ標品に対する傾きの比はそれぞれ、1.533、1.161となった。この結果から、本発明の第一の実施態様の乳酸菌、Ａ社の乳酸菌飲料の抗酸化活性におけるＤＤＭＰの寄与度は約65%(1/1.533)、約86%(1/1.161)と推定された。 Assuming that the antioxidant activity of the product is explained only by DDMP, the slope of this line is 1. The slope ratios of the lactic acid bacteria drink of the first embodiment and the lactic acid bacteria drink of Company A to the DDMP standard were 1.533 and 1.161, respectively. From this result, the contribution of DDMP to the antioxidant activity of the lactic acid bacteria of the first embodiment of the present invention and the lactic acid drink of Company A is estimated to be about 65% (1/1.533) and about 86% (1/1.161). rice field.
[試験例４]乳酸菌飲料の製造工程における加熱する時間を変えた場合の効果 
　第一実施態様の乳酸菌飲料は脱脂粉乳とぶどう糖果糖液糖からなる仕込乳（乳培地）を乳酸菌発酵させることで得られる。また、仕込乳の製造工程においては殺菌のために加熱処理を行う。 [Test Example 4] Effect of changing the heating time in the manufacturing process of lactic acid bacteria beverage
The lactic acid bacteria beverage of the first embodiment is obtained by lactic acid fermentation of milk (milk culture medium) composed of skim milk powder and high-fructose corn syrup. In addition, heat treatment is performed for sterilization in the manufacturing process of the prepared milk.
　ここで仕込乳の加熱殺菌時間を10分とした場合及び120分とした場合（本発明の第一の実施態様の場合に相当）のそれぞれについて得られた仕込乳のＤＤＭＰ量の変化を調査した。ＤＤＭＰピークの面積比から加熱殺菌10分後に対して、加熱殺菌120分後の乳酸菌飲料のＤＤＭＰ量は41.5倍となった(図１３(A))。また、120分の場合の仕込乳に乳酸菌のスターター液（培養液）を接種し、乳酸菌によって発酵させる工程ではＤＤＭＰ量はやや減少した(図１３(B))。これらの実験結果から、ＤＤＭＰは仕込乳の加熱時間が影響することが分かった。尚、図１３の（Ａ）と（Ｂ）は別の実験であるため（Ａ）の加熱殺菌120分と（Ｂ）の加熱殺菌120分 発酵前のピーク面積が異なる。 Here, the change in the amount of DDMP in the prepared milk obtained when the heat sterilization time of the prepared milk was set to 10 minutes and 120 minutes (corresponding to the first embodiment of the present invention) was investigated. . From the area ratio of the DDMP peak, the amount of DDMP in the lactic acid beverage after 120 minutes of heat sterilization was 41.5 times that after 10 minutes of heat sterilization (Fig. 13(A)). In the case of 120 minutes, the amount of DDMP slightly decreased in the step of inoculating the starter solution (culture solution) of lactic acid bacteria into the milk for 120 minutes and fermenting with the lactic acid bacteria (Fig. 13(B)). From these experimental results, it was found that DDMP affects the heating time of the charged milk. Since (A) and (B) of FIG. 13 are different experiments, the peak areas before heat sterilization 120 minutes (A) and heat sterilization 120 minutes (B) before fermentation are different.
[試験例５]　使用する糖原料を変えた場合
　本発明の乳酸菌飲料の製造に使用する糖原料として、ぶどう糖果糖液糖、果糖又はぶどう糖のいずれか一種の糖と、脱脂粉乳から試験的に調製した乳酸菌飲料用の仕込乳についてＤＤＭＰの生成についての試験を行った。各糖の固形分を同一にした表２に示す配合の仕込乳を湯浴中で３時間加熱殺菌した際のＤＤＭＰ濃度を測定した。 [Test Example 5] When the sugar raw material to be used is changed As the sugar raw material to be used in the production of the lactic acid beverage of the present invention, either one of high fructose corn syrup, fructose or glucose, and powdered skim milk are prepared on a trial basis. A test was conducted on the production of DDMP for the prepared milk for the lactic acid bacteria drink. The DDMP concentration was measured when the milk having the composition shown in Table 2, in which the solid content of each sugar was the same, was sterilized by heating in a hot water bath for 3 hours.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　尚、仕込乳から清澄な上清を回収するために、サンプル500μlに60%トリクロロ酢酸(TCA)を100μl添加したのち、遠心分離処理を行った。回収した上清中のＤＤＭＰ濃度をHPLCにより測定した。図１４に示すように３時間加熱殺菌後の仕込乳中のＤＤＭＰ濃度はぶどう糖(1909μM)、ぶどう糖果糖液糖(1585μM)、果糖(734μM)の順で高い値となった。
　このように本発明においては、仕込乳の糖類としては、ぶどう糖を利用することが好ましいことがわかった。 In order to collect a clear supernatant from the prepared milk, 100 µl of 60% trichloroacetic acid (TCA) was added to 500 µl of the sample, followed by centrifugation. The DDMP concentration in the recovered supernatant was measured by HPLC. As shown in FIG. 14, the DDMP concentration in the milk after heat sterilization for 3 hours was high in the order of glucose (1909 μM), high fructose liquid sugar (1585 μM) and fructose (734 μM).
As described above, in the present invention, it was found that it is preferable to use glucose as the sugar in the milk.
[試験例６]　仕込み乳にリジンを添加した場合の効果
　仕込乳へのリジン添加の効果について検証を行った。試験例５におけるぶどう糖（試験区３）、ぶどう糖果糖液糖（試験区１）に示す試験的な仕込乳へリジンを添加し、加熱時間を変化させて調べた(図15)。
　結果として、ぶどう糖を糖源とした場合にリジンを添加した場合（試験区４）又はぶどう糖果糖液糖を糖源とした場合にリジンを添加した場合（試験区５）のいずれにおいてもリジン濃度依存的にＤＤＭＰが増加した（図１５）
　仕込乳にリジンを添加すると、ＤＤＭＰを増加させることができることがわかった。 [Test Example 6] Effect of adding lysine to milk The effect of adding lysine to milk was verified. Lysine was added to the experimental milk preparation shown in the glucose (test group 3) and the high-fructose corn syrup (test group 1) in Test Example 5, and the heating time was varied to investigate (Fig. 15).
As a result, when lysine was added when glucose was used as the sugar source (test group 4) or when lysine was added when glucose-fructose liquid sugar was used as the sugar source (test group 5), the lysine concentration dependence DDMP increased significantly (Fig. 15)
It was found that DDMP can be increased by adding lysine to the formula.
[試験例７]　仕込み乳にプロテアーゼを添加した場合の効果
　次に仕込乳の加熱殺菌前にプロテアーゼを添加する効果について検討を行った。試験例５における試験区１の仕込乳に対して図１６に示した各種プロテアーゼ（サモアーゼ、ペプチダーゼＲ、プロチンＳＤ、プロテアックス、プロテアーゼＰ、プロテアーゼＭ）を最終濃度0.1%となるように仕込乳に添加し、サモアーゼについては70℃で、その他の酵素は50℃で1 時間処理した後に沸騰水浴中で３時間加熱殺菌を行った。仕込乳の加熱殺菌後のＤＤＭＰ濃度を測定した結果、プロテアーゼ処理によって加熱殺菌後のＤＤＭＰはコントロールに対して高い濃度となり、特にペプチダーゼR処理により高いＤＤＭＰ濃度となった(図１６)。 [Test Example 7] Effect of adding protease to prepared milk Next, the effect of adding protease to prepared milk before heat sterilization was examined. Various proteases (Samoase, Peptidase R, Protin SD, Proteax, Protease P, Protease M) shown in FIG. Samoase was treated at 70°C and other enzymes at 50°C for 1 hour, followed by heat sterilization in a boiling water bath for 3 hours. As a result of measuring the DDMP concentration after heat sterilization of the milk, protease treatment resulted in a higher concentration of DDMP after heat sterilization than the control, and peptidase R treatment resulted in a particularly high DDMP concentration (Fig. 16).

Claims (7)

  1. 　抗酸化作用を有する成分を含有する乳酸菌飲料。
    A lactic acid bacteria drink containing an ingredient having an antioxidant effect.
  2. 　前記成分がＤＤＭＰ（2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one）である請求項１に記載の乳酸菌飲料。
    2. The lactic acid bacteria beverage according to claim 1, wherein said ingredient is DDMP (2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one).
  3. 　ＤＤＭＰを有効成分とする抗酸化用乳酸菌飲料。
    Antioxidant lactic acid bacteria beverage containing DDMP as an active ingredient.
  4. 　ＤＤＭＰを１５０μM以上含有する請求項２又は３に記載の乳酸菌飲料。
    The lactic acid bacteria drink according to claim 2 or 3, containing 150 µM or more of DDMP.
  5. 　前記乳酸菌飲料が、原料として脱脂乳及び糖類を含む仕込乳を熱で殺菌する工程を含む製造工程によって製造される請求項１～４のいずれかに記載の乳酸菌飲料。
    5. The lactic acid beverage according to any one of claims 1 to 4, wherein the lactic acid beverage is produced by a production process including a step of heat sterilizing charged milk containing skim milk and sugars as raw materials.
  6. 　前記糖類がぶどう糖を含有する請求項５に記載の乳酸菌飲料。
    6. The lactic acid bacteria beverage according to claim 5, wherein the saccharide contains glucose.
  7. 　抗酸化作用を有する旨を表示した乳酸菌飲料。 A lactic acid bacteria beverage labeled as having antioxidant properties.
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JP2005278517A (en) * 2004-03-30 2005-10-13 Kumamoto Technology & Industry Foundation Lactic fermented food containing mulberry fruit juice
JP2005304322A (en) * 2004-04-19 2005-11-04 Masanori Furuta Lactobacillus fermented food and drink using stock and/or digesting liquid of bean, and method for producing the same
JP2013192470A (en) * 2012-03-16 2013-09-30 Snow Brand Milk Products Co Ltd Food and drink containing lactobacillus with high survival rate and method for producing the same
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