JP7340560B2 - Lactic acid bacteria drink with antioxidant effect - Google Patents

Description

The present invention relates to a lactic acid bacteria beverage. In particular, the present invention relates to a lactic acid bacteria drink having an antioxidant effect.

Lactic acid bacteria drinks are widely used and have been supported by a wide range of age groups for many years. For example, Pirkul (registered trademark: Nissin 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 have excellent effects such as improving stress, lowering blood sugar, and recovering from fatigue.

On the other hand, this lactic acid bacteria beverage is expected to have useful functionality and effects in addition to the above. For example, with regard to antioxidant properties, it has not been known until now that lactic acid bacteria beverages have antioxidant properties, and that there are no prior patent documents regarding the antioxidant substances.
As a patent document related to the antioxidant properties of lactic acid bacteria drinks, Patent Document 1 describes an antioxidant for lactic acid bacteria drinks. However, the main purpose of this patent document is to include docosahexaenoic acid (DHA) in a dairy product lactic acid bacteria beverage, and it merely describes that an antioxidant is added as an incidental component.

JP 1995-327593

Therefore, the present inventors set it as a task to study the antioxidant properties of lactic acid bacteria beverages. Another challenge was to discover its active ingredients.

As a result of intensive research by the present inventors, it was confirmed that lactic acid bacteria drinks have an antioxidant function, and the present invention was completed.
That is, the first invention of the present application is
"A lactic acid bacteria drink containing ingredients with antioxidant effects."

Next, the above-mentioned component having an antioxidant effect 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 the component is DDMP (2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one)."

Next, the applicant also intends to develop an antioxidant lactic acid bacteria beverage containing DDMP as an active ingredient. That is, the third invention of the present application is
"Antioxidant lactic acid bacteria drink containing DDMP as an active ingredient."

Next, it is preferable that the lactic acid bacteria beverage contains 150 μM (μmol/L) or more of the DDMP.
That is, the fourth invention of the present application is
"The lactic acid bacteria drink according to claim 2 or 3, which contains 150 μM or more of DDMP."

Next, it is preferable that the lactic acid bacteria beverage utilizes skim milk and saccharides as raw materials and includes a step of sterilizing the milk containing the raw materials with heat.
That is, the fifth invention of the present application is
"The lactic acid bacteria drink according to any one of claims 1 to 4, which is produced by a manufacturing process that includes a step of heat-sterilizing milk containing skim milk and sugars as raw materials."

Next, in the fourth invention of the present application, it is preferable that the saccharide contains glucose.
That is, the sixth invention of the present application is:
"The lactic acid bacteria beverage according to claim 5, wherein the saccharide contains glucose."

Next, the present applicant also intends to develop a lactic acid bacteria beverage that is labeled on the product package, etc., 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 antioxidant effects."

The lactic acid bacteria beverage of the present invention contains DDMP as an antioxidant component and has an antioxidant effect. This can bring about various useful effects.

FIG. 2 is a diagram comparing the DPPH radical scavenging activity 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. 2 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 fermentation liquor in Test Example 2. FIG. 4 is a diagram showing the antioxidant activity of each fraction in FIG. 3. FIG. 4 is a diagram showing a column-separated chromatography chart of the XAD4-25% ethanol elution fraction in FIG. 3 and the DPPH scavenging activity of each fraction. FIG. 6 is a chromatographic chart showing fraction 7 in FIG. 5 further purified using an ODS column. 7 is a chart showing an absorption spectrum of a peak portion in FIG. 6. FIG. 7 is a chart of the results of LC/MS analysis of the peak portion in FIG. 6. FIG. FIG. 7 is a chart showing the results of identifying the peak portion in FIG. 6 by GC/MS. It is an LC chart figure which measured DDMP contained in the first embodiment of this application and other lactic acid bacteria drinks on the market in Test Example 3. FIG. 2 is a diagram comparing the antioxidant power of the lactic acid bacteria beverage of the first embodiment of the present invention with that of ascorbic acid using the slope of the regression line. 12 is a diagram comparing the antioxidant power of other lactic acid bacteria drinks of Company A using the slope of the regression line, as in the case of FIG. 11. FIG. 4 is an LC chart showing changes in the amount of DDMP depending on the heating time of the milk in Test Example 4. FIG. 4 is a diagram comparing the amount of DDMP produced when the sugar raw material is changed in Test Example 5. FIG. 6 is a diagram showing the effect of adding lysine in Test Example 6. FIG. 7 is a diagram showing the effects of adding various proteases in Test Example 7.

The content of the present invention will be explained below.
─Lactic acid bacteria drink─
The lactic acid bacteria beverage referred to in the present invention is prepared by the following steps. In other words, first, in the raw material mixing process, skimmed milk, water and milk are mixed together to prepare the milk mixture (milk medium), and then in the sterilization/cooling process, the milk mixture is heat sterilized at high temperature and fermented. Cool to the required temperature.
Next, as a lactic acid bacteria inoculation step, separately prepared seed bacteria (in which lactic acid bacteria have been cultured (preculture)) are added to the heated milk mixture. Next, in the fermentation process, fermentation is carried out while maintaining a constant temperature in a tank. Next, after cooling, as a mixing/dilution step, syrup, fruit juice, etc. are added to the fermented liquid after culturing, and adjustments are made with dilution water as necessary. This homogenized product is filled into containers to complete the lactic acid bacteria beverage.

In addition, the types of lactic acid bacteria drinks include "dairy products lactic acid bacteria drinks" and "lactic acid bacteria drinks." First, "dairy lactic acid bacteria drinks" are those that contain 3.0% or more of non-fat milk solids (milk after removing milk fat and water) and have a lactic acid bacteria count or yeast count of 10 million/ml or more. There are live bacteria type and sterilized type.
Next, "lactic acid bacteria drinks" refer to those with non-fat milk solid content of less than 3.0% and the number of lactic acid bacteria or yeast of 1 million/ml or more.
The lactic acid bacteria beverage in the present invention includes both the above-mentioned "dairy products lactic acid bacteria beverage" and "lactic acid bacteria beverage".

─Preferable 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 produce the lactic acid bacteria beverage of the present invention generally as follows. However, the present invention is not limited to the following manufacturing method.

(1) Breeding milk dissolution (preparation of milk medium)
Prepare the milk (milk medium) so that the milk raw material, mainly skim milk, is 5 to 30% by weight and the sugar content is 2 to 20% by weight. Preferably, the milk raw material content is 15 to 20% by weight, and the sugar content is 10 to 15% by weight.
In addition, it is preferable that the sugar in the prepared milk contains 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) Heating Heat the milk mixture at 80°C to 100°C for about 30 to 180 minutes. The heating time is preferably 60 minutes to 160 minutes. Furthermore, the time period is more preferably 90 minutes to 140 minutes. Most preferably, the time is between 100 and 120 minutes.

(3) Fermentation liquid (preculture)
A culture solution (starter solution) in which lactic acid bacteria (Lactobacillus, Lactococcus, Pediococcus, Leuconostoccus, Streptococcus, Enterococcus, etc.) have been cultured is added to the above-mentioned heated milk medium and incubated at a predetermined temperature of about 30°C to 40°C. A fermented liquid can be obtained by culturing until the lactic acid acidity reaches .

(4) Syrup liquid A syrup liquid base containing sugar, glucose, fructose, etc. is prepared, heat sterilized and cooled to obtain a syrup liquid.

(5) Mixing/Homogenization The fermented liquid and syrup liquid are mixed at a weight ratio of 1:1 or 1:5 to 5:1 and homogenized to complete a lactic acid bacteria beverage.

As described above, in the present invention, as a preferred method for producing a lactic acid bacteria beverage, after heating a skim milk powder and a milk mixture containing saccharides, the heated milk mixture and a culture solution of lactic acid bacteria are mixed, and after fermentation for a predetermined period of time, A production method is adopted in which a syrup solution is mixed with the fermented liquid after the fermentation. but,
In this step, it is preferable to heat the skim milk powder and the milk containing sugars for the heating time as described above.

─Antioxidant properties─
Antioxidant properties are said to have various functions in living organisms, and for example, it is said to be able to suppress excessively active enzymes, which are the cause of fatigue (and aging).
That is, free radicals including active oxygen are reported to be the cause of aging, cancer, and lifestyle-related diseases. The free radical theory of aging proposed by Harman states that the cause of aging is that free radicals produced mainly in mitochondria as byproducts of energy metabolism oxidize DNA, proteins, lipids, etc. ((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 in oxygen into their bodies through breathing, produce ATP through the electron transport chain in mitochondria, and obtain the energy necessary for life activities. Various reactive oxygen species are generated during this process. Active oxygen has extremely high reactivity (Shigeo Nakamura, Chemistry of Active Oxygen and Antioxidants, Journal of the Japan Medical College Medical Association 2013 9 164-169). It has been reported that active oxygen reacts with lipids to generate lipid peroxides, which can cause arteriosclerosis and myocardial infarction (JL Witztum, D Steinberg, Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest.
1991 88(6) 1785-92).

In addition, when active oxygen reacts with nucleic acids, it can cause cancer due to DNA strand breaks and DNA mutations due to oxidative modification of nucleobases (Miral Dizdaroglu, Pawel Jaruga, Mechanisms of free radical-induced damage to DNA. Free Radic Res 2012 46(4) 382-419).
Living organisms are equipped with a mechanism to remove active oxygen generated in living organisms. Hydrogen peroxide removal enzymes such as superoxide dismutase (SOD) and catalase render active oxygen generated in living organisms harmless. On the other hand, there are many low-molecular-weight compounds that have antioxidant effects in nature, and living organisms protect themselves from oxidative damage caused by active oxygen by biosynthesizing these or taking them in from food. Typical natural antioxidants include polyphenols such as ascorbic acid (vitamin C), α-tocopherol (vitamin E), catechins found in green tea, and resveratrol found in red wine (Shigeo Nakamura, Active Oxygen and Antioxidants) Chemistry of Matter Journal of the Japan Medical College Medical Society 2013 9 164-169).

Although there are currently many unknowns about the causes of chronic fatigue, it has been reported that people with chronic fatigue produce more free radicals and that oxidative stress may be the cause (AC Logan, C Wong, Chronic fatigue syndrome : oxidative stress and dietary modifications.
Med Rev 2001 6(5) 450-9). It has been reported that continuous intake of a beverage containing 400 mg of imidazole dipeptide, which has an antioxidant effect, reduces fatigue in healthy individuals who are aware of fatigue during daily work (Keichiro Shimizu, Masahiro Fukuda, Haruaki Yamamoto) , The usefulness of continuous intake of imidazole dipeptide-containing drinks for healthy people who are aware of fatigue during daily work (Pharmacology and Treatment) vol.37 no.3 2009 37(3) 255-637). In this way, daily intake of foods containing antioxidants is expected to have the effect of reducing fatigue.

Here, yogurt and lactic acid bacteria drinks contain lactic acid bacteria, and have intestinal regulation effects (Iva Hojsak, Probiotics in Functional Gastrointestinal Disorders. Adv Exp Med Biol 2019 1125 121-137.) and immunomodulatory effects (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), prevention of allergies such as hay fever and atopic dermatitis (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), improvement of ulcerative colitis (Maria Jose Saez-Lara, 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 2015 2015:505878 ) Various physiological activities have been reported.

On the other hand, there are few reports on the antioxidant activity of lactic acid bacteria drinks, and of course, the antioxidant active substances contained in lactic acid bacteria drinks have not been identified. This time, the present inventors evaluated the antioxidant activity of dairy lactic acid bacteria drinks using the DPPH radical scavenging method, separated and purified the antioxidants contained in dairy lactic acid bacteria drinks, and identified DDMP.

─DDMP─
In the present invention, the antioxidant ability of the lactic acid bacteria beverage was evaluated by the DPPH radical scavenging method, and the antioxidant substance was separated and purified and found to be DDMP. Here, DDMP is known as a substance produced by the Maillard reaction (Tetrahedron Letters No. 15, pp. 1243-1246, 1970.) In particular, the lactic acid bacteria beverage of the present invention contains 150 μM or more of DDMP. It is preferable.

Examples of the present invention are described below.

[Test Example 1] Comparison of DPPH radical scavenging activity of lactic acid bacteria drinks of the present invention and lactic acid bacteria drinks of other companies As a first embodiment of the present invention, lactic acid bacteria drinks were prepared as follows. The lactic acid bacteria drink was tested.
─Production method of lactic acid bacteria drink according to the first embodiment of the present invention─
Prepare a milk medium containing 8% by weight of skim milk powder and 5% by weight of high-fructose corn syrup, heat sterilize it at 100°C for about 120 minutes, and add Lactobacillus paracasei starter solution (culture solution) to the heated milk medium. The cells were inoculated and cultured at 37° C. until a predetermined lactic acid acidity was reached to obtain a fermentation liquid.
On the other hand, a syrup liquid base containing sugar and high-fructose corn syrup was prepared, and heat sterilized and cooled to obtain a syrup liquid.
Thereafter, 500 ml of the above syrup liquid was mixed and homogenized with 500 ml of the above fermented liquid together with a small amount of flavoring agent to obtain 1000 ml of a lactic acid bacteria beverage containing live lactic acid bacteria at 3×10 ⁸ /ml or more.

─DPPH radical scavenging activity evaluation method─
The evaluation method for DPPH radical scavenging activity was performed as follows. First, the following reagents were used.
(1)200 mM MES (2-morpholinoethanesulphonic acid) buffer (pH6.0)
(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. Dissolution took 30 minutes to 1 hour in the dark. Since the DPPH solution has low stability, it was prepared immediately before use.
(3)2.0mM Trolox stock solution and Trolox solution for calibration curve
12.51 mg of Trolox (manufactured by Wako, 209-18881) was dissolved in a 50% aqueous ethanol solution, and the volume was adjusted to 25 mL. Trolox stock solution was dispensed into 200 μL portions and stored at -40°C. A 100 μM Trolox solution was prepared immediately before use by adding 50% ethanol aqueous solution (3.9 mL) to 2.0 mM Trolox stock solution (200 μL). A 100 μM Trolox solution was diluted with a 50% ethanol aqueous solution to prepare 80, 60, 40, and 20 mM Trolox solutions.

The sample was dissolved in 50% ethanol aqueous solution. After 100 μL of samples at various concentrations were dispensed into a 96-well plate containing 50 μL of 200 mM MES buffer, 50 μL of 400 μM DPPH solution was added to start the reaction. After reacting for 20 minutes at room temperature in the dark, absorbance at 520 nm or 495 nm was measured using a spectrophotometer or microplate reader. A DPPH solution was added to the Trolox solution having the above concentration and the reaction was carried out in the same manner. The DPPH radical scavenging activity was determined using the slope of the regression line created with Trolox, as the amount of Trolox corresponding to the amount added to the analysis sample, according to the following calculation formula.
DPPH radical scavenging activity (nmol-Trolox equivalent amount/mol or mg) = slope of analysis sample (A520 or A495/(μL or μg/assay))/Slope of Trolox (A520 or A495/(nmol/assay))

─Test method─
Antioxidant activity of samples of the lactic acid bacteria beverage 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 liquid 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 x g for 10 minutes at 4°C, and the supernatant from which lactic acid bacteria were removed was used for measurement. The results are shown in Figure 1.

─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.

─Evaluation of DPPH radical scavenging activity for other lactic acid bacteria drinks on the market─
In order to compare with the lactic acid bacteria drink of the first embodiment of the present invention, other commercially available lactic acid bacteria drinks and fermented milks (Company A, Company B-1, Company B-2, Company B-3, Company C, D Company) was purchased on the market.
The above-mentioned DPPH radical scavenging activity evaluation method was performed on the lactic acid bacteria drinks and fermented milk of each company. The measurement results are shown in Figure 1.

It was confirmed that other commercially available dairy products lactic acid bacteria beverages and fermented milk also have DPPH radical scavenging activity. On the other hand, the lactic acid bacteria beverage obtained by the manufacturing method of the first embodiment of the present invention showed higher antioxidant activity compared to other commercially available dairy products lactic acid bacteria drinks and fermented milk in the antioxidant activity evaluation method using the DPPH method. Ta.

─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 Trolox amount corresponding to the added amount of the analysis sample using the slope of the regression line created with Trolox. I asked for it according to.
DPPH radical scavenging activity (nmol-Trolox equivalent amount/mol or mg) = slope of analysis sample (A520 or A495 / (μL or μg/assay)) / slope of Trolox (A520 or A495 / (nmol/assay)). The results are shown in FIG. 2.

Antioxidant power was calculated as the amount equivalent to Trolox from the slope of the straight line obtained from the relationship between the amount of supernatant added to the lactic acid bacteria drink of the first embodiment and the amount of DPPH radical scavenged evaluated by absorbance at 520 nm. As a result, the antioxidant power of the supernatant of the lactic acid bacteria drink of the first embodiment was calculated to be 2.46 (nmol-Trolox equivalent amount/mg).

[Test Example 2] Separation and Identification of Antioxidant Substances from Lactic Acid Bacteria Beverages An attempt was made to separate and identify antioxidant substances in lactic acid bacteria drinks 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 lactic acid bacteria beverage manufacturing method of the first embodiment of the present invention. The fermentation solution was centrifuged to remove lactic acid bacteria, and the supernatant was passed through an adsorption resin XAD4, sequentially eluted with distilled water, 25%, 50%, and 100% ethanol, and each fraction was collected. Each collected fraction was dried by removing the solvent using a rotary evaporator or freeze drying. Each sample was dissolved in distilled water to a predetermined concentration, and the DPPH radical scavenging activity was evaluated. The fractionation scheme and the antioxidant activity of each fraction are shown in Figures 3 and 4.

The 25% ethanol eluted fraction showed a relatively high DPPH radical scavenging activity of 56.68 (nmol-Trolox equivalent/mg), and the recovered amount was larger 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/separation conditions─
Column: Shodex Asahipak GS320 HQ
Mobile phase: 150mM Sodium phosphate buffer (pH2.5)
Flow rate: 0.6 mL/min
Detection wavelength: 260nm
Column temperature: 35℃
Injection volume: 50μL

After sample injection, the eluent was collected at 5-minute intervals until 60 minutes later (12 fractions in total), and the DPPH radical scavenging activity was evaluated. FIG. 5 shows a chromatographic chart and the DPPH scavenging activity of each fraction.

In FIG. 5, the DPPH scavenging activity 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 separation conditions of the column are as follows.
─Column/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: 50μL

DPPH scavenging activity was observed at the peak around 8 min in the chromatographic chart shown in FIG.
Next, the absorption spectrum of the peak was measured, and as shown in FIG. 7, an absorption maximum was observed at 295 nm.
Furthermore, analysis of this peak by LC/MS showed m/z 145 in positive ion mode (Figure 8).

Furthermore, this peak was analyzed by GC/MS, and as a result of library search based on the mass spectrum pattern of the obtained target peak, it was identified as DDMP (CAS No. 28564-83-2) (FIG. 9). It was thus discovered that the lactic acid bacteria beverage of the present invention contains DDMP as an antioxidant.

[Test Example 3] Amount of DDMP contained in other products DDMP was identified as a substance having DPPH radical scavenging activity that was separated and purified in the production method of the present invention. Next, regarding three types of products (Company A-1, Company A-2, Company A-3) that can be purchased in the market of other Company A, the present invention and Company A-1's DDMP concentration was measured. A chromatogram of each sample separated using an ODS column (for Company A, only Company A-1) is shown in FIG. Table 1 shows the DDMP concentration of each sample calculated from the DDMP calibration curve. The lactic acid bacteria beverage produced by the production method of the present invention exhibited a DDMP concentration approximately twice as high as that of another company's lactic acid bacteria beverage (Company A).

─ Concerning the degree of contribution of DDMP to the DPPH radical scavenging activity of the lactic acid bacteria beverage 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 preparation, it showed an antioxidant power of 0.397 (mol-Trolox/mol). This antioxidant power was calculated to be about 35% relative to ascorbic acid 1.141 (mol-Trolox/mol) (Figure 11).

Next, the degree of contribution of DDMP to the DPPH scavenging activity was calculated 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. As shown in Figure 11, the slope of the straight line obtained when plotting the absorbance at 492 nm, which indicates DPPH radical scavenging against the amount of DDMP, for the lactic acid bacteria drink of the first embodiment and the lactic acid bacteria drink of Company A is compared with the slope of the DDMP sample. The contribution was calculated from the ratio (Figure 12).

Assuming that the antioxidant activity of the product is explained only by DDMP, the slope of this line will be 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 bacteria drink of Company A is estimated to be approximately 65% (1/1.533) and approximately 86% (1/1.161). Ta.

[Test Example 4] Effect of changing the heating time in the manufacturing process of lactic acid bacteria beverages
The lactic acid bacteria beverage of the first embodiment is obtained by fermenting milk (milk medium) consisting of skim milk powder and high-fructose liquid sugar with lactic acid bacteria. In addition, in the manufacturing process of milk powder, heat treatment is performed for sterilization.

Here, changes in the amount of DDMP in the milk mixture obtained when the heat sterilization time of the milk mixture was set to 10 minutes and 120 minutes (corresponding to the first embodiment of the present invention) were investigated. . Based on the area ratio of the DDMP peak, the amount of DDMP in the lactic acid bacteria drink after 120 minutes of heat sterilization was 41.5 times that after 10 minutes of heat sterilization (Figure 13(A)). In addition, in the step of inoculating a lactic acid bacteria starter solution (culture solution) into the milk for 120 minutes and fermenting it with the lactic acid bacteria, the amount of DDMP decreased slightly (Figure 13(B)). From these experimental results, it was found that DDMP was affected by the heating time of the milk mixture. In addition, since (A) and (B) in FIG. 13 are different experiments, the peak areas before fermentation after 120 minutes of heat sterilization in (A) and 120 minutes of heat sterilization in (B) are different.

[Test Example 5] When the sugar raw material used is changed The sugar raw material used in the production of the lactic acid bacteria beverage of the present invention was experimentally prepared from any one of glucose, fructose liquid sugar, fructose, or glucose, and skim milk powder. A test was conducted on the production of DDMP using the prepared milk for lactic acid bacteria drinks. The DDMP concentration was measured when the milk formulations shown in Table 2, in which the solid content of each sugar was the same, were heat sterilized in a hot water bath for 3 hours.

In order to recover a clear supernatant from the milk preparation, 100 μl of 60% trichloroacetic acid (TCA) was added to 500 μl of the sample, and then centrifugation was performed. The DDMP concentration in the collected supernatant was measured by HPLC. As shown in FIG. 14, the DDMP concentrations in the milk after heat sterilization for 3 hours were highest in the order of glucose (1909 μM), high-fructose corn syrup (1585 μM), and fructose (734 μM).
Thus, in the present invention, it has been found that it is preferable to use glucose as the saccharide in the milk preparation.

[Test Example 6] Effect of adding lysine to mixed milk The effect of adding lysine to mixed milk was verified. Lysine was added to the experimental milk preparations shown in Test Example 5 for glucose (test group 3) and high-fructose corn syrup (test group 1), and the heating time was varied to investigate (Figure 15).
As a result, lysine concentration dependence was found in both cases when lysine was added when glucose was used as the sugar source (test group 4) or when lysine was added when glucose and fructose corn syrup were used as the sugar source (test group 5). DDMP increased (Figure 15)
It has been found that DDMP can be increased by adding lysine to the milk formula.

[Test Example 7] Effect of adding protease to milk preparation Next, the effect of adding protease to milk preparation before heat sterilization was investigated. Various proteases (Samoase, Peptidase R, Protin SD, Proteax, Protease P, Protease M) shown in FIG. 16 were added to the milk of Test Group 1 in Test Example 5 at a final concentration of 0.1%. After treatment at 70°C for samoase and 50°C for other enzymes for 1 hour, heat sterilization was performed in a boiling water bath for 3 hours. As a result of measuring the DDMP concentration of the milk after heat sterilization, protease treatment resulted in a higher DDMP concentration than the control, and Peptidase R treatment resulted in a particularly high DDMP concentration (Figure 16).

Claims (1)

An antioxidant lactic acid bacteria drink containing DDMP (2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one) as an active ingredient.