Beverage containing lactic acid bacteria fermented liquid for inhibition of hyaluronidase activity and method for producing lactic acid bacteria fermented liquid

Abstract

To investigate the inhibition of hyaluronidase activity by a beverage comprising lactic acid bacteria fermented liquid, to find an active ingredient thereof, and to identify a method for producing the beverage capable of increasing the content of such an active ingredient.SOLUTION: A beverage includes a lactic acid bacteria fermented liquid for hyaluronidase activity inhibition, wherein α-ketoglutaric acid serves as an active ingredient. In order to produce a lactic acid bacteria fermented liquid with increased content of α-ketoglutaric acid, fermentation treatment is carried out with protease-treated milk or milk with peptides added.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] The present invention relates to a beverage containing a lactic acid bacteria fermentation liquid, and more particularly to a beverage containing a lactic acid bacteria fermentation liquid that has the effect of inhibiting hyaluronidase activity. [Background technology]

[0002] Lactic acid bacteria drinks are widely used and have been popular with people of all ages for many years. Examples include Pilcle (registered trademark: Nissin York Co., Ltd.) and Yakult (registered trademark: Yakult Honsha Co., Ltd.). Lactic acid bacteria drinks are widely known for their ability to improve the intestinal environment. Some types are also known to have excellent effects such as stress relief, blood sugar reduction, and fatigue recovery.

[0003] Drinks containing this lactic acid bacteria fermentation liquid are expected to have other useful functions and effects in addition to those mentioned above. For example, one of the expected effects is the prevention of skin aging. One of the causes of skin aging is thought to be the hydrolysis of hyaluronic acid by hyaluronidase. However, until now, there have been no known patent documents regarding the hyaluronidase activity inhibition of lactic acid bacteria drinks.

[0004] On the other hand, Patent Documents 1 and 2 disclose that lactic acid fermentation products of grapes have the ability to inhibit hyaluronidase activity. However, these patent documents use grape seeds and / or skins as raw materials, and do not describe at all the lactic acid fermentation of dairy raw materials such as milk. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2013-100243 [Patent Document 2] Japanese Patent Application Laid-Open No. 2010-280602 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, the present inventors aimed to investigate the hyaluronidase activity inhibition of lactic acid bacteria beverages, to identify the active ingredient, and to find a production method that increases the amount of the active ingredient. [Means for solving the problem]

[0007] In order to solve the above problems, the present invention is characterized by a beverage containing a lactic acid bacteria fermentation liquid for inhibiting hyaluronidase activity, which contains α-ketoglutaric acid as an active ingredient.

[0008] In addition, in order to solve the above problems, the present invention is characterized in that the method for producing a lactic acid bacteria fermentation liquid with increased production of α-ketoglutaric acid is characterized in that the fermentation treatment is carried out using protease-treated milk.

[0009] Furthermore, in order to solve the above problems, the present invention is characterized in that the method for producing a lactic acid bacteria fermentation liquor with increased production of α-ketoglutaric acid is characterized in that the method for producing a lactic acid bacteria fermentation liquor comprises fermentation treatment using milk to which peptides have been added. [Effects of the Invention]

[0010] The beverage containing the lactic acid bacteria fermentation broth of the present invention contains α-ketoglutaric acid as a hyaluronidase activity inhibitor, which can provide a variety of useful effects. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a graph comparing the inhibitory effects on hyaluronidase activity in lactic acid bacteria fermentation broths in Test Example 1. [Figure 2] FIG. 1 shows the hyaluronidase activity inhibitory effect of each fraction fractionated in Test Example 2. [Figure 3]FIG. 3 shows a chromatogram of each fraction separated by column for the XAD4-25% ethanol elution fraction in FIG. 2. [Figure 4] FIG. 3 shows the hyaluronidase activity inhibitory effect of each fraction separated by column for the XAD4-25% ethanol elution fraction in FIG. 2. [Figure 5] FIG. 4 shows chromatograms of each fraction obtained by further purifying fraction 2 in FIG. 3 using an InertSustain C18 column. [Figure 6] This figure shows the hyaluronidase activity inhibitory effect of each fraction obtained by further purifying fraction 2 in Figure 3 using an InertSustain C18 column. [Figure 7] FIG. 1 shows the hyaluronidase activity inhibition rate at each concentration of α-ketoglutaric acid. [Figure 8] FIG. 1 shows the inhibitory effect of α-ketoglutaric acid on hyaluronidase activity in human cells. [Figure 9] FIG. 1 is a graph comparing the hyaluronidase activity inhibitory effects of the fermented milk of the present invention with those of a commercially available lactic acid bacteria beverage and fermented milk. [Figure 10] FIG. 1 is a graph showing the change in the amount of α-ketoglutaric acid produced when various proteases are added to brewed milk. [Figure 11] FIG. 1 is a graph showing the change in the amount of α-ketoglutaric acid produced when various peptides were added to brewed milk. DETAILED DESCRIPTION OF THE INVENTION

[0012] The contents of the present invention will be explained below. -Beverage containing lactic acid bacteria fermentation liquid- The beverage containing the lactic acid bacteria fermentation liquid of the present invention is prepared by the following steps: First, in the raw material mixing step, the raw materials skim milk, water, and milk are mixed to prepare a milk stock (a milk culture medium), and then, in the sterilization and cooling step, the milk stock is sterilized by heating at a high temperature and cooled to the temperature required for fermentation. Next, in the lactic acid bacteria inoculation process, a separately prepared seed culture (lactic acid bacteria cultured (pre-culture)) is added to the heated milk. Next, in the fermentation process, the temperature is maintained constant in the tank and fermentation takes place. After cooling, in the mixing and dilution process, syrup, fruit juice, etc. are added to the cultured fermented liquid, and if necessary, dilution water is added. This is then homogenized and filled into containers to complete the beverage containing the lactic acid bacteria fermented liquid.

[0013] There are two types of lactic acid bacteria drinks: "dairy lactic acid bacteria drinks" and "lactic acid bacteria drinks." First, "dairy lactic acid bacteria drinks" are those that contain 3.0% or more non-fat milk solids (components of milk excluding milk fat and water) and have a lactic acid bacteria or yeast count of 10 million / ml or more, and are available in live bacteria and pasteurized types. Next, "lactic acid bacteria beverage" 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 million / ml or more. Fermented milk is defined as milk that contains 8.0% or more non-fat milk solids (components remaining after removing milk fat and water) and has a lactic acid bacteria or yeast count of 10 million / ml or more, and is available in live bacteria and pasteurized types. In the present invention, the beverage containing a lactic acid bacteria fermentation liquid includes any of the above-mentioned "dairy lactic acid bacteria beverage," "lactic acid bacteria beverage," and "fermented milk."

[0014] --Method for producing a beverage containing a preferred lactic acid bacteria fermentation liquid of the present invention-- Although the general method for producing a beverage containing a lactic acid bacteria fermentation liquor has been described above, it is particularly preferable to produce a beverage containing a lactic acid bacteria fermentation liquor of the present invention generally as follows, although the present invention is not limited to the following production method.

[0015] (1) Dissolving milk (preparing milk culture medium) The milk feed (milk medium) is prepared so that the milk ingredients, mainly skim milk, are 5 to 30% by weight and sugars are 2 to 20% by weight, and preferably the milk ingredients are 10 to 20% by weight and sugars are 2 to 15% by weight. The sugars in the milk feed preferably contain glucose or sucrose. Specifically, it is preferable to use glucose, glucose-fructose liquid sugar, sucrose, or sucrose-type liquid sugar.

[0016] (2)Heating The milk is heated at 80°C to 130°C for about 15 seconds to 180 minutes.

[0017] (3) Fermentation liquid (pre-culture) A culture liquid (starter liquid) in which lactic acid bacteria (Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, Streptococcus, Enterococcus, etc.) have been cultured can be added to the heated milk medium and cultured at approximately 30°C to 40°C until the desired lactic acid acidity is reached, thereby obtaining a fermentation liquid.

[0018] In the present invention, a milk medium previously treated with a protease or a milk medium supplemented with a peptide may be used for pre-culture. As will be described later, the amount of α-ketoglutaric acid produced can be increased by treating the medium with a protease or adding a peptide.

[0019] The protease that can be used for the protease treatment is not particularly limited. Conventional methods can be used as the method for the protease treatment, and it is preferable to perform the heat treatment at a temperature and for a time that maximizes the protease activity according to the characteristics of each protease.

[0020] The peptide to be added is not particularly limited, but milk peptide is preferred.

[0021] (4) Syrup A syrup liquid base containing sugar, glucose, sucrose, fructose, etc. is prepared, and the syrup liquid is obtained by heat sterilization and cooling.

[0022] (5) Mixing / homogenization The fermented liquid and the syrup liquid are mixed in a weight ratio of 1:1 or about 1:5 to 5:1 and homogenized to complete a beverage containing the lactic acid bacteria fermented liquid.

[0023] Thus, in the present invention, a preferred method for producing a beverage containing a lactic acid bacteria fermentation liquid is to heat a milk preparation containing skim milk powder and sugars, mix the heated milk preparation with a lactic acid bacteria culture liquid, ferment for a predetermined period of time, and then mix the fermented liquid with a syrup liquid to prepare the beverage. In this step, it is preferable to heat the milk feed containing skim milk powder and sugars for the time period described above. [Example]

[0024] Examples of the present invention are described below.

[0025] [Test Example 1] Confirmation of the hyaluronidase activity inhibitory effect in lactic acid bacteria fermentation liquid To confirm whether lactic acid bacteria fermentation liquid has an inhibitory effect on hyaluronidase activity, a lactic acid bacteria fermentation liquid was prepared and tested. ─Production method of lactic acid bacteria fermentation liquid─ A milk medium containing 8% by weight of skim milk powder and 5% by weight of glucose-fructose corn syrup was prepared and sterilized by heating at 100°C for at least 30 minutes. After that, a starter liquid (culture liquid) of Lactobacillus paracasei or Lactobacillus paracasei and Streptococcus thermophilus was inoculated into the heated milk medium and cultured at 37°C until the specified lactic acid acidity was reached, and a fermentation liquid was obtained. 3 x 10 live lactic acid bacteria 8 A lactic acid bacteria fermentation broth containing lactic acid bacteria was obtained.

[0026] -Method for evaluating hyaluronidase activity inhibition- The inhibition of hyaluronidase activity was evaluated as follows.

[0027] First, the reagents were prepared. Hyaluronidase: Hyaluronidase (Hyaluronidase from bovine tests Type IV-S: manufactured by Sigma-Aldrich) was dissolved in 0.1 M acetate buffer (pH 4.0) and adjusted to 10 mg / mL. Substrate solution: Sodium hyaluronate (Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 0.1 M acetate buffer (pH 4.0) and adjusted to 1 mg / mL. Hyaluronidase activator: 20 mg of Compound 48 / 80 and 73.5 mg of calcium chloride dihydrate were dissolved in 10 mL of 0.1 M acetate buffer (pH 4.0) to prepare the solution. p-DAD: 5 g of p-DAD reagent (p-Dimethylaminobenzaldehyde), 6 mL of 10 N HCl, and 44 mL of acetic acid were mixed and diluted 10-fold with acetic acid immediately before use.

[0028] Hyaluronidase activity inhibition was evaluated as follows. After centrifugation (10,000 × g, 4°C, 10 minutes), the supernatant was collected and filtered through a filter paper. 10 μL of the filtered lactic acid fermentation broth was mixed with 5 μL of hyaluronidase adjusted to 10 mg / mL and incubated at 37°C for 20 minutes. Next, 5 μL of the adjusted hyaluronidase activator was added and incubated at 37°C for 20 minutes. 25 μL of the adjusted substrate solution was then added and incubated at 37°C for 40 minutes. To stop the enzymatic reaction of hyaluronidase, 10 μL of 0.4 N NaOH and 10 μL of a buffer solution (pH 9.1) adjusted to a final concentration of 0.8 M boric acid and 1 M KOH were added, followed by heating at 95°C for 3 minutes. After incubation at 37°C for 10 minutes, 600 μL of the adjusted p-DAD was added and incubated at 37°C for 20 minutes. After the reaction, 200 μL of the sample solution was taken and the absorbance at 600 nm was measured using a microplate reader. Note that only the solvent (ion-exchanged water) was added as a control sample. The results are shown in Figure 1.

[0029] ─Results─ Each of the prepared lactic acid bacteria fermentation liquids was confirmed to have the ability to inhibit hyaluronidase activity.

[0030] [Test Example 2] Isolation and identification of hyaluronidase inhibitors from lactic acid bacteria fermentation broth Next, we attempted to isolate and identify hyaluronidase inhibitors from the prepared lactic acid bacteria fermentation liquid. -Search for hyaluronidase activity inhibitors- The following experiment was conducted using the Lactobacillus paracasei fermentation broth prepared in Test Example 1. The fermentation broth was centrifuged to remove lactic acid bacteria, and the resulting supernatant was passed through the adsorption resin XAD4. The supernatant was eluted sequentially with distilled water, 25%, 50%, and 100% ethanol, and each fraction was collected. The collected fractions were then dried by rotary evaporation or freeze-drying to remove the solvent. Each sample was dissolved in distilled water to a predetermined concentration, and hyaluronidase activity inhibition was evaluated. The hyaluronidase activity inhibition of each fraction is shown in Figure 2.

[0031] High hyaluronidase activity was observed in the 25% ethanol elution fraction. Therefore, the XAD4-25% ethanol elution fraction (XAD4-25% EtOH Fraction) was further separated and purified by HPLC. XAD4-25% EtOH Fraction was separated using an InertSustain C18 column (10 x 250 mm, 5 μm) under the separation conditions described in the experimental procedures. ─Column and separation conditions─ Column: InertSustain C18 (10 x 250 mm, 5 μm) Mobile phase: acetonitrile / distilled water Eluent conditions: Acetonitrile concentration 0 min 2% → 10 min 2% Flow rate: 5.0mL / min Detection: UV 210nm Column temperature: 25℃

[0032] After sample injection, the eluate was collected at retention times of approximately 2-3.5 minutes (Fr. 1), 3.5-5 minutes (Fr. 2), and 7-8 minutes (Fr. 3) where peaks were observed (a total of three fractions), and hyaluronidase activity inhibition was evaluated. The chromatogram and hyaluronidase activity inhibition of each fraction are shown in Figures 3 and 4.

[0033] In Figure 4, hyaluronidase activity inhibition was calculated from the difference in absorbance at 600 nm between the control and sample. Relatively high levels of hyaluronidase activity inhibition were observed in fraction 2 (Fr. 2), collected at retention times of 3.5 to 5 minutes. Therefore, Fr. 2 was further purified using an InertSustain C18 (4.6 × 250 mm, 5 μm) column.

[0034] The column separation conditions were as follows: ─Column and separation conditions─ Column: Inertsil ODS-2 5 μm (4.6 × 150 mm) Mobile phase: acetonitrile / 0.1% formic acid Gradient conditions: Acetonitrile concentration 0 min 0% → 10 min 10% → 20 min 95% → 22 min 95% Flow rate: 1.0 mL / min Detection wavelength: 295 nm Column temperature: 40℃ Injection volume: 50μL

[0035] After sample injection, eluents were collected at retention times of approximately 1.5–3.5 min (Fr. 2-1), 3.5–7 min (Fr. 2-2), 7–9 min (Fr. 2-3), 9–10.5 min (Fr. 2-4), 10.5–19 min (Fr. 2-5), 19–22 min (Fr. 2-6), and 22–30 min (Fr. 2-7) (7 fractions in total) and evaluated for hyaluronidase activity inhibition. Chromatographic charts and the hyaluronidase activity inhibition of each fraction are shown in Figures 5 and 6.

[0036] In Figure 6, hyaluronidase activity inhibition was calculated from the difference in absorbance at 600 nm between the control and sample. Relatively high hyaluronidase activity inhibition was observed in Fraction 2-2 (Fr. 2-2), collected between retention times of 3.5 and 7 minutes. Therefore, Fr. 2-2 was analyzed by LC / TOF-MS, and a library search was performed based on the mass spectral pattern of the target peak. The peak shape of the hit compound was compared with that of a standard sample, and the compound was identified as α-ketoglutaric acid (CAS No. 328-50-7). Thus, it was found that the lactic acid bacteria beverage of the present invention contains α-ketoglutaric acid as a hyaluronidase activity inhibitor.

[0037] [Test Example 3] Confirmation of the inhibitory effect of α-ketoglutaric acid on hyaluronidase activity First, the reagents were prepared. α-Ketoglutaric acid: α-Ketoglutaric acid (trade name "2-oxoglutaric acid": manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in ion-exchanged water to prepare solutions with concentrations of 1, 11, 12, 13, 14, 15, and 20 mM.

[0038] Ten μL of α-ketoglutaric acid adjusted to each concentration and 5 μL of hyaluronidase adjusted to 10 mg / mL were mixed and incubated at 37°C for 20 minutes. Next, 10 μL of the adjusted hyaluronidase activator was added and incubated at 37°C for 20 minutes. 25 μL of the adjusted substrate solution was then added and incubated at 37°C for 40 minutes. To stop the hyaluronidase enzymatic reaction, 10 μL of 0.4 N NaOH and 10 μL of a buffer solution (pH 9.1) adjusted to a final concentration of 0.8 M boric acid and 1 M KOH were added, followed by heating at 95°C for 3 minutes. After incubation at 37°C for 10 minutes, 600 μL of the adjusted p-DAD was added and incubated at 37°C for 20 minutes. After the reaction, 200 μL of the sample solution was taken and the absorbance at 600 nm was measured using a microplate reader. In addition, only the solvent (ion-exchanged water) was added to the control sample.

[0039] The hyaluronidase inhibition rate (%) was calculated using the following formula. Inhibition rate (%)=[1-(S / C)]×100...(1) Here, S is the absorbance of the solution to which each component was added as a sample and corrected with the blank, and C is the absorbance of the solution to which only the solvent was added as a sample and corrected with the blank. Furthermore, the blank is the absorbance of the reaction solution to which each sample was added but no enzyme was added.

[0040] The hyaluronidase inhibition rate (%) of the aqueous α-ketoglutaric acid solution added at each concentration was calculated according to the above formula (1), with the hyaluronidase inhibition rate of ion-exchanged water set at 0%. The results are shown in Figure 7.

[0041] As is clear from Figure 7, when the substrate solution is mixed and the reaction is initiated, the hyaluronidase inhibition rate begins to increase when the α-ketoglutaric acid concentration exceeds 0.2 mM. In particular, the curve showing the inhibition rate rises sharply at α-ketoglutaric acid concentrations of 2.2 mM to 2.8 mM. It can also be seen that the hyaluronidase inhibition rate exceeds 50% when 2.6 mM or more is added. On the other hand, at 3 mM or more, the slope of the curve showing the inhibition rate levels off, suggesting that equilibrium has been reached. These findings suggest that hyaluronidase activity can be inhibited by increasing the α-ketoglutaric acid concentration to 0.2 mM or higher.

[0042] [Test Example 4] Confirmation of the inhibitory effect of α-ketoglutaric acid on hyaluronidase activity using human cells Next, we examined the inhibitory effect of α-ketoglutarate on hyaluronidase activity using normal human dermal fibroblasts (NHDFs). Normal human dermal fibroblasts (NHDFs) were subcultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

[0043] The cell culture conditions for quantifying hyaluronic acid were as follows: NHDF were cultured in a 35 mm dish at 1.0 × 10 5The cells were seeded at 100 cells / dish and cultured in 10% FBS-DMEM at 37°C and 5% CO2 for 5 days. After 5 days of culture, the culture medium was removed and the cells were washed with 1 mL of phosphate-buffered saline (PBS(-)). 500 μL of PBS(-) was then added. The PBS(-)-added dishes were either not irradiated with UV-B or irradiated with UV-B (lamp: AS ONE Handy UV Lamp LUV-4) at 30 mJ / cm. 2 The cells were then irradiated at an intensity of 100 μM. The PBS(-) was then removed, and 1 mL of DMEM (0% FBS) containing water, sodium cromoglycate (final concentration 100 μM), or α-ketoglutaric acid (final concentration 100 μM) was added to the UV-B-irradiated cells. DMEM with water was designated UV(+), DMEM with sodium cromoglycate was designated UV(+) Cont., and DMEM with α-ketoglutaric acid was designated UV(+) AKG. 1 mL of DMEM (0% FBS) with water was added to the UV-B-unirradiated cells (hereafter simply referred to as "UV(-)"). All cells were cultured for 3 days at 37°C under 5% CO2 conditions. After 3 days of culture, the cell supernatant was collected and diluted 100-fold to prepare the samples. The hyaluronan content of each sample was quantified according to the protocol of the HA ELISA assay kit (R&D Systems Inc., Minneapolis, MN, USA). The results are shown in Figure 8.

[0044] Figure 8 shows the mean ± SD for n = 5 data points. Significant differences between the UV(-) and UV(+) samples, between the UV(+) and UV(+) AKG samples, and between the UV(+) and UV(+) Cont. samples were calculated by t-test with p<0.05 (indicated by "*" in the figure) and p<0.01 (indicated by "**" in the figure). As is clear from Figure 2, when comparing the UV(+) and UV(-) samples in water-added medium, the amount of hyaluronic acid in the UV(+) sample is reduced. This suggests that UV irradiation activates hyaluronidase, resulting in the degradation of hyaluronic acid.

[0045] Next, a comparison was made between the UV irradiation groups. It was found that the decrease in hyaluronic acid content was significantly suppressed in sample UV(+)Cont., which contained sodium cromoglycate, known to have strong hyaluronidase inhibitory activity, and sample UV(+)AKG, which contained α-ketoglutaric acid, compared to sample UV(+) which contained water. This suggests that α-ketoglutaric acid inhibits the hyaluronidase activity activated by UV irradiation. Furthermore, it was suggested that the inhibition of hyaluronidase activity suppresses the degradation of hyaluronic acid.

[0046] [Test Example 5] Amount of α-ketoglutaric acid contained in beverages containing other fermented liquids -Method for producing a beverage (fermented milk) containing the fermented liquid according to the present invention- A syrup base containing sugar and glucose-fructose corn syrup was prepared, followed by heat sterilization and cooling to obtain a syrup. 500 mL of the syrup was then mixed and homogenized with 500 mL of the co-fermentation liquid of Lactobacillus paracasei and Streptococcus thermophilus prepared in Test Example 1, along with a small amount of flavoring. A syrup base containing 3 × 10 live lactic acid bacteria was obtained. 8 1000 mL of fermented milk containing more than 1 / mL of lactic acid bacteria was obtained.

[0047] Next, the amount of α-ketoglutaric acid contained in the prepared fermented milk, other companies' fermented milk beverages, and fermented milk (companies A-1 to E) was quantified. Here, fermented milk refers to milk that meets the standards set forth in the Ministerial Ordinance on Milk and Dairy Products' Compositional Standards (Milk Ordinance), and fermented milk beverages refer to dairy fermented milk beverages and fermented milk beverages specified in the Milk Ordinance.

[0048] The amount of α-ketoglutaric acid was quantified using the following method. Ten mL of each lactic acid bacteria beverage and fermented milk was centrifuged (10,000 × g, 4°C, 10 minutes), and the supernatant was collected and filtered through a filter paper. This was then used as a sample for LC / MS / MS analysis. For LC / MS / MS analysis, α-ketoglutaric acid standards were dissolved in distilled water at various concentrations (0.2, 0.5, 1.5, 7.5, 10, and 20 μg / mL). The internal standard, α-ketoglutaric acid (1,2,3,4-13C4), was dissolved at a concentration of 2 μg / mL. Each sample and standard were analyzed using the column and separation conditions described below. A calibration curve was created from the peak area of ​​the standard at each concentration, and the α-ketoglutaric acid concentration in each sample was calculated. If the peak area of ​​each sample exceeded the calibration curve range, the sample was diluted appropriately, and the internal standard was added to the sample solution to achieve a concentration of 2 μg / mL.

[0049] LC conditions Analytical column: Intrada Organic Acid (2.0 x 150mm, 3 μm) Mobile phase: A) Acetonitrile:water:formic acid = 10:90:0.1 B) Acetonitrile:100 mM ammonium formate = 10:90 Gradient conditions (B%): 0 min (0%) → 1 min (0%) → 10 min (100%) → 12 min (100%) Flow rate: 0.25 mL / min Column temperature: 40 °C Injection volume: 2 μL

[0050] MS / MS conditions Ionization method: ESI(-) Measurement ion (m / z): α-Ketoglutaric acid: quantitative ion (145 → 101), qualitative ion (145 → 57), (145 → 73) α-Ketoglutaric acid (1,2,3,4-13C4): (149→105) Drying temperature: 400℃ Capillary voltage: 4500 V Curtain Gas(CUR):50 Collision Gas (CAD): 9 Ion Source Gas 1:70 Ion Source Gas2:70

[0051] The amount of α-ketoglutaric acid in each sample calculated from the α-ketoglutaric acid calibration curve is shown in FIG.

[0052] The lactic acid bacteria beverages produced by the production method of the present invention exhibited α-ketoglutaric acid levels that were 4.2 to 394 times higher than those of other companies' lactic acid bacteria beverages (Companies A-1 to E). Although the data is not shown here, even when the fermentation liquid of Lactobacillus paracasei prepared in Test Example 1 was used, an α-ketoglutaric acid level of at least 10 μg / mL was confirmed.

[0053] [Test Example 6] Effect of adding protease to milk Next, we investigated the effect of adding proteases to the milk feed before heat sterilization. A milk medium containing 10% skim milk powder was prepared. Various proteases (M Amano SD, Orienidase OP, Protin SD-NY10, Peptidase R, ADMIL, and Orienidase 22BF) were added to the milk feed to a final concentration of 0.05%. Peptidase R was treated at 37°C, Orienidase 22BF at 65°C, and the other proteases and protease-free medium at 50°C for 1 hour. The protease reaction was then terminated by heat sterilization (121°C, 15 minutes). After cooling to room temperature, 0.3% Streptococcus thermophilus starter solution (culture solution) was added to each medium, which was then cultured at 30°C for 24 hours. The bacterial solution was harvested after 24 hours of culture. Measurement of α-ketoglutaric acid revealed that the protease treatment resulted in a higher concentration than the control, and in particular, protin SD-NY10 treatment resulted in a higher amount of α-ketoglutaric acid (FIG. 10).

[0054] [Test Example 7] Effect of adding peptides to milk Next, we investigated the effect of adding peptides to feed milk. First, a milk medium containing 10% skim milk powder was prepared. Either a non-supplemented medium (milk medium) or a milk medium supplemented with various peptides (LE80GF-US, Morinaga MKP, WGE80GPA) at a final concentration of 0.1% was prepared and sterilized by heating. Next, 0.3% Streptococcus thermophilus starter solution (culture solution) was added to each medium after cooling to room temperature, and the medium was cultured. The bacterial solution was harvested after 24 hours of culture. Measurement of α-ketoglutaric acid revealed that the addition of peptides resulted in higher concentrations than the control, with the addition of WGE80GP resulting in particularly higher α-ketoglutaric acid levels (Figure 11).

[0055] Although data for Test Examples 6 and 7 are not shown, similar results were confirmed when the fermentation liquid of Lactobacillus paracasei was used.

Claims

1. A beverage containing a lactic acid bacteria fermentation liquid that inhibits hyaluronidase activity and contains α-ketoglutaric acid as an active ingredient.

2. A method for producing a lactic acid bacteria fermentation liquid with an increased production of α-ketoglutaric acid, comprising: A method for producing a lactic acid bacteria fermentation liquid, which involves fermenting milk treated with protease.

3. A method for producing a lactic acid bacteria fermentation liquid with an increased production of α-ketoglutaric acid, comprising: A method for producing a lactic acid bacteria fermentation liquid, which involves fermenting milk to which peptides have been added.

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