JP7638505B2 - Antioxidant Composition - Google Patents

Description

IPOD FERM BP-10896 IPOD FERM BP-11115 IPOD FERM BP-11116 IPOD FERM BP-11117 IPOD FERM BP-11118

The present invention relates to an antioxidant composition.

Oxidative stress is involved in the aging process, which is said to be closely related to the formation of reactive oxygen species (ROS). These reactive oxygen species (ROS) are highly reactive and can oxidatively damage many biopolymers, such as nucleic acids, proteins, and lipids, which can lead to genetic mutations and cellular senescence.

Higher levels of free radicals have been reported in aged rats. This has been attributed to a decline in antioxidant levels. An age-dependent decline in antioxidant levels has been well documented in rats and humans. For example, mRNA levels of enzymatic antioxidants such as superoxide dismutase, catalase and glutathione peroxidase have been quantified and found to be decreased in the liver of aged rats.

The redox state of cells, which changes during aging, can be modified by diet. Iron and other nutritional deficiencies, which currently affect more than 2 billion people worldwide, have been shown to induce oxidative stress. Previous studies have suggested that ferritin acts as a protective agent against oxidative damage in endothelial cells and mouse and human leukemia cells. We have recently shown that the antioxidant effect of iron-based hydroferrate fluids derived from divalent and trivalent ferrates protects against oxidative stress-induced apoptosis of mouse splenocytes in vitro (Non-Patent Document 1).

On the other hand, lactobacilli are another natural dietary-related substance that have long been thought to have the potential to extend human lifespan. Biologist Eli Metchnikoff suggested this effect in the early 1900s (Non-Patent Document 2). Lactobacilli are often used in the production of fermented milks and foods, and the lactobacilli strains found in fermented milk are normal bacteria found in the gut microbiome. Lactobacilli have been shown to help reduce pathogenic bacteria in the gut while maintaining a healthy balance of probiotic bacteria. In addition, lactobacilli have been shown to have beneficial effects on a variety of diseases, including rheumatoid arthritis, Crohn's disease, and cancer.

Badr El-Din NK, Noaman E, Fattah SM, Ghoneum M. Reversal of age-associated oxidative stress in rats by MRN-100, a hydro-ferrate fluid. In Vivo. 2010; 24(4):525-33. Metchinkoff E and Metchinkoff II: The Prolongation of Life: Optimistic Studies. Putnam, New York, NY, pp109-132, 1908.

Providing new active ingredients for antioxidant compositions would broaden the range of treatment and therapy options. In addition, it would be desirable if the active ingredient were a natural product or its primary processing product, as this would likely pose less risk of side effects.

Therefore, the object of the present invention is to provide a novel antioxidant composition that contains natural products or their primary processed products as active ingredients.

The present inventors have conducted extensive research to achieve the above object and have completed the present invention.
[1] An antioxidant composition comprising a milk fermentation product of Lactobacillus kefiri or a processed product thereof as an active ingredient.
[2] The antioxidant composition described in [1], which is used to reduce oxidative stress.
[3] The antioxidant composition according to [1] or [2], wherein the Lactobacillus kefiri includes Lactobacillus Kefiri P-IF (Accession No. FERM BP-10896).

The present invention provides a novel antioxidant composition that uses a milk fermentation product of Lactobacillus kefiri or a processed product thereof as an active ingredient, and is therefore safe and has antioxidant effects with few side effects.

1(a) is a graph showing the results of evaluating biochemical serum parameters in Test Example 6, in which FIG. 1(a) is a graph showing the results of measuring the enzyme activity of ALT (EC 2.6.1.2) in serum, FIG. 1(b) is a graph showing the results of measuring the enzyme activity of AST (EC 2.6.1.1) in serum, and FIG. 1(c) is a graph showing the results of measuring the concentration of total protein in serum. 2(a) is a graph showing the results of evaluating biochemical serum parameters in Test Example 6, where FIG. 2(a) is a graph showing the results of measuring the concentration of total cholesterol (TC) in serum, FIG. 2(b) is a graph showing the results of measuring the concentration of triglyceride (TG) in serum, FIG. 2(c) is a graph showing the results of measuring the concentration of low-density lipoprotein (LDL), and FIG. 2(d) is a graph showing the results of measuring the concentration of high-density lipoprotein (HDL) in serum.

Lactobacillus kefiri may be any microorganism belonging to the genus Lactobacillus kefiri, and is not particularly limited thereto. Preferably, Lactobacillus Kefiri P-IF (Accession No. FERM BP-10896) is used. Lactobacillus kefiri can be cultured by known methods or methods similar thereto. For example, mass culture is possible by anaerobic static culture using a commercially available MRS medium (trade name "Lactobacilli MRS Broth", manufactured by Difco).

The milk fermentation product of Lactobacillus kefiri can be obtained by inoculating a raw milk material with Lactobacillus kefiri as a starter and exposing it to specified fermentation conditions.

Examples of dairy raw materials for milk fermentation include milk, whey, fermented milk, lactic acid bacteria beverages, skim milk, skim milk powder, prepared milk powder, whole milk powder, concentrated milk, skim concentrated milk, condensed milk, skim condensed milk, sweetened condensed milk, sweetened skim condensed milk, etc. These can be used as raw materials for milk fermentation as they are, or after dissolving or diluting in water or the like as appropriate, or concentrating, and then subjecting them to treatment such as autoclave sterilization as necessary.

Methods for inoculating the fermentation microorganism (starter) include adding a preculture solution such as MRS medium, or adding the remaining milk fermentation product from a previous fermentation. Culture conditions preferably include anaerobic static culture at 20 to 30°C, more preferably 24 to 26°C, for 1 to 10 days, more preferably 3 to 7 days.

As the starter for the above-mentioned milk fermentation, multiple species of bacteria may be used in combination. In this way, milk fermentation occurs due to the symbiotic growth of multiple microorganisms, and a milk fermented product with good taste and appearance can be obtained. For example, at least one yeast selected from the group consisting of Kazachstania turicensis, Kazachstania unispora, and Kluyveromyces marxianus may be used in combination. Alternatively, as Lactobacillus kefiri, in addition to the above-mentioned Lactobacillus Kefiri P-IF (Accession No. FERM BP-10896), Lactobacillus kefiri P-B1 (Accession No. FERM BP-11115) may be used in combination.

In a particularly preferred embodiment, the starter for the above-mentioned milk fermentation is Lactobacillus kefiri P-IF (Accession No. FERM BP-10896) as the main microorganism, and Lactobacillus kefiri P-B1 (Accession No. FERM BP-11115), Kazachstania turicensis P-Y3 (Accession No. FERM BP-11116), Kazachstania unispora P-Y4 (Accession No. FERM BP-11117), and Kluyveromyces marxianus P-Y5 (Accession No. FERM BP-11118) as secondary microorganisms. It is preferable to ferment the above-mentioned milk raw material using a complex microorganism containing at least Lactobacillus kefiri P-IF (Accession No. FERM BP-11118), and to prepare the microbial population of the fermented milk product after fermentation so that Lactobacillus kefiri P-IF (Accession No. FERM BP-10896) accounts for 80% or more, more preferably 90% or more, of the bacterial count, and to use the fermented milk product thus prepared.

In the present invention, the milk fermentation product of Lactobacillus kefiri obtained as described above or a processed product thereof is used as an active ingredient of the antioxidant composition. Here, the "processed product" includes a product prepared by subjecting the milk fermentation product to one or a combination of two or more of the following treatments: heat sterilization, concentration, drying, cell separation, cell removal, cell disruption, dilution with an aqueous solvent or a hydroalcoholic solvent, and extraction into an aqueous solvent or a hydroalcoholic solvent. It also includes a product prepared by subjecting an extract extracted with an aqueous solvent or a hydroalcoholic solvent to one or a combination of two or more of the following treatments: heat sterilization, concentration, drying, and removal of solids.

For example, Lactobacillus kefiri can be killed by subjecting the cells to a heat treatment at 60 to 120°C, more preferably 95 to 120°C. Thus, the milk fermentation product can be powdered by spray drying, and may be killed by heat added at the same time. Alternatively, the milk fermentation product may be powdered by freeze-drying. In this way, it is possible to provide Lactobacillus kefiri in a live state by reconstituting the freeze-dried milk fermentation product with water or the like.

In the antioxidant composition of the present invention, there is no particular restriction on the addition of other materials in addition to the above-mentioned active ingredients, and the composition can be used in the form of tablets, granules, capsules, pills, powders, liquids, powders, jelly-like agents, candies, injections, inhalants, topicals, etc., by adding pharma- ceutically acceptable base materials or carriers as necessary and using known formulation methods.

The antioxidant composition of the present invention may be administered to humans or animals, and there are no particular limitations on the form of administration. Examples include oral administration, intravenous administration, local administration in the brain, intraperitoneal administration, inhalation, and nasal administration. Among these, oral administration is preferred from the viewpoints of reducing the burden on the recipient and ease of administration.

When the antioxidant composition of the present invention is orally administered, the daily dosage is preferably 0.01 (g/kg body weight) to 10 (g/kg body weight), more preferably 0.05 (g/kg body weight) to 5 (g/kg body weight), and even more preferably 0.1 (g/kg body weight) to 1 (g/kg body weight), calculated as the amount of the fermented milk product of Lactobacillus kefiri in a dry state. Alternatively, the amount converted into the number of bacteria of Lactobacillus kefiri contained in the milk fermentation product is preferably 1×10 ¹³ (cells/kg body weight) to 1×10 ¹⁶ (cells/kg body weight), more preferably 5×10 ¹³ (cells/kg body weight) to 5×10 ¹⁵ (cells/kg body weight), and even more preferably 1×10 ¹⁴ (cells/kg body weight) to 1×10 ¹⁵ (cells/kg body weight). If the dosage is less than this range, it is difficult to obtain a sufficient effect, and if the dosage is more than this range, the risk of some side effects occurring increases.

The number of Lactobacillus kefiri bacteria contained in the dried milk fermentation product of Lactobacillus kefiri is preferably 5 x 10 ¹² (cells/g) to 3 x 10 ¹⁵ (cells/g), more preferably 5 x 10 ¹³ (cells/g) to 2 x 10 ¹⁵ (cells/g), and even more preferably 5 x 10 ¹⁴ (cells/g) to 1 x 10 ¹⁵ (cells/g).

The antioxidant composition of the present invention can be preferably used, for example, to improve oxidative stress associated with age. Typically, it can be preferably used to improve oxidative stress associated with age in humans aged 30 years or older, more typically 40 years or older, even more typically 50 years or older, and most typically 60 years or older. It may also be applied to pet animals, and can be preferably used to improve oxidative stress associated with age in elderly pet animals that correspond to the above-mentioned human ages. For example, in the case of dogs, it can be preferably used to improve oxidative stress associated with age in dogs aged 4 years or older, more typically 6 years or older, even more typically 8 years or older, and most typically 10 years or older. For example, in the case of cats, it can be preferably used to improve oxidative stress associated with age in cats aged 4 years or older, more typically 6 years or older, even more typically 9 years or older, and most typically 11 years or older.

The product form for administering the antioxidant composition of the present invention is not particularly limited, so long as it does not impair its action and effect. For example, it can be used in various product forms, such as medicines, quasi-drugs, health foods, functional foods, nutritional supplements, and supplements, or in combination with such products. It may also be an animal product for pet animals, etc.

The present invention will be described in detail below with reference to examples, but these examples are not intended to limit the scope of the present invention.

Materials and Methods
(1) Preparation of Antioxidant Composition Lactobacillus kefiri P-IF (Accession No. FERM BP-10896) was inoculated into an MRS liquid medium and pre-cultured at 25° C., and the resulting medium was used as the primary microorganism. In addition, Lactobacillus kefiri P-B1 (Accession No. FERM BP-11115), Kazachstania turicensis P-Y3 (Accession No. FERM BP-11116), Kazachstania unispora P-Y4 (Accession No. FERM BP-11117), and Kluyveromyces marxianus P-Y5 (Accession No. FERM BP-11118) were used as secondary microorganisms. A complex microorganism containing at least Lactobacillus kefiri P-IF (Accession No. FERM BP-11118) was used as a starter, inoculated into a skim milk medium (a medium prepared by mixing 10 parts by weight of skim milk powder with 90 parts by weight of ion-exchanged water and sterilizing it in an autoclave), and fermented by anaerobic static culture at 25°C for 5 days. The obtained fermented culture was a yellowish brown yogurt-like liquid in which Lactobacillus kefiri P-IF (Accession No. FERM BP-10896) accounted for 90% or more of the bacterial count. This was heat-treated, freeze-dried, and used in the following animal experiments. The bacterial concentration in the dried product was approximately 1 x 1015/g. For simplicity, this preparation will be referred to as "PFT" below.

(2) Animals and Experiments 10-month-old male Swiss albino mice (body weight about 23-28 g: old mice) and 2-month-old male Swiss albino mice (body weight about 11-22 g: young mice) were used. The mice were classified into group 1 (normal young mice), group 2 (normal old mice), and group 3 (PFT-treated group). In the PFT-treated group, PFT was orally administered at a dose of 2 mg/kg body weight every day for 6 weeks with free access to water. After 6 weeks, the animals were anesthetized after fasting overnight, and blood was collected from the abdominal aorta using a syringe, and serum was separated and stored at -80°C until analysis. In addition, liver and brain tissues were quickly excised, washed with ice-cold 0.9% NaCl, frozen in liquid nitrogen, and similarly stored at -80°C.

For samples for analysis, tissues were homogenized in PBS, 0.1 M, pH 7.4 (9 mL/g tissue), centrifuged at 6000 rpm at 4°C for 20 min, and the supernatant was collected.

[Test Example 1]
Assessment of lipid peroxidation and nitric oxide levels
Lipid peroxidation levels were evaluated by analyzing the malondialdehyde (MDA) content in the form of thiobarbituric acid reactive substances (TBARS). Specifically, 500 μL of sample was added to 1 mL of trichloroacetic acid (TCA, 15%) and centrifuged at 3000 rpm for 10 minutes. 1 mL of the supernatant was mixed with 500 μL of thiobarbituric acid (TBA, 0.7%), heated in a boiling water bath for 10 minutes, cooled, and the absorbance was measured with light of a wavelength of 532 nm. TBARS levels were calculated according to the following formula and expressed in units of the amount (nmol/mL) in the measured sample volume (BROWN WD, TAPPEL AL "Fatty acid oxidation by carp liver mitochondria." Arch Biochem Biophys. 1959 Nov; 85: pp149-58.).

TBARS level (nmol/mL) = sample absorbance 532nm/156

Nitric oxide (NO) levels were measured using the Griess reaction as follows: 100 μL of sample was added to 100 μL of acidic Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid), mixed, and the absorbance was measured at a wavelength of 540 nm. NO levels were calculated in units of concentration (μM) in the measured sample dose by fitting the measurement results to a standard curve covering different concentrations created using a separate standard substance.

As shown in Table 1, the following (1) and (2) became clear.
(1) TBARS levels In aged mice, the TBARS levels in liver, brain, and serum tissues were significantly increased compared to young mice. In contrast, in PFT-treated group, the TBARS levels in each tissue were significantly decreased compared to aged mice, by 55% in liver, 81% in brain, and 73% in serum.

(2) NO levels In aged mice, NO levels in the liver, brain, and serum were significantly increased compared to young mice, whereas in the PFT-treated group, NO levels in each tissue were significantly decreased (47% in the liver, 60% in the brain, and 82% in the serum) compared to aged mice.

These results demonstrate that administration of PFT counteracts age-related increases in TBARS and NO levels; in other words, PFT reduces oxidative stress.

[Test Example 2]
Assessment of reduced glutathione levels and glutathione S-transferase activity
Reduced glutathione (GSH) levels were measured as follows: 100 μL of sample was mixed with 100 μL of sulfosalicylic acid (4%). The mixture was then kept at 4°C for at least 1 hour and centrifuged at 1200 rpm for 10 minutes at 4°C. 100 μL of the supernatant was mixed with 2.7 mL of phosphate buffer (0.1 M, pH 7.4) and 0.2 mL of DTNB reagent (5,5'-Dithiobis(2-nitrobenzoic acid)) and incubated for 5 minutes. The absorbance of the yellow product was measured at a wavelength of 412 nm. GSH levels were calculated in units of the amount of protein in the measured sample (mg mg protein) by fitting the measurement results to a standard curve covering different concentrations of a standard substance.

Glutathione S-transferase (GST) activity was measured using the binding of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GSH) as follows. First, 100 μL of GSH (5 mM), 10 μL of CDNB (1 mM in ethanol), and 25 μL of sample were added to 1.365 mL of phosphate buffer (0.1 M, pH 6.5) and incubated at room temperature for 20 minutes. Then, absorbance was measured at a wavelength of 310 nm, and GST activity (μmol·min-1·mg-1 protein) was calculated by a standard formula (Habig WH, Pabst MJ, Jakoby WB "Glutathione S-transferases. The first enzymatic step in mercapturic acid formation." J Biol Chem. 1974; 249: pp7130-39.).

As shown in Table 2, the following (1) and (2) became clear.
(1) GSH levels In aged mice, GSH levels in liver, brain, and serum tissues were significantly decreased compared to young mice, whereas in PFT-treated mice, GSH levels in liver, brain, and serum tissues were significantly increased (122%, 126%, and 167%) compared to aged mice.

(2) GST activity GST activity in the liver, brain, and serum of aged mice was significantly decreased compared to that in young mice, whereas GST activity in each tissue of the PFT-treated group was significantly increased (205% in the liver, 164% in the brain, and 255% in the serum) compared to that in aged mice.

These results demonstrate that administration of PFT counteracts age-related increases in GSH levels and GST activity, in other words, that PFT reduces oxidative stress.

[Test Example 3]
<Evaluation of superoxide dismutase activity, catalase activity, and glutathione peroxidase activity>
Superoxide dismutase (SOD) activity was measured using the inhibition rate of pyrogallol oxidation as follows. First, 20 μL of sample and 10 μL of 20 mM pyrogallol (10 mM hydrochloric acid aqueous solution) were added to 1 mL of buffer solution to prepare a test solution. The absorbance of the test solution was measured with light of a wavelength of 420 nm after 30 seconds and 90 seconds. The inhibition rate of pyrogallol oxidation was calculated according to the following formula.

Inhibition rate (%) = [(100 - ΔAt) / ΔAo] x 100
(ΔAt is the change in absorbance 420n per unit time (1 min) and unit dose (1 mL) of the test solution, and ΔAo is the change in absorbance 420n per unit time (1 min) and unit dose (1 mL) of the reference solution.)

Catalase (CAT) activity was measured by mixing 0.1 mL of sample with 0.5 mL of 0.2 M sodium phosphate buffer at pH 7.6 and 0.3 mL of 0.5% H2O2 in a sample cuvette, and diluting the mixture with distilled water to a total of 3 mL. The decomposition of H2O2 was measured by the absorbance of light at a wavelength of 240 nm, and the rate of change in absorbance per minute (absorbance change rate % min-1) was calculated as the enzyme activity.

Glutathione peroxidase (GPx) activity was measured by adding 50 μL of sample and 100 μL of GSH to 750 μL of Tris-HCl solution (50 mM, pH 7.6) and incubating at 37°C for 10 minutes. Next, 1 mL of TCA (15% by mass) and 100 μL of H2O2 were added and centrifuged at 3000 rpm for 20 minutes. 2 mL of Tris-HCl solution (0.4 M, pH 8.9) and 100 μL of DTNB reagent (5,5'-Dithiobis(2-nitrobenzoic acid) were added to 1 mL of the supernatant and incubated for 5 minutes. The absorbance of the obtained test solution was measured with light at a wavelength of 412 nm, and the activity of GPx was calculated using the following formula.

GPx activity (mol min-1 mg-1 protein amount) = (At-Ao) x 6.2 x [100/13.1] x 0.05 x 10
(At is the absorbance of the test solution at 412 nm, A0 is the absorbance of the reference solution at 412 nm)

As shown in Table 3, the following (1) to (3) became clear.
(1) GPx activity In aged mice, GPx activity in liver, brain, and serum tissues was significantly decreased compared to young mice. In contrast, in the PFT-treated group, GPx activity in each tissue was significantly increased (136% in liver, 142% in brain, and 135% in serum) compared to aged mice.

(2) CAT activity In aged mice, CAT activity in liver, brain, and serum was significantly decreased compared to young mice, whereas in PFT-treated mice, CAT activity in liver, brain, and serum was significantly increased (141%, 155%, and 140%, respectively) compared to aged mice.

(3) SOD activity In aged mice, SOD activity in liver, brain, and serum was significantly decreased compared to young mice, whereas in PFT-treated mice, SOD activity in liver, brain, and serum was significantly increased by 289%, 170%, and 212%, respectively, compared to aged mice.

These results demonstrate that administration of PFT counteracts the age-related decline in GPx activity, CAT activity, and SOD activity; in other words, PFT reduces oxidative stress.

[Test Example 4]
<Evaluation of total antioxidant capacity>
The total antioxidant capacity (TAC) level was measured as follows: 1 mL of sample was added to 1 mL of a solution containing sulfuric acid (0.6 M), sodium phosphate (28 mmol) and ammonium molybdate (4 mmol), and the mixture was incubated at 95° C. for 90 minutes. After cooling to room temperature, the absorbance was measured at a wavelength of 695 nm. The TAC level was calculated according to the following formula:

TAC (%) = [(A0-At)/A0] x 100
(A0 is the absorbance of the reference solution at 695 nm, At is the absorbance of the test solution at 695 nm)

As shown in Table 4, the TAC levels in the liver, brain, and serum tissues of old mice were significantly decreased compared to young mice. In contrast, in the PFT-treated group, the TAC levels in each tissue were significantly increased compared to old mice, by 126% in the liver, 142% in the brain, and 144% in the serum.

These results demonstrate that administration of PFT counteracts the age-related decline in TAC levels; in other words, PFT reduces oxidative stress.

[Test Example 5]
<Evaluation of anti-hydroxyl radical activity>
Anti-hydroxyl radical (AHR) activity was measured as follows: 10 μL of sample was added to a reaction mixture of 100 μL H2O2, 100 μL FeSO4, 100 μL 2-deoxyribose-D-ribose, and 2.7 mL phosphate buffer (pH 7.4) and incubated at 37°C for 60 minutes. Then, 0.2 mL EDTA and 2 mL TBA reagent (5.2 mL perchloric acid, 1.5 g thiobarbituric acid, 60 g trichloroacetic acid) were added, and the absorbance of the pink complex formed was measured at 532 nm wavelength. The results were expressed in units of "(U mg-1 protein amount)" according to the standard formula (Halliwell B1, Gutteridge JM, Aruoma OI, "The deoxyribose method: a simple "test-tube" assay for determination of rate constants for reactions of hydroxyl radicals." Anal Biochem. 1987; 165(1): pp215-219.).

As shown in Table 5, AHR activity in the liver, brain, and serum tissues of old mice was significantly decreased compared to young mice. In contrast, AHR activity in each tissue of the PFT-treated group was significantly increased compared to old mice, by 119% in the liver, 118% in the brain, and 119% in the serum.

These results demonstrate that administration of PFT counteracts the age-related decline in AHR activity; in other words, PFT reduces oxidative stress.

[Test Example 6]
Evaluation of biochemical serum parameters
The enzyme activities of ALT (EC2.6.1.2) and AST (EC2.6.1.1), which are indicators of liver function, and the concentration of total protein were measured. In addition, the concentrations of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), and high density lipoprotein (HDL) were measured as serum lipid profiles. These biochemical serum parameters were analyzed using a commercially available kit according to the kit manufacturer's instructions. In addition to the PFT-treated group for old mice, the test groups included a PFT-treated group for young mice, and serum was collected and subjected to analysis.

As a result, as shown in Figure 1(a), the serum concentration of ALT (EC2.6.1.2), an indicator of liver function, was higher in old mice than in young mice, whereas in the PFT-treated old mice, the concentration was reduced to the same level as in young mice.

As shown in Figure 1(b), the serum concentration of AST (EC2.6.1.1), an indicator of liver function, was higher in old mice than in young mice, whereas the concentration was significantly reduced in the PFT-treated old mice.

In addition, as shown in Figure 1(c), no changes were observed in the total protein concentration in serum between the test groups.

Based on the above, it is believed that PFT treatment of aged mice brought about an antioxidant effect in the liver and improved liver function in the aged mice.

As shown in Figure 2(a), the serum concentration of total cholesterol (TC) was higher in old mice than in young mice, whereas in the PFT-treated old mice, the concentration was reduced to the same level as in young mice.

As shown in Figure 2(b), serum triglyceride (TG) concentrations were higher in aged mice than in young mice, whereas the concentrations were lower in the PFT-treated aged mice.

As shown in Figure 2(c), the serum concentration of low-density lipoprotein (LDL) was higher in aged mice than in young mice, whereas the concentration was significantly reduced in the PFT-treated aged mice.

As shown in Figure 2(d), the serum concentration of high-density lipoprotein (HDL) was lower in old mice compared to young mice, whereas in the PFT-treated old mice, the concentration increased to the level of young mice.

Based on the above, it is believed that PFT treatment of aged mice brought about an antioxidant effect in the liver, improving liver function in aged mice and thereby improving serum lipid profiles.

Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the gist of the invention. These embodiments and their modifications are included within the scope and gist of the invention, and are included in the scope of the invention and its equivalents as set forth in the claims.

Claims (2)

An antioxidant composition comprising a milk fermentation product of a plurality of microorganisms or a processed product thereof as an active ingredient , the plurality of microorganisms being Lactobacillus Kefiri P-IF (Accession No. FERM BP-10896), Lactobacillus kefiri P-B1 (Accession No. FERM BP-11115), Kazachstania turicensis P-Y3 (Accession No. FERM BP-11116), Kazachstania unispora P-Y4 (Accession No. FERM BP-11117), and Kluyveromyces marxianus P-Y5 (Accession No. FERM BP-11118). BP-11118). The antioxidant composition according to claim 1, which is used to reduce oxidative stress.

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