antioxidants

Abstract

To screen lactic acid bacteria that have high antioxidative action even as heated and killed cells or dried powder of them while having high safety and wide availability to food and provide an antioxidant containing the lactic acid bacteria as an active ingredient.SOLUTION: An antioxidant contains Lactococcus lactis subsp. lactis biovar. diacetylactis) N7 (FERM P-18217) as an active ingredient. The killed cells of the N7 strain and dried powder of them indicated significant antioxidant activity even in an evaluation system using zebrafish.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an antioxidant containing a specific lactic acid bacterium as an active ingredient, and more particularly to an antioxidant containing Lactococcus lactis subsp. lactis biovar diacetylactis N7 as an active ingredient. [Background technology]

[0002] While active oxygen, which is produced during life activities, has a bactericidal effect, if produced in large quantities it is thought to cause diseases and cellular aging, such as by generating lipid peroxides in the body. For this reason, substances with antioxidant properties (such as vitamin C and carotenoids) are currently being actively taken as supplements.

[0003] Meanwhile, lactic acid bacteria, known as probiotics (live microorganisms that contribute to maintaining the health of the host when ingested in appropriate amounts), have also been reported to have antioxidant properties (see, for example, Non-Patent Document 1). Lactic acid bacteria are safe microorganisms with a wide range of uses; live bacteria are used in the production of yogurt, pickles, etc., and dead bacteria are added to foods such as sweets and beverages.

[0004] The present inventors have been conducting research into the functionality of Lactococcus lactis subsp. lactis biovar diacetylactis N7 (hereinafter sometimes referred to as "N7 strain") and have previously reported its cholesterol-reducing and -removing effects (see Patent Document 1). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Japanese Journal of Lactic Acid Bacteria,Vol.29,No.2,p.69-78 [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2002-65203 Summary of the Invention [Problem to be solved by the invention]

[0007] One of the mechanisms of the antioxidant properties of Lactococcus lactis subsp. cremoris H61 described in Non-Patent Document 1 is thought to be due to an enzyme (superoxide dismutase: SOD) that removes reactive oxygen species. When the bacterial cells are heat-treated, the enzyme is thermally denatured, and its activity decreases. Therefore, there has been a demand for lactic acid bacteria that have high antioxidant properties even in the form of heat-killed bacterial cells or their dried powder.

[0008] The objective of the present invention is to select lactic acid bacteria that have high antioxidant activity even in the form of heat-killed cells or dried powder thereof, are highly safe, and can be widely used in foods, and to provide an antioxidant containing the lactic acid bacteria as an active ingredient. [Means for solving the problem]

[0009] As a result of extensive research aimed at solving the above problems, the present inventors have found that, among Lactococcus lactic acid bacteria, Lactococcus lactis subsp. lactis biovar diacetylactis N7 has significantly higher antioxidant activity (DPPH radical scavenging activity) than the aforementioned H61 strain in in vitro tests (see Example 1). Furthermore, they have demonstrated that the activity of the N7 strain does not decrease even after heat treatment (see Example 2).

[0010] Furthermore, the present inventors have demonstrated that killed cells and dried powder of the N7 strain have high antioxidant activity even at the living body level using a zebrafish antioxidant function evaluation system, and have found that this lactic acid bacterium is highly practical as an antioxidant. Thus, the present invention was completed.

[0011] That is, the present invention provides an antioxidant containing Lactococcus lactis subsp. lactis biovar. diacetylactis N7 as an active ingredient.

[0012] Here, the N7 strain may be in the form of live or killed cells, or a dried powder thereof.

[0013] The antioxidant may also be in the form of a food composition, a beverage, an animal feed, a pharmaceutical, a veterinary drug, a cosmetic, or a quasi-drug. [Effects of the Invention]

[0014] The present invention provides an antioxidant using Lactococcus lactis subsp. lactis biovar diacetylactis N7. The N7 strain is a lactic acid bacterium that has been used in dairy product production for many years and can be safely ingested. Furthermore, the antioxidant activity of the N7 strain is observed even in heat-killed cells and dried powder, making it suitable for use as an antioxidant in a variety of food products. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a diagram illustrating an antioxidant function evaluation system using zebrafish. [Figure 2] This graph shows the effect of adding killed N7 strain cells (suspension) on the viability (vertical axis: %) of zebrafish larvae treated with hydrogen peroxide (2.8 mM HO) (N = 24 or more). The horizontal axis represents the treatment time (unit: hours) of the oxidizing agent hydrogen peroxide solution, and the vertical axis represents the viability (unit: %), with the value at the start of treatment taken as 100. The solid line represents the results for larvae pretreated with a suspension of N7 strain OD620 = 0.125 or OD620 = 0.25, and the dashed line represents the results for larvae not pretreated. [Figure 3]This graph shows the effect of adding killed N7 strain cells (lyophilized cell solution) on the viability (vertical axis: %) of zebrafish larvae treated with hydrogen peroxide (2.8 mM HO) (N = 24 or more). The horizontal axis represents the treatment time (unit: hours) of the oxidizing agent hydrogen peroxide solution, and the vertical axis represents the viability (unit: %), with the value at the start of treatment taken as 100. The solid line represents the results for larvae pretreated with a 250 μg / mL solution of the N7 strain, the dashed-dotted line represents the results for larvae pretreated with a 125 μg / mL solution of the N7 strain, and the dotted line represents the results for larvae not pretreated. DETAILED DESCRIPTION OF THE INVENTION

[0016] Hereinafter, embodiments of the present invention will be described in detail. The antioxidant according to this embodiment is characterized by containing Lactococcus lactis subsp. lactis biovar diacetylactis N7 as an active ingredient.

[0017] The N7 strain has been deposited at the Patent Organism Depositary of the National Institute of Technology and Evaluation under accession number FERM P-18217. This strain can be cultured according to standard methods for culturing lactic acid bacteria, for example, by culturing it in MRS medium or TYG medium under facultative anaerobic conditions for about 1 to 2 days.

[0018] As described below, the N7 strain demonstrated significant DPPH radical scavenging activity in in vitro tests. Furthermore, the killed cells and dried powder of the N7 strain were also confirmed to have high antioxidant activity at the living body level using a zebrafish evaluation system. Given its long history of use as a food, the N7 strain is considered to be safe for the human body and is therefore considered to be extremely useful as an antioxidant.

[0019] The antioxidant according to this embodiment contains N7 strain cells (live or killed cells) as an active ingredient. Here, the "cells" of the N7 strain may be the culture itself or cells that have been subjected to conventional treatments such as concentration, washing, purification, sterilization, pH adjustment, crushing, or powdering.

[0020] The antioxidant of this embodiment may contain components other than the N7 strain that have antioxidant activity, but since the N7 strain alone is sufficiently effective, it is desirable to contain only the N7 strain as the active ingredient.

[0021] The "dry powder" of the N7 strain is not particularly limited, but examples include freeze-dried powder, spray-dried powder, fluidized-bed dried powder, etc. For example, the cells obtained by collecting cells from a culture of the N7 strain by centrifugation or the like can be washed with physiological saline or the like, heat-sterilized, and then freeze-dried to obtain a dry powder of killed cells of the N7 strain.

[0022] The antioxidant of this embodiment can be in the form of a food composition, beverage, animal feed, and can be used by being contained in, for example, any food composition, beverage, animal feed, etc. Here, examples of "animals" include fish such as zebrafish, medaka, goldfish, etc.; mammals such as mice, rats, guinea pigs, hamsters, ferrets, monkeys, cows, pigs, goats, sheep, chickens, dogs, cats, etc.; birds such as parakeets, etc.

[0023] By ingesting this antioxidant to humans or animals, it is expected that it will exert its antioxidant effect in the body, suppressing the production of lipid peroxides, preventing cell damage, and suppressing the production of active oxygen.In addition, it can also have the effect of suppressing the oxidation of food compositions to which the antioxidant is added.

[0024] Examples of food compositions or beverages include, but are not limited to, dairy products such as yogurt, confectioneries, various processed foods, supplements, lactic acid bacteria beverages, soft drinks, tea beverages, nutritional drinks, etc. In the case of yogurt, antioxidant yogurt containing the N7 strain can be produced by adding the N7 strain to raw materials such as raw milk and fermenting the mixture.

[0025] The content of the active ingredient in a food composition, drink, or animal feed varies depending on the intake form, but can be an amount that provides an antioxidant effect. Specifically, when it is contained as a dried bacterial cell powder, the content can be such that the daily intake is, for example, 1 mg to 1 g, preferably 10 mg to 100 mg. When it is contained as live bacterial cells, the daily intake is, for example, 10 9 ~10 12 cfu, preferably 10 10 ~10 11 The content can be adjusted to give cfu.

[0026] Therefore, for example, in the case of yogurt with a serving size of 100 g, the content of dry bacterial powder is 0.001 to 1 mass % (preferably 0.01 to 0.1 mass %), and the content of live bacterial cells is 10% per 100 g. 9 ~10 12 cfu (preferably 10 10 ~10 11 cfu).

[0027] The antioxidant of the present embodiment can also be in the form of a pharmaceutical product, a veterinary drug, a cosmetic product, or a quasi-drug, which can be produced by a conventional method.

[0028] When administered orally as a pharmaceutical, veterinary drug, or quasi-drug, the dosage form may be, for example, tablets, powders, fine granules, granules, capsules, syrup, etc. When the pharmaceutical, veterinary drug, cosmetic, or quasi-drug is used as an external preparation, it may be in the form of, for example, a liquid, gel, cream, ointment, etc.

[0029] In addition, the pharmaceuticals, veterinary drugs, cosmetics or quasi-drugs may contain other ingredients that are acceptable for formulation (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.) or other medicinal ingredients.

[0030] The content of the active ingredient in the above-mentioned pharmaceuticals, veterinary medicines, cosmetics or quasi-drugs varies depending on the form, but can be an amount that can obtain an antioxidant effect. Specifically, in the case of an oral preparation containing the active ingredient as a dried bacterial cell powder, the content can be set so that the daily intake is, for example, 1 mg to 1 g, preferably 10 mg to 100 mg. In the case of an oral preparation containing the active ingredient as a live bacterial cell, the daily intake is, for example, 10 9 ~10 12 cfu, preferably 10 10 ~10 11 cfu. In the case of an external preparation, the content can be, for example, 0.0125% by mass or more, preferably 0.0125% by mass to 0.025% by mass, in terms of dry bacterial cell powder. The dosage of the preparation can be appropriately determined taking into consideration the symptoms, age, weight, sex, etc. of the patient.

[0031] The administration method can be, for example, a method in which an effective dose of the pharmaceutical or veterinary pharmaceutical is administered orally or parenterally to the target human or animal, for example, once to several times per day or once every 2 to 3 days.

[0032] The N7 strain is a lactic acid bacteria strain belonging to Lactococcus lactis subsp. lactis, which is present in a wide range of foods, including dairy products and pickles, and has a long history of use in the production of dairy products. Therefore, the antioxidant according to this embodiment can be safely ingested daily over a long period of time as a food, beverage, animal feed, pharmaceutical, or veterinary drug. [Example]

[0033] The present invention will be specifically explained below with reference to examples and comparative examples, but the present invention is not limited to these.

[0034] [Example 1] Comparison of DPPH radical scavenging activity of lactic acid bacteria The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of various Lactococcus species was compared.

[0035] (1) Sample preparation The lactic acid bacteria used in this study were the N7 (NITE FERM P-18217), G50 (NITE FERM P-18415), and 527 (MAFF 400103) strains belonging to Lactococcus lactis subsp. lactis, and the H61 (NITE P-92) and ATCC 19257 strains belonging to Lactococcus lactis subsp. cremoris.

[0036] The above lactic acid bacteria were cultured overnight by conventional methods, and the MRS culture solution was inoculated into MRS medium (Becton Dickinson and Company) at 0.5% (v / v) and cultured overnight at 30°C. The culture was centrifuged at 13,000 g for 10 minutes to collect the cells. The resulting cells were washed three times with water and then suspended in a 50% (v / v) aqueous ethanol solution to obtain a cell suspension of 100 mg / mL (wet cell weight w / v). This cell suspension was centrifuged at 13,000 g for 10 minutes, and the collected supernatant was used as the test sample.

[0037] (2) Measurement of DPPH radical scavenging activity 30 μL of the test sample prepared above was mixed with 90 μL of freshly prepared DPPH reaction solution and incubated at room temperature for 20 minutes. The degree of DPPH fading was then measured at 520 nm. In a blank test, 50% (v / v) ethanol solution was used instead of the test sample. The DPPH reaction solution was prepared as follows.

[0038] Preparation of DPPH reaction solution 1. 400 μM DPPH / ethanol Weigh out 1 mg of DPPH and dissolve it in 6.5 mL of ethanol. 2. 0.2M MES (2-morpholinoethanesulfonic acid) (pH6.0) Dissolve 8.53 g of MES in water, adjust the pH to 6.0 with NaOH solution, and then make up to 200 mL. 3. Preparation of DPPH reaction solution A DPPH reaction solution was prepared by mixing equal amounts of the 400 μM DPPH / ethanol from 1. above, the 0.2 M MES from 2. above, and a 50% (v / v) aqueous ethanol solution.

[0039] (3) Results The measurement results are shown in Table 1. The DPPH radical scavenging activity was calculated using the following formula.

number

[0040] [Table 1] *Results are expressed as the mean value of triplicates ± standard deviation. There was a significant difference between the different codes (p<0.05).

[0041] Table 1 shows that the N7 strain has the highest antioxidant activity (DPPH radical scavenging activity) among live Lactococcus lactic acid bacteria. Furthermore, it was found that the N7 strain has more than twice the antioxidant activity of Lactococcus lactis subsp. cremoris H61 described in Non-Patent Document 1.

[0042] [Example 2] DPPH radical scavenging activity of N7 strain after heat treatment The N7 strain, which exhibited the highest antioxidant activity among the live bacteria in Example 1, was subjected to heat treatment to measure its DPPH radical scavenging activity as follows.

[0043] Specifically, the N7 strain was cultured overnight in MRS medium, and the resulting culture was centrifuged at 13,000 g for 10 minutes to harvest the cells. The harvested cells were washed three times with water and then suspended in water to obtain a cell suspension of 100 mg / mL (wet cell weight w / v). This cell suspension was heated at 95°C for 15 minutes, cooled, and then mixed with an equal volume of 100% ethanol. The resulting mixture was centrifuged at 13,000 g for 10 minutes, and the collected supernatant was used as the test sample. Unheated cells were treated in the same manner and used as the test sample.

[0044] The test samples prepared as described above were measured for DPPH radical scavenging activity in the same manner as in Example 1. The results are shown in Table 2.

[0045] [Table 2] *The results for DPPH radical scavenging activity (%) are expressed as the average value ± standard deviation of triplicates.

[0046] Table 2 shows that the antioxidant activity of the N7 strain does not decrease even after heat treatment. It has been reported that many Lactococcus lactic acid bacteria, such as the H61 strain described in Non-Patent Document 1, possess SOD. Because SOD is an enzyme, it was thought that the antioxidant activity of Lactococcus lactic acid bacteria would decrease after heat treatment. Under these circumstances, it was an unexpected discovery that the N7 strain, even when heat-killed, possesses the same high antioxidant activity as live cells.

[0047] [Example 3] Evaluation of antioxidant function of killed N7 strain bacteria using zebrafish In Examples 1 and 2, the antioxidant activity of the N7 strain was demonstrated in vitro. However, in vitro results often do not coincide with in vivo results. Therefore, in this Example, the antioxidant activity of the N7 strain at the in vivo level was measured using an antioxidant function evaluation system using zebrafish.

[0048] First, we will explain the antioxidant function evaluation system using zebrafish with reference to Figure 1. First, zebrafish parent fish were mated (night of day 1), and fertilized eggs were collected (morning of day 2). These fertilized eggs were hatched to prepare 3.5-day-old fertilized zebrafish fry (night of day 5). Next, eight of these 3.5-day-old fertilized fry were placed in each well of a 24-well plate, and a solution (0.5 mL) of the target lactic acid bacteria (food component) was added to initiate pretreatment (night of day 5). After 12 hours, the lactic acid bacteria solution was removed (end of pretreatment), and the plates were washed with water. An oxidant treatment was then added to initiate oxidant treatment (morning of day 6). The oxidant used was 2.8 mM hydrogen peroxide (0.5 mL). The oxidant treatment was continued for 48 hours, and the number of surviving fish was counted every 12 hours from the start of treatment.

[0049] The advantages of using zebrafish in this evaluation system are that it poses fewer ethical issues than mammals and can be used for toxicity testing and drug discovery screening. In addition, zebrafish hatch and begin feeding two and a half days after fertilization, allowing for simple, rapid, and large-scale analysis of food-related behavior at the small, easily manageable juvenile stage.

[0050] The test method was as described above (Fig. 1). The lactic acid bacteria solution used was prepared as follows. First, the N7 strain was cultured overnight in MRS medium, and the resulting culture was centrifuged at 1800g for 15 minutes to collect the bacteria. Next, the collected bacteria were washed twice with physiological saline and suspended in water to obtain a bacterial cell suspension. This bacterial cell suspension was heat-treated at 121°C for 15 minutes to obtain a suspension (stock solution) of heat-killed N7 strain bacteria. This was diluted with water to a predetermined concentration and used as the lactic acid bacteria solution. The concentration of the bacterial cell suspension measured with an absorbance meter is shown in Fig. 2. For the strain, the OD 620 =1.0 is 1.5 x 10 9 Equivalent to cfu / mL.

[0051] The results are shown in Figure 2. In Figure 2, the horizontal axis represents the treatment time of the oxidizing agent, hydrogen peroxide solution, and the vertical axis represents the survival rate (%) of zebrafish, with the survival rate at the start of treatment being set at 100%. The survival curves were analyzed using the Kaplan-Meier method, and significant differences between different conditions were tested using the log-rank method. Each analysis was performed on 8 fish x 3 wells, and multiple analyses were performed, so all measurements were performed using N = 24 or more zebrafish fry.

[0052] As a result, the survival rate of the control juveniles (dotted line) that had not been pretreated decreased to 22.5% 12 hours after the start of treatment, while the OD 620 =0.125 suspension and OD 620 The survival rate of larvae pretreated with the suspension of killed N7 strain bacteria was 100% even 48 hours after the start of treatment. -29 The results showed that lethality to hydrogen peroxide was significantly reduced compared to the control. Therefore, it was revealed that the N7 strain (killed bacteria) has antioxidant activity at the in vivo level, which can improve the reduced survival rate of zebrafish due to oxidant treatment.

[0053] [Example 4] Evaluation of antioxidant function of freeze-dried powder of killed N7 strain bacteria using zebrafish In this example, the antioxidant capacity of freeze-dried powder of killed cells of the N7 strain was measured at the living body level using an antioxidant function evaluation system using zebrafish.

[0054] The test was carried out as described in Example 3 (Fig. 1). The lactic acid bacteria solution used was prepared by powdering the suspension (stock solution) of heat-killed N7 strain cells prepared in Example 3 by freeze-drying to obtain freeze-dried powder of N7 strain, which was then dissolved and suspended in water at a predetermined concentration. The concentrations of the lactic acid bacteria solution are as shown in Fig. 3.

[0055] The results are shown in Figure 3 and Table 3. In Figure 3, the horizontal axis represents the treatment time with the oxidizing agent hydrogen peroxide solution, and the vertical axis represents the survival rate (%) of zebrafish, with the survival rate at the start of treatment set at 100%. The survival curve analysis method, significance test method, and number of analyses were the same as in Example 3 (Figure 2). Table 3 also shows the survival rate (%) of zebrafish in each test group by treatment time.

[0056] [Table 3] *The figures are the average survival rates (%) of zebrafish.

[0057] The results showed that larvae pretreated with 250 μg / mL (thick solid line) and 125 μg / mL (dotted-dash line) solutions of the N7 strain freeze-dried powder were significantly less susceptible to lethality from hydrogen peroxide compared to control larvae (dotted line). Even in the widely used form of freeze-dried killed bacterial powder, the N7 strain possesses antioxidant activity at the in vivo level, which can ameliorate the decline in zebrafish survival caused by oxidant treatment.

[0058] Although the embodiments and examples of the present invention have been described in detail above, the above-mentioned embodiments and examples are merely illustrative of the present invention, and the present invention is not limited to the configurations of the above-mentioned embodiments and examples. Design changes and the like that do not deviate from the gist of the present invention are also included in the present invention. [Industrial Applicability]

[0059] The antioxidant containing the lactic acid bacteria strain N7 according to the present invention as an active ingredient is expected to be used not only in foods and beverages, but also in the production of animal feed, pharmaceuticals, veterinary medicines, cosmetics, and quasi-drugs. The N7 strain is a lactic acid bacterium that has been used for many years in the production of dairy products, and is highly safe, allowing it to be safely consumed as a food on a daily basis over a long period of time.

Claims

1. An antioxidant containing Lactococcus lactis subsp. lactis biovar. diacetylactis N7 (FERM P-18217) as an active ingredient.

2. The antioxidant according to claim 1, wherein the N7 strain is in the form of live or killed cells, or a dried powder thereof.

3. A food composition, beverage, animal feed, pharmaceutical, veterinary drug, cosmetic or quasi-drug, comprising the antioxidant described in claim 1 or 2.

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