JP2004018469A - Antiallergic agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antiallergic agent selected from lactic bacteria of the genus Lactococcus which are the lactic bacteria suitable for the production of a milk product, having excellent survival in the intestines, and exhibiting the effects at an organism level. <P>SOLUTION: The antiallergic agent contains Lactococcus lactis subspecies lactis G50 strain (FERM P-18415) as an active ingredient. The fermented food contains the antiallergic agent. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an antiallergic agent, and more particularly to an antiallergic agent containing a specific Lactococcus lactic acid bacterium as an active ingredient.
[0002]
[Prior art]
In recent years, allergic patients are increasing due to changes in eating habits and lifestyle habits. Allergic diseases are classified from type I to type IV according to their mechanism of action. Among them, type I allergy includes atopic dermatitis, food allergies, etc., and people of all ages from children to adults are allergic. Suffering from symptoms.
Until now, methods for removing causative substances from food have been mainly used for the prevention and treatment of food allergies. However, removal of the causative substance involves not only the burden on the person but also the burden on the cooking side, and there are many problems.
[0003]
In type I allergic reactions, immunoglobulin E (IgE) antibody production is said to play an important role. Therefore, to suppress type I allergic reaction, suppression of IgE antibody production is important, and so far the immune system regulating action such as the suppression effect of IgE antibody production by Enterococcus lactic acid bacteria, Lactobacillus lactic acid bacteria, and Bifidobacterium It has been reported.
By the way, since lactic acid bacteria and bifidobacteria can be ingested not only as pharmaceuticals but also as foods, it is considered that there is little resistance to daily ingestion to prevent allergies.
[0004]
However, for Enterococcus lactic acid bacteria, not all of the strains belonging to this group are safe, and there are strains that have pathogenicity, so it is necessary to carefully select the strain to be used.
In addition, Lactobacillus lactic acid bacteria are known to be a highly safe group of lactic acid bacteria, but these strains generally have poor growth in milk. In order to grow the strain sufficiently, it must be mixed with milk-derived lactic acid bacteria or added with glucose, yeast extract and the like.
On the other hand, bifidobacteria are highly safe bacteria, but bifidobacteria are obligate anaerobic bacteria that cannot grow at all in the presence of oxygen. Therefore, equipment such as an anaerobic glove box is required for handling them. The
[0005]
On the other hand, Lactococcus lactic acid bacteria are a group of lactic acid bacteria that are not only highly safe, but also have good growth in milk and are suitable for dairy production. Moreover, since this bacterium is facultative anaerobic bacterium, handling is also easy. Therefore, a novel strain, particularly a strain having an inhibitory effect on IgE antibody production, has been demanded for the development of dairy products with high added value in Lactococcus lactic acid bacteria. However, so far, studies on immunomodulatory effects using Lactococcus lactic acid bacteria have only been reported at the test tube level. It is dangerous to estimate the effect in the living body from the results obtained at the test tube level, and there is a problem that the reliability is low because careful and sufficient consideration is required. Therefore, analysis at the biological level is indispensable for practical use.
[0006]
Lactococcus lactic acid bacteria have hardly been evaluated for their probiotic functions, including immunoregulatory action. “Probiotics” was originally defined as “a microbial additive that works beneficially for the host animal by improving the balance of the intestinal flora of the host.” Lactic acid bacteria have sufficient functions as probiotics. In order to demonstrate, it has been desirable to reach the intestine alive.
For this reason, probiotic research has been mainly conducted using Lactobacillus lactic acid bacteria and bifidobacteria derived from the intestines of animals. On the other hand, most of the Lactococcus lactic acid bacteria are derived from milk and have been considered to have low intestinal survival until now, so research as probiotics has not progressed.
[0007]
Recently, the term probiotics is often used in a broad sense as “microorganisms that have a beneficial effect on maintaining the health of the host”, and there is an idea that even dead cells can be included in probiotics. . However, reaching the digestive tract with viable bacteria is advantageous for exhibiting functions such as antibacterial action, and a combined effect with other physiological actions can be expected.
Moreover, if the period which exists in intestines as a living microbe is long, it is thought that it is more advantageous than the dead microbe which an effect is transient at the point of the lasting effect.
The above-mentioned Lactococcus lactic acid bacteria that have been reported to have an immunomodulatory effect at the test tube level, particularly the Lactococcus lactis subspecies Cremolis ATCC 19257 strain, the Lactococcus plantarum ATCC 43199 strain, the Lactococcus raffinolactis ATCC 43920 strain, are all intestinal Since the residual property is low, even if taken orally, it is expected that only an action as a dead cell, that is, a transient action can be expected.
[0008]
The properties of lactic acid bacteria are mostly strain-specific, including intestinal survival, and intestinal survival may differ even for lactic acid bacteria belonging to the same species. Therefore, there is a need for a lactic acid strain that belongs to the Lactococcus lactic acid bacterium that plays an important role in dairy production, has excellent intestinal survival, and has an antiallergic activity, particularly an IgE antibody production inhibitory effect at the biological level. It has been.
[0009]
[Problems to be solved by the invention]
An object of the present invention is to provide an antiallergic agent that is excellent in intestinal survival and can be seen at the living body level, among lactic acid bacteria belonging to the genus Lactococcus, which are lactic acid bacteria suitable for dairy production. is there.
[0010]
[Means for Solving the Problems]
The present inventors have made extensive studies to achieve the above object, among Lactococcus, Lactococcus lactis subsp. Lactis (Lactococcu s lactis subsp. Lactis) G50 strain, intestines remain viable The present invention was completed by finding that it has an action of suppressing IgE antibody production in mice by oral administration.
[0011]
The present invention according to claim 1 is an antiallergic agent comprising Lactococcus lactis subspecies lactis G50 strain (FERM P-18415) as an active ingredient.
The present invention according to claim 2 is the antiallergic agent according to claim 1, wherein the antiallergic agent is based on an IgE antibody production inhibitory action.
The present invention according to claim 3 is the antiallergic agent according to claim 1, wherein the Lactococcus lactis subspecies lactis G50 strain (FERM P-18415) is a living cell.
The present invention according to claim 4 is a fermented food comprising an antiallergic agent containing Lactococcus lactis subspecies lactis G50 strain (FERM P-18415) as an active ingredient.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
The Lactococcus lactis sub-species lactis G50 strain according to the present invention can be selected from Lactococcus lactic acid bacteria by the following method.
That is, the cytokine production amount of the macrophage strain when the test lactic acid bacteria and the macrophage strain which is one of the immunocompetent cells are co-cultured can be selected as an index. The macrophage strain used at this time includes the J774.1 strain (ATCC TIB-67) derived from BALB / c mice.
The selected Lactococcus lactis subspecies lactis G50 strain has been deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, and its deposit number is FERM P-18415.
[0013]
This strain has an effect of suppressing an increase in the amount of IgE antibody in serum when orally administered to a mouse immunized with egg white ovomucoid or whey β-lactoglobulin, which is a typical food allergen. Moreover, when it was orally administered to mice, it was found from the stool as viable bacteria, so that it was found that it reached the intestine as it was. From this, after ingestion, this strain can overcome specific environmental conditions in the digestive tract (low pH of gastric acid, presence of bile acids in the small intestine, etc.), reach the intestinal tract, and exert a sustained effect Probability is high. This strain is a microorganism having important characteristics as probiotics.
[0014]
This strain is cultured under arbitrary conditions according to a conventional method for culturing lactic acid bacteria, and collected from the culture by solid-liquid separation means such as centrifugation, once or twice, preferably twice, It can be washed with sterilized water or the like and used for the purposes of the present invention. There is no restriction | limiting in particular also about the form of formulation, What is necessary is just to determine suitably according to the intended purpose, such as freeze-drying powder, spray-drying powder, and suspension in a liquid, if desired. In addition, fermented foods can be produced using this bacterium as a starter or the like and used in a state containing the bacterium.
[0015]
Oral administration of this strain is desirable for suppression of IgE antibody production according to the present invention. In experiments with mice (body weight 20 g), since a suppressive effect was observed when administered at 0.171 to 0.22 mg / 0.2 ml / day in terms of cell weight, in the case of humans, 8 cell weight per kg body weight. A sufficient effect is produced by administration of about 55 to 11 mg / day, but a larger amount may be taken.
What is necessary is just to divide said microbial cell amount once a day or in several times. Since this strain grows well even in milk, it can be ingested in the form of a dairy product such as fermented milk produced using this strain as described above.
[0016]
【Example】
EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.
Example 1
BALB / c mice (average weight 20 g) were reared with commercial feed (MM-3, Funabashi Farm, Chiba Prefecture) and water for 12 days for habituation after delivery.
Lactococcus lactis subspecies Lactis G50 strain (FERM P-18415) or Lactobacillus acidophilus JCM1132 strain (RIKEN) was cultured overnight in advance according to a conventional method. Next, the lactic acid strain was washed twice with 0.85% saline and suspended in 10% nonfat milk (Snow Brand Skimmed Milk, Snow Brand Milk Products Co., Ltd.) so as to obtain 0.171 to 0.22 mg per 200 μl.
[0017]
200 μl of a 10% skim milk suspension of the lactic acid bacteria live cells were orally and continuously administered to the 6 BALB / c mice once a day for 19 days. In the control group, only 10% nonfat milk was orally administered in the same manner.
On day 5, 19 and 43, 50 μg of ovomucoid (OVM) and 2-3 mg / aluminum hydroxide suspended in physiological saline were intraperitoneally administered on the 5th, 19th and 43rd days. Blood was collected, and total IgE value and OVM-specific IgE value in serum were measured by enzyme immunoassay (ELISA). The total IgE value was measured using an OptEIA mouse IgE measurement kit (manufactured by Pharmingen). Relative values are shown with the control as 100%.
[0018]
The measurement result of the total IgE value is shown in FIG. 1, and the measurement result of the OVM-specific IgE value is shown in FIG. As is clear from FIG. 1, in the group administered with Lactococcus lactis subspecies lactis G50, the total IgE value was significantly lower than that in the control group. In the figure, ** indicates a significance level of less than 1% (p <0.01). In addition, the OVM-specific IgE value tended to decrease in the G50 strain administration group as compared to the control group (FIG. 2).
On the other hand, in the group administered with Lactobacillus acidophilus JCM1132, the total IgE value decreased compared to the control group, but was not significant (FIG. 1). Further, as shown in FIG. 2, the OVM-specific IgE value in the group administered with the JCM1132 strain did not tend to decrease as compared with the control group.
[0019]
Example 2
BALB which was cultured in the same manner as in Example 1 and was preliminarily raised in the same manner as in Example 1 was obtained by washing G50 strain (FERMP-18415) or Lactobacillus acidophilus JCM1132 strain (RIKEN) obtained by washing with saline twice. / C mice (average body weight 20 g) were orally administered for 15 days. During the administration period, β-lactoglobulin (BLG), a milk allergen, was orally administered (1 mg × 5 / animal) and immunized (BLG 50 μg + aluminum hydroxide 2-3 mg / animal), and antibody response to BLG (counting) , × 10 ⁻³ ). The results are shown in FIGS. 3 (A) and 3 (B). (A) shows the BLG non-administered group, and (B) shows the BLG administered group. In the figure, saline indicates physiological saline,-indicates an average value, and * indicates a significance level of less than 5% (p <0.05).
As is clear from FIGS. 3 (A) and 3 (B), the G50 strain administration group has a strong IgE antibody inhibitory effect compared to the JCM1132 strain administration group and the control group regardless of whether or not BLG is administered. Admitted.
Here, it should be noted that when BLG is orally administered in advance, the immune response to BLG is suppressed, that is, oral immune tolerance is induced. IgE antibody production is significantly reduced compared to the control (administered only saline) group. This suggests that the G50 strain has a function of efficiently inducing oral tolerance.
[0020]
Example 3
In this example, the intestinal survival of G50 strain (FERM P-18415) orally administered to mice was examined. First, a rifampicin resistant strain of G50 strain was prepared. The G50 strain was cultured all day and night according to a conventional method and applied to a GM17 agar medium containing rifampicin (Sigma, 100 μg / ml). After culturing at 30 ° C., rifampicin resistant strains were obtained by catching the grown colonies. This resistant strain was cultured all day and night, washed 4 times with 0.85% saline, and suspended in 10% nonfat milk to 10 ⁹ cells per 200 μl.
200 μl of a 10% skim milk suspension of rifampicin-resistant G50 strain live cells was forcibly orally administered to three BALB / c mice preliminarily raised in the same manner as in Example 1. After administration, stool was collected over time, suspended in sterilized 0.85% saline, a part of which was serially diluted and applied to a rifampicin-containing agar medium. This was cultured in an incubator at 30 ° C. for 1-2 days, and the number of grown colonies was measured.
No colonies growing on the rifampicin-containing medium were detected from stool before administration of lactic acid bacteria. After administration of the G50 strain, the number of viable bacteria of the genus Lactococcus detected from the stool is 7.30 ± 0.329 (logarithmic value) after 12 hours and 4.75 ± 0.426 after 24 hours, It became clear that the G50 strain can pass through the digestive tract of mice alive.
[0021]
Test example 1
The G50 strain (FERM P-18415), the Lactococcus lactis subspecies Cremolis ATCC 19257 strain, the Lactococcus raffinolactis ATCC 43920 strain, and the Lactococcus plantarum ATCC 43199 strain were cultured in advance overnight according to a conventional method.
Dehydrated bile (Oxygal, manufactured by Difco) was added to MRS liquid medium (Difco) at 0.3%, and the cells were inoculated. Thereafter, in order to adjust to the intestinal environment, culture was performed at 37 ° C., and the absorbance at 620 nm was measured after 24 hours. As a control, the cells were inoculated in an MRS liquid medium without added dehydrated bile and cultured in the same manner.
As a result, when cultured in a dehydrated bile-free medium, the G50 strain, ATCC 19257 strain, ATCC 43920 strain and ATCC 43199 strain showed turbidity of 1.69, 0.6, 0.98 and 0.62, respectively. When cultured in a dehydrated bile-added medium, only the G50 strain showed the same turbidity as the control, and the ATCC 19257 strain, the ATCC 43920 strain, and the ATCC 43199 strain did not grow.
[0022]
Test example 2
The G50 strain (FERM P-18415), the Lactococcus lactis subspecies Cremolis ATCC19257 strain, and the Lactococcus raffinolactis ATCC 43920 strain were cultured in advance overnight according to a conventional method. Next, these lactic acid bacterial strains were washed twice with 0.85% saline and suspended in a phosphate buffer having a pH of 7.0. Dehydrated bile was added to this bacterial solution so as to be 0.3%, and the mixture was kept at 37 ° C. for 3 hours.
Next, this bacterial solution was serially diluted, applied to M17 (Difco) agar medium, cultured for 1-2 days in a 30 ° C. incubator, and then the number of colonies grown on the agar was counted. As a result, the viable cell count after treatment with bile acid was 4.1 × 10 ^{6 for} the G50 strain, 5.8 × 10 ^{2 for} the ATCC 19257 strain, and 5.7 × 10 ² for the ATCC 43920 strain. From this, it was found that the G50 strain can reach the intestinal tract as viable bacteria without being destroyed by bile acids even under specific environmental conditions in the digestive tract.
[0023]
【The invention's effect】
According to the present invention, the Lactococcus lactic acid bacterium, Lactococcus lactis subspices Lactis G50 strain, which is highly safe and suitable for dairy production among lactic acid bacteria among the lactic acid bacteria, is contained as an active ingredient. An antiallergic agent is provided.
[0024]
Furthermore, the invention according to claim 4 provides a fermented food comprising an antiallergic agent containing Lactococcus lactis subspecies lactis G50 strain as an active ingredient. For example, the function can be utilized by producing a dairy product using this strain as a starter and ingesting a fermented food containing the strain.
[0025]
Since the G50 strain according to the present invention can reach the intestine alive, it can be expected to have an effect as a living bacterium, that is, an inhibition of harmful bacteria due to production of antibacterial substances and competition of nutrients, and an antiallergic effect due to an IgE antibody suppressing effect A comprehensive physiological effect can be expected.
In this study, egg white ovomucoid and whey β-lactoglobulin were used among food allergens, but the effect of oral administration of the G50 strain is expected on other foods that induce allergies such as wheat and peanuts. Is done. Moreover, the administration effect of G50 strain | stump | stock is anticipated not only in food allergy, but also in relief of the hay fever and atopic dermatitis which belong to a type I allergy.
[Brief description of the drawings]
FIG. 1 shows the effect of oral administration of G50 strain and JCM1132 strain on mice immunized with ovomucoid on IgE antibody production in serum.
FIG. 2 shows the effects of oral administration of G50 strain and JCM1132 strain on ovomucoid-specific IgE antibody production.
FIG. 3 shows the effects of oral administration of G50 strain and JCM1132 strain on β-lactoglobulin (BLG) -specific IgE antibody production. (A) shows BLG non-administered group, (B) shows BLG The administration group is shown.

Claims (4)

An antiallergic agent characterized by containing Lactococcus lactis subspecies Lactis G50 strain (FERM P-18415) as an active ingredient. The antiallergic agent according to claim 1, wherein the antiallergic agent is based on an inhibitory effect on IgE antibody production. The antiallergic agent according to claim 1, wherein Lactococcus lactis subspecies Lactis G50 strain (FERM P-18415) is a viable bacterium. A fermented food comprising an antiallergic agent containing Lactococcus lactis subspecies Lactis G50 strain (FERM P-18415) as an active ingredient.

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