JP2007131550A - Immunity function regulator - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicine effective as a preventing and treating agent of various immunity disorders and a beverage, food and feed blended with the same by finding out a new action mechanism of milk fat globule membrane and/or ganglioside GM3, associated with the immunity function, and medicinal uses by utilizing them. <P>SOLUTION: This immunity function regulator contains the milk fat globule membrane and/or ganglioside GM3 as an active ingredient. The ganglioside GM3 is preferably derived from the milk. The immunity function regulator can be blended with the beverage, food or feed. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an immune function regulator comprising milk fat globule membrane and / or ganglioside GM3 as an active ingredient, and food and drink and feed containing the same.

  Milk fat globule membrane is contained in a large amount in butter milk produced in the butter production process, and is rarely used although there are a few examples of its use as a food material. However, the fat globule membrane contains many proteins and lipids, and some of them, such as mucins, have been suggested to be involved in biological defense. The use of the spherical film is expected in the future.

  Patent Document 1 discloses an antiallergic nutritional composition containing an enzyme-treated milk-derived fat globule membrane component as an active ingredient. In the same document, milk-derived fat globule membrane components are used as emulsifiers. That is, it is used as an emulsifier for other components in the antiallergic nutritional composition by enzymatically treating the fat globule membrane component to improve the emulsifying ability.

On the other hand, ganglioside GM3 is a glycosphingolipid containing N-acetylneuraminic acid and exists on every cell membrane. As its physiological action, neutralizing activity of cytotoxin, receptor activity of interferon, hormone and the like are known. Ganglioside is a component contained in milk, and ganglioside contained in milk is mainly GM3 and GD3. GM3 induces differentiation of myeloid leukemia cell line HL-60 and monocytic leukemia cell line U937. It is known to have an action (Non-Patent Document 1). Patent Document 2 discloses that GM3 matures the intestinal mucosal cells of infants. By maturation, allergens are prevented from passing through the gaps of mucosal epithelial cells, or on mucous membranes. It is disclosed that IgA production that binds to allergens and prevents their entry can be increased. However, unlike infants in which the allergens easily enter the body because the gastrointestinal tract is immature, no effective means has been found for those who have a sufficiently mature gastrointestinal tract.
JP-A-8-214836 JP-A-8-109133 Nojiri et al. Biol. Chem. , 263, 7443, (1988)

Cytokines are proteinaceous factors produced mainly from cells of the immune system, and are known to exert various physiological effects not only on immune system cells but also on many cells. Among cytokines, TNF-α is a representative of inflammatory cytokines that play a central role in biological defense, inflammatory response, and the like. Factors that affect the ability to produce TNF-α are expected to have a biological defense ability and an action that regulates an inflammatory response.
IL-12 is a representative cytokine that regulates cellular immunity, and is known to promote the differentiation of Th0 type T cells into Th1 type. IL-12 has homo and heterodimers composed of two subunits, p40 and p35. The heterodimer consisting of p40 and p35 acts as active IL-12 as p70. In addition, p80, a p40 homodimer, is known to act as an inhibitor that inhibits the activity of p70. However, since p40 is constitutively produced, the regulation of IL-12 activity is regulated by p35. It is thought to be done by changing the production.
IL-4 is a representative cytokine that controls humoral immunity, and is considered to promote the proliferation of Th2 cells.
Therefore, by analyzing changes in the production ability of IL-12 and IL-4 in the tissues of the immune system, the existing T cells are predominantly Th1-type, that is, whether cellular immunity is dominant, or Th2-type dominant That is, it is possible to infer whether humoral immunity is in a dominant situation.

  The present invention has found a novel mechanism of action relating to the immune function of milk fat globule membrane and / or ganglioside GM3 and a pharmaceutical use using the same, and a pharmaceutical effective as a preventive or therapeutic agent for various immune diseases and a combination thereof It is an object of the present invention to provide a food and drink and feed.

  In order to solve the problems, the present inventors have studied various functions of milk fat globule membrane and / or ganglioside GM3 against cytokines. These substances reduce the production amount of TNF-α, and activate IL- The present inventors have found that the production of 12 can be promoted or suppressed, and that the immune function can be regulated using these substances, and the present invention has been completed based on this finding.

That is, the present invention is an immune function regulator comprising milk fat globule membrane and / or ganglioside GM3 as an active ingredient.
The present invention also provides the above-mentioned immune function regulator, characterized in that ganglioside GM3 is derived from milk.
The present invention also provides foods and drinks and feeds containing the immune function regulator.

  By using the immune function regulator of the present invention, the production of TNF-α is reduced using milk fat globule membrane and / or ganglioside GM3, and the production of active IL-12 is promoted or suppressed, thereby Function can be adjusted. The immune function regulator of the present invention can be effectively used for prevention and treatment of diseases associated with immune functions such as food allergies and atopic dermatitis. In particular, when a milk fat globule membrane is used, it is effective for treatment of a disease related to immune function, and when ganglioside GM3 is used, it is useful for prevention of the disease.

Hereinafter, the present invention will be described in detail.
The milk fat globule membrane used as an active ingredient in the present invention is a membrane that coats milk fat globules, and is mainly composed of proteins, phospholipids and glycolipids. The milk fat globule membrane plays a role of maintaining an emulsified state between the highly hydrophobic fat globule and the surrounding aqueous phase by a surface active action.
The milk fat globule membrane used in the present invention may be obtained by any method. Examples of the raw milk include cow milk, goat milk, sheep milk and human milk, with milk being particularly preferred.
In the present invention, as the milk fat globule membrane, butter serum as a by-product when producing butter oil from butter or milk cream, or butter milk as described in JP-A-5-292880, pH 4. Using a complex lipid-containing powder obtained by adjusting to 4-4.6, performing isoelectric precipitation to remove protein precipitation, ultrafiltration or microfiltration of the resulting supernatant, and drying Also good.

  Ganglioside GM3 used as an active ingredient in the present invention may be obtained by any method. That is, a product that has been separated and purified from a beast's brain or the like by a conventional method may be used, or a product obtained by utilizing a method for efficiently recovering from milk (Japanese Patent Laid-Open No. 63-269992). It may be used. According to the method described in the publication, it is possible to produce in large quantities on an industrial scale. For reference, the method for separating and extracting gangliosides from milk described in the publication is as follows. That is, by reacting milk raw materials such as milk, buttermilk, whey and skim milk with acids such as hydrochloric acid and lactic acid or proteolytic enzymes such as trypsin and pepsin, the protein is decomposed, and the resulting proteolytic solution is obtained. Prepare by concentrating gangliosides by ultrafiltration, gel filtration or dialysis. The ganglioside thus prepared is composed of GM3 and GD3. In the present invention, GM3 is an active ingredient.

The immune function regulator of the present invention comprises milk fat globule membrane and / or ganglioside GM3 as an active ingredient, but can be used as a preparation having various dosage forms, and the dosage form is not particularly limited. Therefore, the milk fat globule membrane and / or ganglioside GM3, which are active ingredients, can be used in various dosage forms such as tablets, capsules, powders, liquids, etc. using various excipients and carriers. . Moreover, the immune function regulator of this invention may contain the other pharmaceutical which has the effect | action which regulates an immune function.
In addition, the type of food and drink of the present invention is not particularly limited, and food and drink such as milk, processed milk, milk drink, yogurt, soft drink, coffee drink, juice, jelly, wafer, biscuit, bread, noodle, sausage and the like It can mix | blend with a nutritional supplement and the composition for a nutritional supplement. Moreover, the kind of feed of this invention is not specifically limited.
In addition, the immune function regulator of this invention, food-drinks, and feed can be manufactured by a conventional method except containing a milk fat globule membrane and / or ganglioside GM3.

In the present invention, in order to exert an immune function-modulating effect, when a milk fat globule membrane is used as an active ingredient, pharmaceuticals, foods and drinks are generally taken so that about 10 to 500 mg per day can be ingested as the milk fat globule membrane. It is sufficient to adjust the amount added to the feed or feed. Moreover, when using ganglioside GM3 as an active ingredient, what is necessary is just to adjust the compounding quantity etc. to a pharmaceutical, food-drinks, and a feed so that about 10-500 mg can generally be ingested as ganglioside GM3 per day.
In addition, in the case where the milk fat globule membrane is used as an active ingredient or in the case where ganglioside GM3 is used as the active ingredient, the administration target is not limited. However, when ganglioside GM3 is used as the active ingredient, it is used for a person with a sufficiently digestive tract. And more effective.

(Test example)
Hereinafter, the present invention will be described in more detail with reference to test examples. Unless otherwise specified, “%” in the test examples means “% by weight”.
In all the test examples, Balb / C male mice were used.
The administered milk fat globule membrane (hereinafter referred to as “MFGM”) was dissolved in water at a concentration of 1 mg / ml or 2 mg / ml and subjected to micelle formation by sonication for 30 minutes. As a control, whey protein isolate (WPI) dissolved at the same concentration was used.
Ganglioside GM3 was dissolved in RPMI 1640 medium supplemented with 10% fetal bovine serum at a concentration of 10 μg / ml and sonicated for 30 minutes to form micelles.

(Test Example 1: Effect of MFGM on ability to produce TNF-α by intraperitoneal and splenic macrophages)
6 weeks old Balb / C male mice were preliminarily raised for 1 week, then divided into 2 groups so that their body weights were equal, and 1 ml of 1 mg / ml solution per mouse was orally administered to 1 group for 4 consecutive days did. In another group, the same amount of WPI was orally administered as a control. The mice were sacrificed 4 hours after oral administration on the 4th day, and intraperitoneal macrophages were separated and the spleen was collected. Intraperitoneal macrophages were separated by washing the mouse abdominal cavity with RPMI 1640 medium, and spleen cells were separated by spleen spleen in a culture dish, finely crushed with a syringe, and passed through a mesh. In both cases, adherent macrophages were isolated by culturing in a medium (10% FCS / RPMI1640) supplemented with fetal bovine serum to 10% in RPMI1640 medium for 2 hours and then washed twice in the same medium.

The isolated macrophages were further cultured for 3 hours in 10% FCS / RPMI1640 medium supplemented with LPS to 1 μg / ml, or the same medium without LPS added. After completion of the culture, the medium was collected and the cells were solubilized with 0.1% SDS solution. The TNF-α concentration in the collected medium was measured by a bioassay method using L929 mouse fibroblasts. The amount of DNA in the solubilized cells was measured by DAPI fluorometry, and TNF-α secretion per amount of DNA was determined. The results of intraperitoneal macrophages are shown in FIG. 1A, and the results of spleen macrophages are shown in FIG. 1B.
From the results shown in FIGS. 1A and 1B, in the MFGM administration group, the ability to produce TNF-α is decreased in both the absence and presence of LPS compared to the WPI administration group as the control group. The tendency to do was seen. The effect of reducing TNF-α production ability was more prominent in splenic macrophages (B) than in intraperitoneal macrophages (A).
The ability to produce TNF-α by oral administration of MFGM suggests that MFGM acts anti-inflammatory when taken orally.
In this test example, MFGM was orally administered in a micellar state, but it was absorbed from the small intestine as chylomicron via lymphatic vessels, and after absorption, it first reached the mesenteric lymph node and then transported by the liver, spleen and bloodstream It is expected to be done. It is presumed that the effect on spleen macrophages was more remarkable than that on intraperitoneal macrophages because the amount of MFGM reached in the spleen was larger than that in the abdominal cavity.
Although TNF-α production ability in mesenteric lymph nodes was also measured, TNF-α secretion at a level that could be measured by either LPS stimulation or mitogen stimulation was not observed. From this, it is presumed that in the mesenteric lymph node, T cells and B cells are main, and there are not many macrophages and monocyte cells.

(Test Example 2: Influence of MFGM on IL-4 and IL-12 production ability by spleen cells and mesenteric lymph node cells)
Animal breeding and sample oral administration were carried out in the same manner as in Test Example 1. However, in this experiment, the sample concentration was 2 mg / ml, and 0.5 ml per mouse was orally administered. The mice were sacrificed 4 hours after oral administration on the fourth day, and spleen and mesenteric lymph nodes were collected. The collected spleen and mesenteric lymph nodes were shredded in the same manner as in Test Example 1, and then strongly crushed into pieces, and each cell was separated by passing through a mesh. Cells were seeded in 10% FCS / RPMI 1640 medium. In this test example, since the whole spleen cells were cultured, the washing operation after 2 hours was not performed. On the second day from the start of culture, Contanavallin A, which is a mitogen, was added to a concentration of 5 μg / ml and further cultured for 4 days. After completion of the culture, the medium was collected and the cells were lysed. The concentrations of IL-4, IL-12p40, and IL-12p70 in the medium were each measured by sandwich ELISA. The DNA concentration in the cells was measured, and the amount of cytokine secretion per amount of DNA was determined. The results for spleen cells are shown in FIG. 2, and the results for mesenteric lymph node cells are shown in FIG.

As shown in FIG. 2, in spleen cells, there was no difference in the IL-4 production ability, but the production ability of the active form IL-12p70 was significantly increased compared to the control group. In addition, IL-12p40 secretion ability was significantly reduced.
Furthermore, as shown in FIG. 3, in the mesenteric lymph node cells, the IL-4 production ability was significantly reduced, and the IL-12p70 production ability was significantly increased.
Thus, the fact that MFGM increased the ability to produce IL-12p70 indicates that MFGM has an action of suppressing allergy, and it is clear that MFGM is effective in the treatment of immune diseases such as allergies. is there.
In addition, although the production ability of IL-12p40 has fallen, this fall is not statistically significant. As described above, IL-12p40 is constitutively secreted and secreted, and it is known that the amount of active IL-12p70 changes due to changes in IL-12p35 production ability. In fact, the amount of IL-12p70 in mesenteric lymph node cells is only a few percent of the amount of IL-12p40, and even if the amount of IL-12p40 changes slightly, it does not affect the ability to produce IL-12p70. . That is, it is considered that the physiological significance of IL-12p40 production ability change is not great in the spleen and mesenteric lymph nodes.
As described above, these results shown in FIG. 2 and FIG. 3 suggest that an orally administered MFGM induces a Th1-type dominant state in the spleen and mesenteric lymph nodes.

The fact that the Th1-inducing action in Test Example 2 was more prominent in the mesenteric lymph nodes than in the spleen is a result consistent with the prediction of the absorption process of MFGM in Test Example 1.
Looking at the Th1 / Th2 balance in the body, it is said that Th2 predominates in allergic diseases such as food allergies and atopic dermatitis. Therefore, if induced to a state predominantly Th1, the symptoms can be suppressed, that is, it is considered effective for the treatment of allergic diseases.
In this test example, the fact that MFGM showed a Th1 type-inducing action by oral administration is that daily ingestion of MFGM induces the immune system of the living body to be Th1-type, that is, cellular immunity is dominant. The possibility is strongly suggested, and the possibility that MFGM can be used as a food material having an antiallergic action is shown.

(Test Example 3: Effect of ganglioside GM3 on TNF-α production ability of intraperitoneal macrophages)
Intraperitoneal macrophages were isolated by washing the peritoneal cavity of 10-week-old Balb / C male mice with RPMI1640 medium. Subsequently, after culturing for 2 hours in a medium (10% FCS / RPMI1640) supplemented with fetal bovine serum to 10% in RPMI1640 medium, the adhered macrophages were isolated by washing twice with the same medium.
To the isolated intraperitoneal macrophages, 10% FCS / RPMI1640 medium supplemented with ganglioside GM3 at 10 μg / ml or RPMI1640 medium (control) to which nothing was added was added and cultured for 2 hours. Thereafter, LPS was added to 1 μg / ml, and further cultured for 3 hours. After completion of the culture, the medium was collected and the cells were solubilized with 0.1% SDS solution. The TNF-α concentration in the collected medium was measured by a bioassay method using L929 mouse fibroblasts. The amount of DNA in the solubilized cells was measured by DAPI fluorometry, and TNF-α secretion per amount of DNA was determined. FIG. 4 shows the TNF-α secretion activity of the control group as 100% and the activity of the ganglioside GM3 addition group.
From the results shown in FIG. 4, the ganglioside GM3 addition group had a much lower TNF-α production ability than the control group.
Suppressing the ability to produce TNF-α by adding ganglioside GM3 directly to the medium suggests that ganglioside acts anti-inflammatory.

(Test Example 4: Effect of ganglioside GM3 on IL-4 and IL-12 production ability by mesenteric lymph node cells)
Separation of cells was performed in the same manner as in Test Example 2. That is, the collected mesenteric lymph nodes were shredded in the same manner as in Test Example 2, and then strongly crushed into pieces, and then the cells were separated by passing through a mesh. The cells were seeded in 10% FCS / RPMI1640 medium or 10% FCS / RPMI1640 medium supplemented with GM3 at 10 μg / ml. On the second day from the start of culture, Contanavallin A, which is a mitogen, was added to a concentration of 5 μg / ml and further cultured for 3 days. After completion of the culture, the medium was collected and the cells were lysed. The concentrations of IL-4, IL-12p40, and IL-12p70 in the medium were each measured by sandwich ELISA. The DNA concentration in the cells was measured, and the amount of cytokine secretion per amount of DNA was determined. The results are shown in FIG.

As shown in FIG. 5, IL-4 production ability tended to increase in mesenteric lymph node cells, and IL-12p70 production ability significantly decreased.
In addition, although the production ability of IL-12p40 is also increasing, this fall is not statistically significant. As described above, IL-12p40 is constitutively secreted and secreted, and it is known that the amount of active IL-12p70 changes due to changes in IL-12p35 production ability. In fact, the amount of IL-12p70 in mesenteric lymph node cells is only a few percent of the amount of IL-12p40, and even if the amount of IL-12p40 changes slightly, it does not affect the ability to produce IL-12p70. . That is, it is considered that the physiological significance of IL-12p40 production ability change is not great.
These results suggest that Th1 suppression is induced in the mesenteric lymph nodes by the addition of ganglioside GM3.

Looking at the Th1 / Th2 balance in the body, it is said that Th1 predominates at the initial stage of allergic diseases such as food allergies and atopic dermatitis. Therefore, it is considered that the suppression of Th1 can suppress the progression of allergic disease, that is, it is effective in preventing allergic disease.
In this test example, ganglioside GM3 showed a Th1 type inhibitory action because the daily ingestion of ganglioside GM3 may induce Th1 type, that is, cellular immunity, in the suppression direction. This suggests that ganglioside GM3 can be used as a food material having an allergy-preventing action by suppressing the initial stage of allergy development.

  Hereinafter, the present invention will be described in more detail with reference examples and examples. In Examples, “%” means “% by weight” unless otherwise specified.

(Example 1: Preparation of milk fat globule membrane)
Dissolve 10 kg of butter at 75 ° C., add 2 kg of warm water at the same temperature, stir well, and centrifuge in a continuous centrifuge (5,000 × g) to fractionate into butter oil and buttersarum. . 2 kg of the fraction collected as buttersalam was freeze-dried to obtain 140 g of milk fat globule membrane. The milk fat globule membrane thus obtained can be used as it is as an immune function regulator of the present invention.

(Example 2: Preparation of milk powder using milk fat globule membrane)
200 g of the milk fat globule membrane prepared in Example 1 was dissolved in 700 kg of water together with 6.8 kg of casein, 70.0 kg of whey powder, and 1 kg of vitamin and mineral components. Furthermore, 23.9 kg of vegetable fats and oils were mixed and homogenized, and then sterilized, concentrated, and dried, and 100 kg of milk powder containing the immune function regulator of the present invention was obtained. The milk fat globule membrane content in 100 g of the obtained milk powder was 200 mg.

(Example 3: Preparation of drink using milk fat globule membrane)
Using the milk fat globule membrane prepared in Example 1, a drink which is an immune function regulator of the present invention was prepared by the following formulation.
20g milk fat globule membrane
Sodium citrate 2.8g
Sucrose 360g
40g whole milk powder
Nonfat dry milk 50g
130g roasted coffee powder
2g of salt
Baking soda 2.5g
Sugar ester 2.5g
Fragrance 6g
The above components were dissolved in water to 5 L. This solution was homogenized at a pressure of 160 kg / cm ² , sterilized by holding at 120 ° C. for 3 seconds with a plate sterilizer, and then cooled to 5 ° C. The obtained drink was filled into a 200 ml paper container. This drink contained 400 mg of milk fat globule membrane per 100 ml.

(Example 4: Preparation of tablet using milk fat globule membrane)
The milk fat globule membrane prepared in Example 1 was filled in a soft capsule made of gelatin so as to be 100 mg per tablet by a conventional method to obtain an immune function regulator (tablet) of the present invention.

(Example 5: Preparation of ganglioside GM3)
0.5 kg of Bacillus subtilis protease is added to a solution obtained by dissolving 20 kg of buttermilk powder in 180 L of water, reacted at pH 7.6, temperature of 40 ° C. for 15 hours to decompose the protein, and heated at 90 ° C. for 10 minutes. Sterilized and inactivated enzyme. Next, this solution was subjected to a temperature of 40 ° C., a flow rate of 15 L / min, and an average pressure of 0 using an ultrafiltration membrane device (LAB-20 type module: manufactured by DDS) equipped with a membrane having a membrane area of 0.36 m ^2. Filtration at 6 MPa and concentration of the ganglioside. Concentrated hydrochloric acid was added to 10 L of this ganglioside concentrate to adjust the pH to 2.5, and the mixture was heated at 37 ° C. for 8 hours, and then neutralized with sodium hydroxide. This solution was freeze-dried to obtain 1 kg of ganglioside powder containing 6 g of GM3. The ganglioside powder thus obtained can be used as it is as an immune function regulator of the present invention.

(Example 6: Preparation of milk powder using ganglioside GM3)
200 g of ganglioside powder prepared in Example 5 was dissolved in 700 kg of water together with 6.8 kg of casein, 70.0 kg of whey powder and 1 kg of vitamin and mineral components. Furthermore, 23.9 kg of vegetable fats and oils were mixed and homogenized, and then sterilized, concentrated, and dried, and 100 kg of milk powder containing the immune function regulator of the present invention was obtained. The GM3 content in 100 g of the obtained milk powder was 1 mg.

(Example 7: Preparation of drink using ganglioside GM3)
Using the ganglioside powder prepared in Example 5, a drink which is an immune function regulator of the present invention was prepared by the following formulation.
Ganglioside powder 20g
Sodium citrate 2.8g
Sucrose 360g
40g whole milk powder
Nonfat dry milk 50g
130g roasted coffee powder
2g of salt
Baking soda 2.5g
Sugar ester 2.5g
Fragrance 6g
The above components were dissolved in water to 5 L. This solution was homogenized at a pressure of 160 kg / cm ² , sterilized by holding at 120 ° C. for 3 seconds with a plate sterilizer, and then cooled to 5 ° C. The obtained drink was filled into a 200 ml paper container. This drink contained 2 mg GM3 per 100 ml.

(Example 8: Preparation of tablets using ganglioside GM3)
The ganglioside powder prepared in Reference Example 2 was filled in a soft capsule made of gelatin so as to be 100 mg per tablet by a conventional method to obtain an immune function regulator (tablet) of the present invention.

  The immune function regulator comprising the milk fat globule membrane and / or ganglioside GM3 of the present invention or a food or drink or feed containing the same is useful for prevention or treatment of various diseases related to immune function, improvement of symptoms, etc. It can be used and is very useful. In particular, an immune function regulator comprising a milk fat globule membrane as an active ingredient is effective for treating various immune diseases. Moreover, when ganglioside GM3 is used as an active ingredient, it is useful for preventing various immune diseases.

6 is a graph showing the results of Test Example 1. 10 is a graph showing the results of spleen cells of Test Example 2. 10 is a graph showing the results of mesenteric lymph node cells of Test Example 2. 10 is a graph showing the results of Test Example 3. 10 is a graph showing the results of Test Example 4.

Claims (4)

An immune function regulator comprising milk fat globule membrane and / or ganglioside GM3 as an active ingredient. 3. The immune function regulator according to claim 1 or 2, wherein ganglioside GM3 is derived from milk. A food or drink containing the immune function regulator according to claim 1 or 2. The feed which mix | blended the immune function regulator of Claim 1 or 2.

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