JP5177728B2 - NK cell activator - Google Patents

Description

  The present invention relates to a composition having a natural killer (hereinafter also referred to as “NK”) cell activation effect.

As the name suggests, NK cells have the ability to quickly detect and kill virus-infected cells and cancer cells, and are attracting attention as a deciding factor in preventing influenza and other infectious diseases. However, the activity (destructive capacity) of NK cells decreases with aging, and the higher the incidence of cancer with increasing age, it is said to be related to the decline in activation of NK cells. In addition, since NK cells are affected by autonomic nerves, when sympathetic nerves become dominant under stress, NK cell activity (destructive ability) decreases drastically. Thus, in order to maintain health, activation of NK cells is indispensable. And although the substance which promotes NK cell activation is searched, most are utilized as a chemical | medical agent. As materials that can be used as food, polysaccharides (lentinan, chitin, chitosan, maitake D-fraction, nigerooligosaccharide, etc.) and bacterial cells (Lactobacillus casei shirota, Bifidobacterium lactis HN019, etc.) are known (Non-Patent Documents 1 to 4). 6, Patent Document 1) cannot always obtain sufficient activity. However, if it is derived from a material used as a food, it is expected to be safely and easily ingested in daily life and enhance immunity.
Yukio Akiyama, Jun Hamuro, Protein Nucleic Acid Enzyme, 26 (3), 208-224 (1981) Nishimura K, Nishimura S, Nishi N, Numata F, Tone Y, Tokura S, Azuma I, Vaccine, 3 (5), 379-84 (1985) Kodama N, Komuta K, Sakai N, Nanba H, Biol Pharm Bull, 25 (12), 1647-50 (2002) Murosak S, Muroyama K, Yamamoto Y, Liu T, Yoshikai Y, Int Immunopharmacol, 2 (1), 151-9 (2002) Hori T, Kiyoshima J, Yasui H, Biosci Biotechnol Biochem, 67 (2), 420-2 (2003) Gill HS, Rutherfurd KJ, Cross ML, Gopal PK., Am J Clin Nutr, 74 (6), 833-9 (2001) JP 2001-31573 A

  Therefore, a food-derived composition that activates NK cells and enhances immunity is desired.

When the present inventors examined the composition derived from the foodstuff which activates NK cell, it discovered that acidic polysaccharide had NK cell activation effect | action, and completed this invention.
The present invention specifically includes:
[1] An NK cell activator comprising an acidic polysaccharide as an active ingredient,
[2] The NK cell activator according to [1] above, wherein the acidic polysaccharide is a phosphorylated polysaccharide,
[3] The NK cell activator according to [1] or [2] above, wherein the polysaccharide is derived from lactic acid bacteria,
[4] The NK cell activator according to any one of [1] to [3] above, which has an NK cell activation action, and is used for NK cell activation Fermented milk with an indication that
Is to provide.

  An NK cell activator comprising an acidic polysaccharide has made it possible to prevent infection such as influenza, prevent cancer, and prevent progression. One of the therapeutic or prophylactic drugs for these diseases without a fundamental treatment can be newly provided.

The NK cell activator of the present invention contains an acidic polysaccharide, preferably a phosphorylated polysaccharide as an active ingredient. Examples of phosphorylated polysaccharides include cultures of lactic acid bacteria that produce polysaccharides, polysaccharides purified from the cultures, polysaccharides obtained by phosphorylating polysaccharides chemically or enzymatically, and the like. The phosphorus content in the phosphorylated polysaccharide is preferably 0.01% or more, and more preferably 0.1% or more. In addition, the polysaccharide referred to in the present invention includes an oligosaccharide obtained by chemically or enzymatically degrading the polysaccharide.
The lactic acid bacteria used in the present invention are not limited as long as they are lactic acid bacteria producing polysaccharides. Acidic polysaccharides may be used alone or in combination of two or more different types.
As lactic acid bacteria, Lactobacillus bulgaricus, Lactococcus lactis ssp. Cremoris, etc. are used alone or in combination. Among these, Lactococcus lactis ssp. Cremoris KVS20 strain (Kitazawa H, Yamaguchi T, Miura M, Saito T, Itoh T, J Dairy Sci, 76, 1514-9 (1993)), Lactobacillus bulgaricus OLL1073R-1 strain (FERM P-17227 ) Is preferred.

The culture may be used as it is, but the neutral polysaccharide may be removed by the method described in JP-A-2000-247895, or a product purified as described below may be used if necessary. I do not care. Note that some of the following steps may be omitted or added.
1. Removal of bacterial cells from the culture by centrifugation 2. Recovery of high molecular weight polysaccharides and proteins as precipitates by ethanol precipitation 3. Removal of proteins
a) Protein precipitation with about 5-10% final trichloroacetic acid, centrifugation
b) Heat denaturation and centrifugation of the protein by boiling
c) After b), protein degradation by proteinase or nucleic acid degradation by DNAase is also performed, dialysis 4, recovery by adsorption and elution of acidic polysaccharides by anion exchange resin, and molecular weight of 1 million or more by gel filtration. Acidic polysaccharides are purified as a substance.
At this time, if proteins with acidic and close molecular weight are mixed, it is possible to add some protein removal operation before or after gel filtration.

  When an acidic polysaccharide is formulated into a pharmaceutical product, the dosage form can be selected according to the therapeutic purpose, administration route, etc., for example, tablets, pills, powders, solutions, suspensions, emulsions, granules Agents, capsules, injections, suppositories, syrups, soaking agents, decoction, tinctures and the like. For formulation, diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants, lubricants and the like can be used as necessary. In addition, a colorant, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other pharmaceuticals may be included in the pharmaceutical preparation.

Examples of foods containing acidic polysaccharides include fermented milk and beverages such as yogurt, and can be used as health foods, foods for specified health use, dietary supplements, foods displaying NK cell activation, etc. it can. Acidic polysaccharides and food hygiene acceptable formulations, for example, stabilizers, preservatives, coloring agents, fragrances, vitamins and the like are added to the phosphorylated polysaccharides as appropriate, mixed, and tableted in a conventional manner. Granules, granules, powders, capsules, liquids, jellies, creams, beverages and other foods can be used.
Since NK cell activation is achieved by ingesting NK cell activation supplements in this form, the food obtained in the present invention can be used to prevent infection such as influenza, cancer, and progression. It is possible to contribute.

  Hereinafter, the present invention will be described, but it goes without saying that the present invention is not limited to these examples.

Production of polysaccharides derived from lactic acid bacteria 1, cultivation of lactic acid bacteria
10% whey was treated with protease at 55 ° C for 8 hours, 0.5% yeast extract was added, and the pH adjusted to 7.0 was inoculated with Lactobacillus bulgaricus OLL1073R-1 strain (FERM P-17227) at 37 ° C for 24 hours Was statically cultured.
2. Purification of polysaccharides
Bacterial cells were centrifuged (22800 × g, 4 ° C., 20 min) from 10-20 L of the culture solution, and then ethanol precipitation (60-70% (v / v)) was performed twice. The precipitate was dissolved in MilliQ water, and protein denaturation with trichloroacetic acid (final 10%) and insoluble matter were removed by centrifugation (22800 xg, 4 ° C, 20 min).
After dialysis against MilliQ water and freeze-drying, dissolve in 0.02M Tris-HCl (pH 8.6) and separate neutral and acidic polysaccharides using DEAE-Sepharose (Amersham Biosciences), an anion exchange resin (FIG. 1). Neutral polysaccharides were collected as the Pass fraction, and acidic polysaccharides were collected by elution with 0-0.5M ionic strength gradient using NaCl.
The collected acidic polysaccharide was subjected to gel filtration using Sephacryl S-400HR (Amersham Biosciences) and separated by molecular weight. Honda sugar molecular weight 1 × 10 ⁶ Da or more fractions and the molecular weight are separated into smaller fractions were collected for partial molecular weight 1 × 10 ⁶ Da or more fractions (Figure 2).
The collected polysaccharide was dialyzed against MilliQ water and freeze-dried to obtain a purified acidic polysaccharide product.
The polysaccharide was detected and quantified using the phenol / sulfuric acid method with glucose as a standard substance.
Further, when the polysaccharide was analyzed by the method described in the literature (Uemura J, Milchwissenschaft, 53 (8), 443-6 (1998)), it was confirmed that it contained a phosphorylated polysaccharide.

NK activity of polysaccharide 1 and oral administration test of polysaccharide to mice 30 mice BALB / c (male, 8 weeks old, SLC) were divided into 3 groups with 10 mice per group, and the following administration was administered for 3 weeks Was administered by oral gavage.
1) Distilled water administration group: 0.5ml / body / day
2) Acid polysaccharide administration group obtained in Example 1: 5 mg / kg / day
3) Acid polysaccharide administration group obtained in Example 1: 30 mg / kg / day
The polysaccharide was dissolved in distilled water at each concentration and administered at 0.5 ml / body / day.
2. Measurement of NK activity After 3 weeks of administration, remove the spleen from each mouse and suspend the cells in RPMI1640 (10% FCS) to obtain a spleen cell suspension (1 × 10 ⁷ cells / ml). Prepared.
NK activity was measured using a fluorescence activated cell sorter (hereinafter referred to as FACS) using the spleen cells as effector cells and YAC-1 cells as target cells. The target cell, YAC-1, was fluorescently stained with 3,3'-dioctadecyloxacarbocyanine perchlorate (hereinafter referred to as Dio), and then used together with spleen cells in a 96-well microplate (round bottom) at 37 ° C and 5% CO ₂ Incubated for 4 hours. Before completion of the culture, Propidium Iodide (hereinafter referred to as PI) was added to the culture solution to stain dead cells. After completion of the culture, cells stained with Dio and PI were detected as dead YAC-1 cells using FACS with reference to Johann's paper (Johann S, Blumel G, Lipp M, Forster R, J Immunol Methods, 185 (2), 209-16 (1995)). The ratio of effector cells to target cells was 40: 1.
As a result, the spleen cell NK activity in the acidic polysaccharide 30 mg / kg / day administration group was significantly higher than that in the control water administration group (P <0.01). In addition, the acidic polysaccharide 5 mg / kg / day administration group showed a higher tendency (P <0.1) than the control (FIG. 3).

Preparation of fermented milk using polysaccharide-producing lactic acid bacteria Fermented milk was prepared using L. bulgaricus OLL1073R-1 which produces an acidic polysaccharide having an NK activity increasing action. Using raw milk, skim milk powder and sugar, a solution prepared to SNF9.7%, FAT3.05%, sugar 3.0% (hereinafter fermented milk Mix) was added to L. bulgaricus OLL1073R-1, S. thermophilus OLS3059 (FERM P-15487 ) As a starter fungus and fermented at 43 ° C. Fermentation was completed at an acidity of 0.7, and a final acidity of 0.78 was obtained by storing at 4 ° C for 1 day. In addition, fermented milk using L. bulgaricus OLL1256 and S. thermophilus OLS3295 as starter fungi as control fermented milk was also produced.

NK activity of fermented milk using polysaccharide-producing lactic acid bacteria 1. Oral administration test of fermented milk using polysaccharide-producing lactic acid bacteria to mice 32 mice BALB / c (male, 11 weeks old, SLC) with 8 mice per group Divided into 4 groups, the following doses were administered by oral gavage with a sonde for 4 weeks.
1) Distilled water administration group 1.0ml / body / day
2) L. bulgaricus OLL1073R-1 fermented milk (Example 3) administration group 200 mg / body / day
3) Control fermented milk (Example 3) administration group 200 mg / body / day
4) Fermented milk mix (Example 3) administration group 200mg / body / day
Fermented milk and fermented milk Mix were lyophilized and suspended in distilled water at 200 mg / ml at the time of administration.
2. Measurement of NK activity After 4 weeks of administration, remove the spleen from each mouse and suspend the cells in RPMI1640 (10% FCS) to obtain a spleen cell suspension (1 × 10 ⁷ cells / ml). Prepared.
NK activity was measured using a fluorescence activated cell sorter (hereinafter referred to as FACS) using the spleen cells as effector cells and YAC-1 cells as target cells. The target cell, YAC-1, was fluorescently stained with 3,3'-dioctadecyloxacarbocyanine perchlorate (hereinafter referred to as Dio), and then used together with spleen cells in a 96-well microplate (round bottom) at 37 ° C and 5% CO ₂ Incubated for 4 hours. Before completion of the culture, Propidium Iodide (hereinafter referred to as PI) was added to the culture solution to stain dead cells. After completion of the culture, cells stained with Dio and PI were detected as dead YAC-1 cells using FACS with reference to Johann's paper (Johann S, Blumel G, Lipp M, Forster R, J Immunol Methods, 185 (2), 209-16 (1995)). The ratio of effector cells to target cells was 40: 1.
As a result, fermented milk administration showed significantly higher NK activity in the fermented milk administration group using L. bulgaricus OLL1073R-1 (p <0.05) compared to the distilled water administration group, which was a control, using fermented milk. A high tendency (p <0.1) was observed for Mix and control fermented milk (FIG. 4).
.

  According to the present invention, since the NK cell activation effect is high, it is not only useful as an immunostimulator, but can also be used as an immunostimulatory food or drink.

It is a figure which shows isolation | separation of neutral polysaccharide and acidic polysaccharide by anion exchange resin DEAE sepharose. It is a figure which shows isolation | separation by gel filtration using Sephacryl S-400HR of acidic polysaccharide. It is a figure which shows NK activity of the spleen cell in the mouse | mouth which orally administered acidic polysaccharide for 3 weeks. (Test method: Student's t-test) It is a figure which shows the NK activity of a spleen cell in the mouse | mouth which orally administered fermented milk using a polysaccharide-producing lactic acid bacterium for 4 weeks. (Test method: Student's t-test)

Claims (6)

A fermented milk having an NK cell activation effect, which produces L. bulgaricus OLL1073R-1 and S. thermophilus OLS3059 as a starter fungus. The fermented milk according to claim 1 , comprising an acidic polysaccharide in an amount effective for NK cell activation. Fermented milk according to claim 2 , wherein the acidic polysaccharide is a phosphorylated polysaccharide. The fermented milk of Claim 3 whose phosphorus content of a phosphorylated polysaccharide is 0.01% or more. The NK cell activator which uses the fermented milk of the said Claims 1-4 as an active ingredient.   A method for producing an acidic polysaccharide having NK cell activation action, comprising adding L. bulgaricus OLL1073R-1 and S. thermophilus OLS3059 to a solution containing whey, raw milk and / or skim milk powder.

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