JP2007290978A - Immunomodulation composition containing extracellular water-soluble polysaccharide of acetic acid bacterium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a food containing a substance having a stronger immunomodulation action with high safety. <P>SOLUTION: An immunomodulation composition contains a water-soluble heteropolysaccharide extracellularly produced by Acetobacter tropicalis in culturing the above bacterium, and the Acetobacter tropicalis is Acetobacter tropicalis SKU 1100 strain (Deposition Strain No.NBRC 101654), and the water-soluble heteropolysaccharide contains a water-soluble heteropolysaccharide composed of at least glucose and rhamnose and having a molecular weight of not less than 400,000. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an immunoregulatory composition containing, as an active ingredient, a water-soluble heteropolysaccharide produced outside the cell by Acetobacter tropicalis, and an immunoregulator comprising the composition, food and drink And feed.

  Lymphocytes include T cells that control cellular immunity, and B cells that play a central role in humoral immunity. T cells play a key role in the regulation of immune activity, as well as being responsible for cellular immunity. Helper T cells are said to be important. Helper T cells have been found to include Th1 cells that secrete interferon (IFN) gamma and interleukin-2 (IL-2), and Th2 cells that secrete IL-4 and IL-10. IFN-gamma secreted by Th1 cells suppresses Th2 cells, whereas IL-4 and IL-10 secreted by Th2 cells suppress Th1 cells. Th1 cells and Th2 cells suppress each other, and both maintain a certain balance.

  In addition, IL-2 and IFN-gamma secreted by Th1 cells enhance cellular immunity, while IL-4, IL-5, and IL-10 secreted by Th2 cells enhance humoral immunity. When Th1 cells are dominant, they are resistant to infectious diseases involving cellular immunity such as tuberculosis and Listeria, and when Th2 cells are dominant, they are easily infectious. In addition, Th1 cell predominates in the development of organ-specific autoimmune diseases involving cellular immunity, and Th2 cell predominates in systemic autoimmune diseases involving large humoral immunity and bronchial asthma involving IgE. Is supposed to be involved.

  IL-4 and IL-10 produced by Th2 cells inhibit the reaction of TH1 cells, whereas IFN-gamma produced by Th1 cells inhibits the reaction of Th2 cells. That is, the production of IFN-gamma is promoted and the production of IL-4 is suppressed, suggesting a transition from a state predominantly Th2 cells to a state predominantly Th1 cells.

  Allergic diseases are thought to develop due to Th2 cell responses to environmental antigens. In this case, conversion of Th2 cell significant responses to allergens into Th1 cell significant responses is expected as a new treatment for allergic diseases. ing. As such food components that significantly change the Th1 / Th2 balance in Th1 cells, lactic acid bacteria (see Patent Document 1), mushroom-derived beta-glucan (see Patent Document 2), and the like are known. Have the ability to induce IFN-gamma and interleukin-12 (IL-12). It has been clarified that induction of IFN-gamma and IL-12 activates cellular immunity including NK cells and the like, and increases resistance to cancer development (see Patent Document 1).

  On the other hand, it has also been clarified that active IFN-gamma production induction suppresses the production of IgE causing allergy (see Patent Document 3). Therefore, it can be said that a food ingredient having a strong IFN-gamma inducing ability has an antitumor action and an antiallergic action.

  Acetobacter acetic acid bacteria are gram-negative microorganisms widely distributed on the surface of petals and fruits. Since ancient times, it has been used worldwide for the production of fermented foods such as vinegar, vinegar, pickles, and yogurt. As a water-soluble polysaccharide outside the cell produced by an Acetobacter acetic acid bacterium, glucose: galactose: mannose: glucuronic acid = 10: 3-6: 0.5-2: 0.5-2 Constituent sugars of glucose: mannose: rhamnose: glucuronic acid = 4: 0.9 to 1.1: 0.9 to 1.1: 0.9 to 1.1 (see Patent Document 4) And acetan (see Non-Patent Document 1) composed of a constituent sugar of glucose: mannose: rhamnose: glucuronic acid = 4: 1: 1: 1.

  In addition, as polysaccharides derived from acetic acid bacteria having allergic regulatory activity, polysaccharides produced outside the cells by Acetobacter polysaccharogenes and Acetobacter xylinum are known (Patent Document 6). reference). The extracellular water-soluble heteropolysaccharide disclosed in the Examples of the present invention has glucose, mannose, and glucuronic acid as constituent sugars, and has the effect of suppressing IgE production and IL-4 production, and Th2 cells. It is said that there is an effect of promoting the production of IL-12 that suppresses the differentiation of the cells.

JP 2002-80364 A JP 2005-97308 A JP 2005-139160 A JP 58-78596 A JP 59-203498 A Carbohydr. Res. , Vol. 245, no. 2, p. 303-310, 1993 JP 2004-59547 A

  However, in order to ingest these substances and to exert their effects, a larger amount of intake and long-term ingestion are required, and it has been difficult for the intaker to obtain a sufficient effect. Therefore, it has been desired to develop a food containing a substance having a stronger immunoregulatory effect while having high safety.

  In order to solve the above-mentioned problems, the present inventors proceeded with studies on the immunomodulatory activity of water-soluble polysaccharides of acetic acid bacteria isolated from the natural world, and liquid-cultured Acetobacter tropicalis under aerobic conditions. Water-soluble heteropolysaccharides produced outside the cells have excellent immunoregulatory activities such as IFN-gamma production-promoting activity and enhance IFN-gamma production by immune cells They have found that they have the effect of improving resistance to cancer and alleviating allergic symptoms such as hay fever and house dust allergies, and have completed the present invention.

  That is, the first aspect of the present invention is an immunoregulatory composition characterized in that it contains a water-soluble heteropolysaccharide produced outside the cell by culturing Acetobacter tropicalis. Preferably, Acetobacter tropicalis is Acetobacter tropicalis SKU1100 strain (deposited strain number NBRC101654), and preferably water-soluble The telopolysaccharide is a water-soluble heteropolysaccharide composed of at least glucose and rhamnose, preferably the water-soluble heteropolysaccharide is a water-soluble heteropolysaccharide having a molecular weight of 400,000 or more, and more preferably, the immune But interferon immune cells - is an immunomodulatory composition is gamma production inducing.

  The second aspect of the present invention is a gist of an immunomodulator comprising the first immunoregulatory composition of the present invention as an active ingredient.

  The third aspect of the present invention is a food / beverage product containing the first immunoregulatory composition of the present invention as an active ingredient and having immunomodulating activity.

  A fourth aspect of the present invention is a feed comprising the first immunoregulatory composition of the present invention as an active ingredient and having immunomodulating activity.

  The immunomodulating composition according to the present invention has a stronger immunomodulating effect than conventional ones, and in particular has the ability to induce IFN-gamma from immune cells. IFN-gamma exerts an antitumor effect by activating NK cells. On the other hand, IFN-gamma activates Th1 cells and inactivates Th2 cells to suppress IgE production, and as a result, can alleviate allergic symptoms involving IgE.

Hereinafter, the present invention will be described in detail.
Acetobacter tropicalis used in the present invention is an acetic acid bacterium that adheres to petals and fruit surfaces and exists widely in nature. As for Acetobacter tropicalis, the NBRC16470 strain and the DSMZ274 strain are already stored in public institutions and are readily available. Among them, Acetobacter tropicalis SKU1100 strain, which is one strain isolated from the natural world by the present inventors, produces a water-soluble heteropolysaccharide outside the cells by liquid culture under aerobic conditions (Microbiology, vol. 151, p. 4111-4120, 2005). This heteropolysaccharide has a molecular weight of 400,000 or more and is composed of glucose, rhamnose, and galactose. In addition, Acetobacter sp. Cell membrane-bound heteropolysaccharide of SKU1100 strain (Biosci. Biotechnol. Biochem., Vol. 65, p777-783, 2002) (see JP 2002-95493 A) and the water-soluble heteropolysaccharide used in the present invention Are the same in that the constituent sugars are composed of glucose, rhamnose, and galactose, but the molecular weight (120,000 for membrane-bound heteropolysaccharide) and the sugar composition ratio are different. The membrane-bound heteropolysaccharide is formed on the surface of the culture solution when Acetobacter tropicalis is statically cultured, while the water-soluble heteropolysaccharide of the present invention is a medium when the strain is subjected to liquid culture under aerobic conditions. Produced in.

  Furthermore, the water-soluble heteropolysaccharide of the present invention includes not only the wild strain of Acetobacter tropicalis SKU1100 strain, but also the Pel- strain lacking polE of the polysaccharide synthesis gene operon, and the polysaccharide synthesis gene operon. It can also be produced using a recombinant gene such as a ΔgalE strain lacking only a galactose synthesis gene (Microbiology, vol. 151, p. 4111-4120, 2005).

  The water-soluble heteropolysaccharide according to the present invention may have at least glucose and rhamnose as its constituent sugars, and may further contain galactose, mannose, glucuronic acid and the like as constituent sugars, preferably from glucose, rhamnose and galactose. Is a water-soluble heteropolysaccharide. Further, the composition ratio is not particularly limited.

  When this strain is cultured to obtain a water-soluble heteropolysaccharide, a known medium composition (for example, a YPG liquid medium containing 0.5% yeast extract, 0.5% polypeptone, 1% glycerin, etc.) and a culture temperature (27- Water-soluble heteropolysaccharide can be produced outside the cells by liquid culture under aerobic conditions such as shaking culture using a pH of 5 to 7 at 40 ° C. The produced water-soluble heteropolysaccharide can be easily separated and recovered by a known method such as precipitation with alcohols or acetone after removing the cells from the culture solution.

   The extracellular water-soluble heteropolysaccharide of Acetobacter tropicalis obtained as described above can be used as it is or as an immunoregulatory composition of the present invention by adding other components as necessary. it can.

  The other components that can be included in the composition can be mixed as long as they do not reduce the immunomodulating activity in the present invention. For example, conventionally used pharmaceutically acceptable surfactants, It can be mixed with additives such as solvents, thickeners, stabilizers, preservatives, antioxidants, flavorings, and the like.

  Examples of the form of the immunomodulating composition of the present invention include powders, tablets, capsules, soft capsules, pastes or troches, gums or drinkable solutions or emulsions, dry oral supplements, wet oral supplements, etc. It is not limited to. Moreover, these forms can be produced by a conventionally known method. The ingestion method can also be administered through general administration routes such as oral administration, rectal administration, intravenous injection, and infusion.

  The immunomodulator of the present invention comprises the above-described immunomodulatory composition of the present invention as an active ingredient. The content of the active ingredient may vary depending on the age, weight, etc. of the subject to be ingested, but it is preferably contained so that it can be taken from 0.01 to 100 mg / kg per day for an adult. kg is preferred, and 0.5-5 mg / kg is most preferred.

  Examples of various additives contained in the immunomodulator of the present invention include surfactants, excipients, colorants, preservatives, coating aids, and combinations thereof. These additives are not particularly limited as long as they are additives in normal pharmaceutical production, and more specific examples include lactose, dextrin, sucrose, mannitol, corn starch, sorbitol, crystalline cellulose, polyvinylpyrrolidone, dextrin, and methylcellulose. , Carboxymethylcellulose salt, stearic acid and its salts, additives such as talc, and combinations thereof. Furthermore, spices, sweeteners and the like may be added. Furthermore, you may add another chemical | medical agent, a food ground material, and a food extract as needed.

  The dosage form of the immunomodulating agent of the present invention is not particularly limited, and is prepared into an appropriate dosage form according to the Japanese Pharmacopoeia. Specifically, it is produced in dosage forms such as capsules, tablets, powders, sustained-release agents.

  The food-drinks which have the immunomodulating activity of this invention contain the composition which has the above-mentioned immunomodulating activity of this invention. The content of the active ingredient may be set according to the form of each food and drink so that the daily intake is 0.01 to 100 mg / kg, more preferably 0.1 to 10 mg / kg. Most preferred is 5 to 5 mg / kg.

  The food-drinks which have the immunomodulating activity of this invention contain the above-mentioned immunoregulatory composition. Examples of other materials that can be mixed in the food and drink of the present invention include those that can be generally used as food materials. Examples include polysaccharides obtained from rice, wheat, corn, potato, kelp, proteins obtained from soybeans and dairy products, animal raw materials, glucose, lactose, fructose, sucrose, mannitol, xylitol, various oligosaccharides, etc. Saccharides, as well as combinations thereof, may be mentioned. Furthermore, spices, colorants, sweeteners, acidulants, edible oils, vitamins and other food crushed products, food extracts and the like may be added. These suitable materials and additives are used alone or in combination. Furthermore, you may process to a desired shape by adding water as needed. Moreover, as long as the immunoregulatory activity of the present invention is not lowered, there is no problem even if other food materials considered to have immunomodulatory activity, for example, beta-glucan derived from mushrooms or lactic acid bacteria are added.

  Specific examples of food and drink include confectionery (gum, candy, caramel, chocolate, cookies, snack confectionery, jelly, gummy, tablet confectionery, etc.), noodles (soba, udon, ramen, etc.), dairy products (milk, ice cream, Yogurt, etc.), seasonings (miso, soy sauce, etc.), soups, beverages (juice, coffee, tea, tea, carbonated drinks, sports drinks, etc.) and general foods and health foods (tablets, capsules, etc.), nutrition Supplementary foods (such as energy drinks) can be mentioned, but are not limited thereto. Moreover, you may add the extract of this invention to an instant foodstuff. For example, a product obtained by spray-drying or freeze-drying an extract together with powdered cellulose can be easily contained in a food or drink by making it into a powder, granule, tablet, or solution.

  The feed having immunomodulating activity of the present invention contains the above-described immunomodulating composition of the present invention, and can be applied to all domestic animals, poultry and pets. Examples of livestock and poultry to which the feed of the present invention can be applied include domestic animals such as cows, pigs, horses, goats, sheep and rabbits, and animals such as chickens, turkeys, ducks, and quails. The pet animals to which the present invention can be applied are animals and birds raised for personal hobbies such as dogs, cats, hamsters and small birds. This may be administered alone as a feed composition, or may be added directly to the feed, and the form of use is not particularly limited. When added to the feed, there are no particular restrictions on the addition method, timing of addition, and the like.

  The content of the active ingredient contained in the feed of the present invention may be set according to the form of each feed so as to be 0.01 to 100 mg / kg as the amount of active ingredient fed per day, and further 0 .1-10 mg / kg is preferred, and 0.5-5 mg / kg is most preferred.

  Other materials that can be mixed in the feed of the present invention include, for example, corn, milo, soybean, soybean meal, wheat, barley, rice, buckwheat, fish meal, skim milk powder, beet pulp pellets, bran pellets, grain screening pellets, alfalfa pellets , Tapioca pellets, corn cob meal, cotton hull pellets, sweet potato chips, homini feed, beer koji, hay cube, mini cube, alfalfa baled hay, sudan grass, rice straw, ryegrass straw, fescue straw, timothy hay, oat hay, Molasses, lupine, canola, rapeseed meal, cottonseed, corn gluten meal, corn gluten feed, soy hull pellets, whey powder, brewer's yeast, cholesterol, and other pharmaceutically acceptable vitamins And minerals, feed additives, veterinary drugs and the like, and are not particularly limited.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these at all.

<Method for recovering and purifying extracellular water-soluble heteropolysaccharide>
After culturing Acetobacter tropicalis SKU1100 wild-type strain, and Acetobacter tropicalis SKU1100 Pel-strain (gene recombinant lacking the polE gene of the wild-type polysaccharide synthesis gene operon) and ΔgalE strain in YPG liquid medium for several days The culture solution was centrifuged (9,000 X g, 10 min) to obtain a culture supernatant from which the cells were removed. Add twice the volume of isopropyl alcohol to this culture supernatant, leave it at -20 ° C for 24 hours (it can be left at 4 ° C overnight), and collect the precipitate by centrifugation (8,000 X g, 30 min) did. This precipitate was suspended in a small amount of distilled water and dialyzed against 25 mM Tris-Hcl (pH 8.5). This dialysate was centrifuged (9,000 X g, 10 min) and then applied to a DEAE-cellulose column equilibrated with the same buffer to obtain a flow-through fraction. This fraction was precipitated with isopropyl alcohol as described above, suspended in 0.1 M NaCl containing a small amount of 0.1% SDS, applied to a gel filtration (Sephacryl S-400) column and eluted with the same solution. The peak fractions containing the target polysaccharide were combined and lyophilized to obtain the final purified polysaccharide.

[Reference example]
<Method for recovering and purifying bacterial membrane-bound heteropolysaccharide>
Acetobacter tropicalis SKU1100 strain was left to stand at 30 ° C. for several days in a YPG liquid medium (0.5% glucose, 0.5% polypeptone, 1% glycerol), and then the cells were collected and phosphate buffer And then suspended in the same buffer. The obtained bacterial cell suspension was sonicated at maximum output for about 10 minutes, and then the supernatant was collected by centrifugation and further subjected to ultracentrifugation (150,000 X g, 90 min). To this supernatant, add DNase and RNase to a concentration of 50 μg / ml, treat at 37 ° C for 24 hours, and continue with protease (Proteinase K) to a final concentration of 0.1 mg / ml. Added and left at 37 ° C. for 24 hours. After dialyzing this solution against distilled water containing 0.1% SDS for 24 hours, this dialysate was centrifuged to remove the precipitate, and twice the amount of isopropyl alcohol was added to the supernatant and left at -20 ° C for 24 hours. Later (may be left overnight at 4 ° C.), the precipitate was recovered by centrifugation (8,000 × g, 30 min). This precipitate (crude polysaccharide fraction) was suspended in a small amount of distilled water, the precipitate was removed by ultracentrifugation (150,000 X g, 90 min), and then charged on a gel filtration (Superdex S-200) column. The column was eluted with 0.1 M NaCl solution containing 0.1% SDS, the peak fractions containing the target polysaccharide were combined, and lyophilized to obtain the final purified polysaccharide.

<Method for measuring immunomodulatory action>
Balb / c mice female 6 weeks old (Nippon Charles River) were intraperitoneally administered with 200 μl of a physiological saline solution in which 20 μg of OVA was absorbed in 2 mg of aluminum hydroxide gel, and the spleen was collected on the 10th day. Spleen cells were collected and adjusted, suspended in RPMI-1640 culture medium containing 20% FBS, and 10 μg of Acetobacter tropicalis SKU1100 bacterial membrane-bound and water-soluble heteropolysaccharide in the presence of 100 μg / ml OVA. It added so that it might become / ml, and it co-cultured. Seven days later, the culture supernatant was collected, and IFN-gamma was quantified by ELISA. (N = 3)

  The IFN-gamma-inducing activity of the polysaccharide produced by Acetobacter tropicalis SKU1100 is shown in FIG. As a result, Acetobacter tropicalis SKU1100 wild strain and Pel-strain produced extracellular water-soluble heteropolysaccharide (consisting of glucose, rhamnose and galactose, molecular weight of 400,000 or more) 33 times that of the negative control The above IFN-gamma-inducing activity is that the extracellular water-soluble heteropolysaccharide (consisting of glucose and rhamnose, molecular weight 400,000) produced by the ΔgalE strain has an IFN-gamma-inducing activity 20.5 times that of the negative control. It was found. On the other hand, no wild-type membrane-bound heteropolysaccharide (consisting of glucose, rhamnose, galactose, molecular weight 120,000) has been found to have IFN-gamma-inducing activity, and strong IFN-gamma-inducing activity is unique to water-soluble polysaccharides. It was. This strain has been deposited with NBRC, and its deposit number is NBRC101654.

  The extracellular water-soluble heteropolysaccharide of Acetobacter tripicalis according to the present invention can be used as a pharmaceutical, food or drink or feed having an immunomodulating action. Therefore, the present invention can be used in the food industry, pharmaceutical industry, and feed industry.

It represents a comparison of IFN-gamma production-inducing activity of water-soluble heteropolysaccharides produced by Acetobacter tropicalis SKU1100.

Claims (8)

  An immunoregulatory composition comprising a water-soluble heteropolysaccharide produced outside the cells by culturing Acetobacter tropicalis.   2. The immunoregulatory composition according to claim 1, wherein Acetobacter tropicalis is Acetobacter tropicalis SKU1100 (deposited strain number NBRC101654).   The immunoregulatory composition according to claim 1 or 2, wherein the water-soluble heteropolysaccharide is composed of at least glucose and rhamnose.   The immunomodulating composition according to any one of claims 1 to 3, wherein the water-soluble heteropolysaccharide has a molecular weight of 400,000 or more.   The immunomodulating composition according to any one of claims 1 to 4, wherein the immunomodulation is induction of interferon-gamma production of immune cells.   An immunomodulator comprising the composition according to any one of claims 1 to 5 as an active ingredient.   A food or drink comprising the composition according to any one of claims 1 to 5 as an active ingredient and having immunomodulating activity. A feed comprising the composition according to any one of claims 1 to 5 as an active ingredient and having immunomodulating activity.