WO2004061090A1

WO2004061090A1 - Composition capable of activating natural killer cells and process for producing the same, composition containing natural killer cells activated by the former composition and process for producing the same

Info

Publication number: WO2004061090A1
Application number: PCT/JP2004/000003
Authority: WO
Inventors: Toshiro Omori; Yasufumi Umemoto; Yoshifumi Furuta; Hideki Hokazono; Mihoko Furutera; Hideki_____________________________________- Ohba
Original assignee: Sanwa Shurui Co., Ltd.; Barley Fermentation Technologies, Inc.; National Institute Of Advanced Industrial Science And Technology
Priority date: 2002-12-27
Filing date: 2004-01-05
Publication date: 2004-07-22
Also published as: JP2005104957A; JPWO2004061090A1

Abstract

A composition capable of activating natural killer cells comprising a fraction which is substantially free from uronic acid, contains polysaccharides comprising xylose, arabinose and glucose and is insoluble in an organic acid solvent. This fraction is obtained by subjecting barley shochu (distilled spirit) stillage to solid-liquid separation to give a liquid, subjecting this liquid to an ion exchange treatment using an ion exchange resin to give a fraction (a) not adsorbed by the ion exchange resin, or subjecting the above-described liquid to an adsorption separation treatment using a synthetic adsorbent to give a fraction (b) not adsorbed by the synthetic adsorbent, then subjecting this fraction (b) to an ion exchange treatment using an ion exchange resin to give the above-described fraction (a), concentrating the thus obtained fraction (a) by ultrafiltration using an ultrafiltration membrane to give a liquid concentrate, and adding the above-described organic solvent to the liquid concentrate. A composition containing natural killer cells having been inactivated with the use of the above composition.

Description

Specification

TECHNICAL FIELD The present invention relates to a composition having an action of activating natural killer cells and a method for producing the same, and a composition containing natural killer cells activated using the composition and a method for producing the same.

The present invention obtains a liquid component by solid-liquid separation of a barley shochu distillation residue (hereinafter abbreviated as “barley shochu distillation residue”) by-produced in the production of shochu using barley as a raw material, The fraction that has not been adsorbed to the synthetic adsorbent, which has been collected by exhausting the fraction or subjecting the liquid to an adsorption separation treatment using a synthetic adsorbent, is subjected to ion exchange treatment using an ion exchange resin. The fraction that is not adsorbed on the ion exchange resin is fractionated, and the fraction that is not adsorbed on the ion exchange resin is subjected to a concentration treatment by ultrafiltration using an ultrafiltration membrane. It is related with the composition which consists of a fraction insoluble in the said organic solvent which has the effect | action which activates a natural killer cell collected by adding an organic solvent to this concentrated liquid, and its manufacturing method, and its manufacturing method. The present invention also includes a composition containing a natural killer cell and a natural killer cell activated by using a composition comprising a natural killer cell and a fraction insoluble in the organic solvent, and a method for producing the same. Background art

Natural killer cells (hereinafter referred to as “spider cells”) are found in the lymphoid tissues of animals (humans, mice, hamsters, rats, chickens, pigs, etc.). These sputum cells are morphologically somewhat large lymphocytes (LGLs) that contain azul granules in the cytoplasm, occupying a few percent of lymphocytes, and without immune memory. It is thought that it plays a part in the defense mechanism of the body against tumor development because it can destroy non-specific tumor cells (mainly hematopoietic tumors). The Furthermore, whereas B cells and T cells are responsible for antigen-specific acquired immunity, sputum cells play an important role in non-antigen-specific innate immunity that acts at the very beginning of the immune response. Even though it does not have a sputum cell receptor, it can accurately identify self and non-self and can cause necrosis of virus-infected cells and tumor cells without prior antigen sensitization. Are known.

By the way, in recent years, in the treatment of intractable diseases (lifestyle-related diseases) such as cancer and diabetes, the treatment methods that depend on synthetic drugs often have problems of side effects that cannot be overcome. As a treatment method that does not have side effects caused by the above, a so-called alternative medicine treatment method that enhances the immunity (self-healing power) inherent in humans by using medicinal foods made from natural materials is attracting attention. Because such alternative medicine is a patient-centered treatment and there are few concerns about side effects, 60% of doctors recommend alternative medicine to patients, especially in the United States, and more than half of cancer patients substitute. It is reported that medical care is provided. In Japan as well, interest in alternative medicine has increased as the first academic council on “alternative medicine” was held in February 1998 in Japan. For these reasons, substances that activate する cells derived from various natural products are gradually being used as pharmaceuticals and medicinal foods as one of the immunity enhancing substances, and in the future, Use seems to be popular.

As described below, several substances that activate sputum cells derived from such natural products are known. For example, Japanese Patent Publication No. 2532899 (hereinafter referred to as “Patent Document 1”) describes a filamentous fungus of the genus Aspergillus or Basidiomycetus as a plant tissue raw material of rice bran, bran, rice straw or bagasse. Describes a method for producing hemicellulose having an immunostimulatory action, characterized by being assimilated in the culture medium. The hemicellulose having an immunostimulatory effect obtained through the production method is described as having main components xylose and arabinose and having an average molecular weight of 550,000 or 600,000. In addition, the rice bran in Patent Document 1, The bran, rice straw, or bagasse is the residue of barley shochu that is a by-product of the distillation of the whitened grains that have undergone whitening to remove the hulls of barley grains, followed by distillation after alcohol fermentation. It is completely different from liquid.

In JP 9-23895 (hereinafter referred to as “Patent Document 2”), water-soluble polysaccharides are obtained by preparing water-soluble polysaccharides of plant tissues such as gramineous plants, especially rice bran. An immunity enhancing substance obtained by preparing an extracellular enzyme of a filamentous fungus to obtain an enzyme complex, mixing the water-soluble polysaccharide and the enzyme complex, and reacting at a predetermined pH for a predetermined time, It is described that it has an effect of activating NK cells. Further, it is described that the substance for enhancing immunity has an average molecular weight of 600,000 or 650,000, and main components are xylose and arabinose. In addition, the rice bran in Patent Document 2 is a by-product generated when whitening the threshed brown rice, and after subjecting the whitened grains that have been whitened to remove the hulls of barley grains to alcohol fermentation, distillation is performed. It is completely different from the barley shochu distillation residue, which is a distillation residue produced as a by-product during the production of shochu.

In Japanese Patent Laid-Open No. 10-146166 (hereinafter referred to as “Patent Document 3”), an aqueous extract of sake lees produced as a by-product when rice is subjected to alcoholic fermentation to produce sake is activated by NK cell activation. It is described to have In addition, the sake lees in Patent Document 3 is a solid content which is a residue after separating the liquid obtained by subjecting rice to alcoholic fermentation and then squeezing it as a sake product. Therefore, the sake lees in Patent Document 3 is a distillation residue produced as a by-product in producing shochu by subjecting the refined grains that have undergone whitening to remove the hulls of barley grains to alcohol fermentation and then distilling them. It is completely different from a barley shochu distillation residue.

Japanese Unexamined Patent Publication No. 2002-255843 (hereinafter referred to as “Patent Document 4”) describes an immunostimulation comprising a water extract of rice bran and comprising an extract having a molecular weight of about 30000 to 40000. A composition for use is described. Patent Document 4 describes that the composition has mitogenic activity and excellent NK cell activation action. Patent The rice bran in Ref. 4 is a by-product generated when whitening the threshed brown rice, and the whitened grains that have undergone whitening to remove the hulls of barley grains are subjected to alcoholic fermentation followed by distillation to produce shochu. It is completely different from the barley shochu distillation residue, which is a by-product distillation residue.

Japanese Unexamined Patent Publication No. 2002-338475 (hereinafter referred to as “Patent Document 5”) discloses a partial decomposition of a hemicell mouthpiece obtained from corn hull, which is commercially available under the trade name “Celuse”. An antitumor composition containing the product as an active ingredient is described. According to Patent Document 5, the partial degradation product of hemicellulose has an average molecular weight of 20,000 to 200,000, and NK cell activity is significantly increased in colon26 cancer-bearing mice administered with the partial degradation product. Furthermore, it is described that the ability to produce cytokines (IL-2 and INF-a) is also significantly increased.

March 1991 (Company) Confectionery Technology Center, published by the Ministry of Agriculture, Forestry and Fisheries, Food Distribution Bureau, Contracted Business No. 8 Effective Use Technology Series for Active Materials for Food and Drink, Water-soluble Corn Fiber I (Arabinoxylan), page 3 (below) (Referred to as “Non-Patent Document 1”), a partially decomposed product of hemicellulose obtained from corn hull marketed under the trade name “Cel Ace” described in Patent Document 5 contains arabinose and It is mainly composed of arabinoxylan consisting of xylose and has a composition of 40.32% xylose, 27.76% arabinose, 11.86% uronic acid, 5.91% galactose and 2.30% glucose. It is described that there is.

By the way, the corn hull, which is the source of the “partial degradation product of hemicellulose obtained from corn hull” described in Patent Document 5, is basically the corn hull that is removed when the corn kernel is refined. There is a barley shochu distillation residue that is a distillation residue by-produced when producing white spirits by subjecting the whitened grains that have undergone whitening to remove the hulls of barley grains to alcohol fermentation and then distilling them. Are completely different.

Apart from the above, JP 2001-145472 A (hereinafter referred to as “Patent Document 6”) The barley shochu distillation residue (i.e., obtained from the distillation residue produced as a by-product in producing shochu by subjecting the whitened grains that had undergone whitening to remove the hull of barley grains to alcohol fermentation and then performing distillation) A composition having a fatty liver inhibitory action different from the activating action on M cells is described, that is, Patent Document 6 obtains a liquid component by solid-liquid separation of the barley shochu distillation residue. Then, Al force is added to the obtained liquid to fractionate the Al force soluble fraction, and the fraction obtained is neutralized with an acid to obtain a neutral soluble fraction. A composition having a fatty liver inhibitory action obtained by adding ethanol to the neutral soluble fraction is described in Patent Document 6. In addition, Patent Document 6 describes the composition described above. , Which contains a molecular weight of 3000 or less as a main component, and the composition contains hemicellulose It is described that the constituent element is xylose, and therefore, in any of the above-mentioned known documents, whether or not the barley shochu distillation residual liquid contains a component having an activating action on M cells. It is clear that there is no suggestion about this.

In view of the above-mentioned state of the art, the present invention further provides a barley shochu distillation residue (hereinafter referred to simply as “barley shochu distillation residue”) produced as a by-product when producing barley shochu. As a result of intensive studies to achieve effective use, it has been completed.

An object of the present invention is to provide a composition having a significant activating effect on natural killer cells (hereinafter abbreviated as “NK cells”) collected from a barley shochu distillation residue and a method for producing the same. There is.

Another object of the present invention is to provide a composition containing NK cells activated using the composition having a significant activating effect on NK cells, and a method for producing the same.

The composition having a significant activating effect on NK cells of the present invention includes the following two embodiments. The composition having a significant activating effect on NK cells of the first aspect is obtained by solid-liquid separation of a barley shochu distillation residual liquid to obtain a liquid component, and the liquid component is subjected to an ion exchange treatment using an ion exchange resin. The fraction that is not adsorbed on the ion-exchange resin is fractionated, and the fraction that is not adsorbed on the ion-exchange resin that has been collected is subjected to a concentration treatment by ultrafiltration using an ultrafiltration membrane. To obtain a concentrated solution, and a fraction insoluble in the organic solvent fractionated by adding an organic solvent to the concentrated solution, and the fraction insoluble in the organic solvent substantially contains uronic acid. It contains polysaccharides consisting of xylose, arabinose, and glucose. The molecular weight distribution is 5% for 100,000 or more, 18% for 30,000 to 100,000, 23% for 10,000 to 30,000, 000 to 10,000 is 31%, 1,000 to 3,000 is 11%, 500 to 1,000 is 3%, and 500 or less is 9%. Has an activating effect. The fraction insoluble in the organic solvent is about 84.1% by weight of polysaccharide, about 54.4% by weight of total arabinose and xylose derived from polysaccharide, about 4.2% by weight of crude protein, organic acid Has a component composition consisting of about 0.3% by weight and about 1.2% by weight of free sugars.

The composition having a significant activating effect on NK cells according to the second aspect is obtained by subjecting the barley shochu distillation residue to solid-liquid separation to obtain a liquid component, which is then subjected to an adsorption separation process using a synthetic adsorbent. And fractionating a non-adsorbed fraction on the synthetic adsorbent, and subjecting the non-adsorbed fraction to the fractionated synthetic adsorbent to an ion exchange treatment using an ion exchange resin. A non-adsorbed fraction is fractionated, and the non-adsorbed fraction is subjected to a concentration treatment by ultrafiltration using an ultrafiltration membrane to obtain a concentrated solution. The fraction insoluble in the organic solvent fractionated by adding an organic solvent to the organic solvent, and the fraction insoluble in the organic solvent is substantially free of uronic acid and consists of xylose, arabinose and glucose. Contains polysaccharides, molecular weight distribution over 100,000 is 11%, 30,000 to 100, 000 is 29, 10,000 to 30,000, 24, 3,000 to 10,000 is 21%, 1,000 to 3,000 is 6%, 500 to 1,000 is 2%, 500 or less is 7% Some have a significant activation effect on NK cells. In the fraction insoluble in the organic solvent, the total content of arabinose and xylose derived from polysaccharide is about 58.6% by weight. It has a component composition consisting of about 3.3% by weight of crude protein, about 0.2% by weight of organic acid, and about 1.5% by weight of free sugars.

The composition having an activating effect on NK cells according to the first and second aspects of the present invention is very remarkable for NK cells, comparable to IL-2 which is a known NK cell activator. It has an activating effect and can be used very suitably for the treatment of cancer including leukemia as a pharmaceutical.

The composition containing the activated NK cell of the present invention includes the following two embodiments.

The composition containing activated NK cells according to the first aspect contains activated NK cells using the composition having a significant activation action on the NK cells according to the first aspect. It is a cell-containing composition.

The composition containing activated NK cells according to the second aspect contains NK cells activated using the composition having a significant activating effect on the M cells according to the second aspect. It is a cell-containing composition.

The activated NK cell-containing composition containing activated NK cells according to the first and second aspects of the present invention can be used very suitably for the treatment of cancer including leukemia as a pharmaceutical. .

The present invention has been completed based on the facts obtained by the present inventors through experiments. The experiment conducted by the present invention will be described below.

The present inventors obtained a liquid component by solid-liquid separation of a barley shochu distillation residue produced as a by-product in the production of shochu using barley as a raw material, and an organic solvent (preferably ethanol) was added to the liquid component. In view of the fact that the collected fraction insoluble in the organic solvent contains polysaccharide as one of its main components, the polysaccharide contained in the fraction insoluble in the organic solvent contains NK cells. In order to clarify whether or not it is involved in activation, we conducted intensive studies through experiments. That is, components other than polysaccharides contained in the fraction insoluble in the organic solvent, that is, amino acid, peptide, protein, organic acid, or barley-derived For the purpose of removing polyphenols, etc., various ion exchange resins are prepared, and the liquid components obtained by solid-liquid separation of the barley shochu distillation residual liquid are ion exchange treatments using these ion exchange resins individually. To obtain a non-adsorbed fraction on each ion-exchange resin, and add an organic solvent to each of the non-adsorbed fractions on the obtained plurality of ion-exchange resins. A fraction insoluble in the organic solvent collected by fractionation is obtained, and whether each of the obtained fractions insoluble in the organic solvent has an NK cell activation effect or not, has an NK cell activation effect. In this case, the following experiment was conducted to clarify the degree of the NK cell activation effect. Barley shochu was produced for the purpose of the following Experiment 1 to Experiment 14. Barley (70% refined) was used as a raw material.

[Manufacture of firewood]

40% barley water was absorbed, steamed for 40 minutes, allowed to cool to 40 ° C, inoculated with 1 kg of seed perilla (birch) per ton of barley, 38 ° C, RH95¾ for 24 hours, 32 ° C, Barley straw was produced by holding at RH92% for 20 hours.

[Manufacture of steamed barley]

Steamed barley was produced by absorbing 40% by weight of barley, steaming for 40 minutes, and allowing to cool to 40 ° C.

[Production of barley shochu and barley shochu distillation residue]

In the first preparation, add 3.6 ml of water and lkg (wet weight) of cultured cells of shochu yeast as yeast to the barley koji (3 tons as barley) produced by the method described above to obtain primary mash. The obtained primary moromi was subjected to 5 days of fermentation (1st stage fermentation). Next, in the secondary charging, 11.4 kiloliters of water and 7% ton of steamed barley (7 tons of barley) produced by the method described above were added to the primary mash that had been subjected to the first stage fermentation. (Stage fermentation). The fermentation temperature was 25 ° C for both the first and second charge. The second mash after the second stage fermentation was subjected to simple distillation by a conventional method to obtain 10 kiloliters of barley shochu and 15 kiloliters of barley shochu distillation residue. The obtained barley shochu distillation residue was Used for Experiment 1 to Experiment 14.

Experiment 1

From the barley shochu distillation residue obtained in the above-mentioned “Production of barley shochu and barley shochu steamed residue”, a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue is centrifuged under the conditions of 8000 rpm and lOmin to obtain a liquid component, the Brix of the obtained liquid component is adjusted to 10, and the liquid component adjusted to BrixlO is adjusted to 1 L. Ethanol is added so that the concentration becomes 75% by volume, and the mixture is centrifuged at 8000 rpm and lOmin to collect a fraction insoluble in an organic solvent (ethanol). The obtained fraction is lyophilized. 11.8 g of a freeze-dried product was obtained.

Experiment 2

From the barley shochu distillation residue obtained in “Manufacturing barley shochu and barley shochu distillation residue”, a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue is centrifuged at 8000 rpm and lOmin to obtain a liquid component. After adjusting the liquid component to BrixlO, 1 ml of the liquid component adjusted to BrixlO is stored in a volume of 500 ml. The fraction not adsorbed on the ion exchange resin was fractionated through a column packed with Amberlite IRC76 (weakly acidic cation exchange resin) manufactured by Organo Co., Ltd., and the obtained fraction was converted to Brix60. After the adjustment, ethanol was added to a final concentration of 75% by volume, and the mixture was centrifuged under the conditions of 8000ι · ρπι and lOmin, and the fraction insoluble in the organic solvent (ethanol) was collected. Was subjected to lyophilization to obtain 7. lg of lyophilized product.

Experiment 3

From the barley shochu distillation residue obtained in “Manufacturing barley shochu and barley shochu distillation residue”, a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm and lOmin to obtain a liquid component. After adjusting the obtained liquid component to Brixl O, 1 ml of the liquid component adjusted to BrixlO was added to 500 ml. Through a column packed with Amberlite IRA67 (weakly acidic anion exchange resin) manufactured by Organo Co., Ltd., fractions not adsorbed on the ion exchange resin were collected, and the obtained fractions were combined with Brix60. To After the preparation, ethanol was added to a final concentration of 75% by volume, and the mixture was centrifuged at 8000 rpm and lOmin to collect a fraction insoluble in the organic solvent (ethanol), and the obtained fraction was lyophilized. The freeze-dried product 8.2g was obtained.

Experiment 4

From the barley shochu distillation residue obtained in “Manufacturing barley shochu and barley shochu distillation residue”, a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue is centrifuged under the conditions of 8000 rpm and lOmin to obtain a liquid component. After the liquid component thus obtained is adjusted to BrixlO, 1 L of the liquid component adjusted to BrixlO is added to a 500 ml organopolyester. Pass through a column packed with Amberlite IR120B (Strongly Acidic Cation Exchange Resin) manufactured by Co., Ltd., fractionate that is not adsorbed on the ion exchange resin, and adjust the resulting fraction to Brix60 Add ethanol to a final concentration of 75% by volume, centrifuge at 8000n) m, lOmin, fractionate the fraction insoluble in the organic solvent (ethanol), and freeze-dry the obtained fraction To give 7.6 g of lyophilized product.

Experiment 5

From the barley shochu distillation residue obtained in “Manufacturing barley shochu and barley shochu distillation residue”, a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue is centrifuged under the conditions of 8000 rpm and lOmin to obtain a liquid component. After the liquid component thus obtained is adjusted to BrixlO, 1 L of the liquid component adjusted to BrixlO is added to a 500 ml organopolyester. Pass through a column packed with Amberlite 200CT (Strongly Acidic Cation Exchange Resin), and collect fractions not adsorbed on the ion exchange resin, and adjust the resulting fractions to Brix60. Add ethanol to a final concentration of 75% by volume, centrifuge at 8000 rpm and lOmin to collect the fraction insoluble in the organic solvent (ethanol), and subject the resulting fraction to lyophilization. As a result, 5.5 g of a lyophilized product was obtained.

Experiment 6

From the barley shochu distillation residue obtained in “Manufacturing barley shochu and barley shochu distillation residue”, a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley ware 酎 Centrifugation was performed at 8000 rpm and lOmin to obtain a liquid component. After adjusting the resulting liquid component to BrixlO, 1 L of the liquid component adjusted to BrixlO was added to 500 ml capacity Ampo Co., Ltd. Pass through a column filled with Ilite IRA402BL (the strongest basic anion exchange resin) to fractionate the non-adsorbed fraction on the ion exchange resin, adjust the resulting fraction to Brix60, and obtain a final concentration of 75 Ethanol is added so that the volume% is reached, and the mixture is centrifuged at 8000 n) m and lOmin to fractionate the fraction insoluble in the organic solvent (ethanol), and the resulting fraction is subjected to lyophilization. As a result, 3.2 g of a lyophilized product was obtained.

Experiment 7

From the barley shochu distillation residue obtained in “Manufacturing barley shochu and barley shochu distillation residue”, a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue is centrifuged under the conditions of 8000 rpm and lOmin to obtain a liquid component. After adjusting the liquid component to BrixlO, 1 L of the liquid component adjusted to BrixlO Mixed bed ion exchange resin obtained by mixing Amberlite IRC76 (weakly acidic cation exchange resin) and 150 ml capacity Organolite IRA67 (weakly basic anion exchange resin) The non-adsorbed fraction was fractionated into the mixed bed ion exchange resin through a column packed with, and the resulting fraction was adjusted to Brix 60, and ethanol was added to a final concentration of 75% by volume, Centrifugation was performed at 8000 i "pm and lOmin to fractionate the fraction insoluble in the organic solvent (ethanol), and the resulting fraction was freeze-dried to obtain 2.5 g of a lyophilized product. It was.

[Measurement of NK cell activity]

For the fractions (lyophilized product) insoluble in each organic solvent obtained in Experiments 1 to 7, NK cell activity (ie, activation effect on NK cells) was performed in the same manner as described in the Examples below. ) Was measured.

The following facts were found from the measurement results of NK cell activity exhibited by NK cells cultured by adding the insoluble fraction (lyophilized product) to each organic solvent obtained in Experiments 1 to 7. That is, it was obtained in Experiment 1 to Experiment 7 compared to the control in which no fraction insoluble in organic solvent was added. The NK cell activity of all NK cells cultured with each of the fractions (lyophilized product) insoluble in organic solvent added increased, and the cytotoxic activity against K562 cells increased. Among them, the NK cell activity exhibited by the NK cells cultured with the fraction (lyophilized product) insoluble in the organic solvent obtained in Experiment 5 was the highest. On the other hand, the NK cell activity exhibited by the NK cells cultured with the insoluble fraction (lyophilized product) added to the organic solvent obtained in Experiment 1 was the lowest.

Next, the present inventors obtain a liquid component by solid-liquid separation of the barley shochu distillation residual liquid by-produced in the production of shochu using barley as a raw material, and add an organic solvent (preferably ethanol) to the liquid component. In view of the fact that polysaccharides are included as one of the main components in the fraction insoluble in the organic solvent fractionated, the polysaccharides contained in the fraction insoluble in the organic solvent activate M cells. Further studies were conducted through experiments to clarify whether or not they are involved in the process. That is, the following experiments were conducted for the purpose of removing amino acids, peptides, proteins, organic acids, polyphenols derived from barley, etc., which are components other than polysaccharides contained in the fraction insoluble in the organic solvent. . Specifically, the liquid content obtained by solid-liquid separation of the barley shochu distillation residue was synthesized for the purpose of removing polyphenol derived from barley contained in the liquid content of the barley shochu distillation residue. A fraction that is not adsorbed to the synthetic adsorbent is separated by an adsorption separation process using an adsorbent, and then the amino acids and peptides contained in the fraction that is not adsorbed to the synthesized adsorbent. In order to remove proteins, proteins and organic acids, the ion-exchange resin is obtained by subjecting the non-adsorbed fraction to the synthetic adsorbent to ion exchange treatment using various ion-exchange resins. Individual fractions of non-adsorbed fractions were obtained, and fractions insoluble in the organic solvent were fractionated by adding an organic solvent to each of the non-adsorbed fractions obtained in the ion-exchange resin. Fractions insoluble in organic solvents The following experiments were conducted in order to determine with the activating effects such as. Experiment 8

Barley shochu distillation residue obtained in “Manufacture of barley shochu and barley shochu distillation residue” above From the above, a fraction insoluble in an organic solvent was collected by the method shown below. That is, the barley shochu distillation residue is centrifuged under the conditions of 8000 rpm and lOmin to obtain a liquid component, the Brix of the obtained liquid component is adjusted to 10, and the liquid component adjusted to BrixlO is adjusted to 1 L. Ethanol is added so that the concentration is 75% by volume, and the mixture is centrifuged at 8000 rpm and lOmin to collect a fraction insoluble in the organic solvent (ethanol), and the resulting fraction is freeze-dried. As a result, 11.8 g of a freeze-dried product was obtained.

Experiment 9

From the barley shochu distillation residue obtained in “Manufacturing barley shochu and barley shochu distillation residue”, a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm and lOmin to obtain a liquid component, and the resulting liquid component was packed with a synthetic adsorbent Amberlite XAD-16 manufactured by Organo Corporation. The fraction that was not adsorbed to the synthetic adsorbent consisting of a flow-through solution exhibiting non-adsorbability with respect to the synthetic adsorbent of the column was fractionated. The obtained fraction was adjusted to BrixlO, and 1 L of the fraction adjusted to BrixlO was passed through a column packed with 500 ml of Amberlite IRC76 (weakly acidic cation exchange resin) manufactured by Organo Corporation. The fraction not adsorbed on the ion exchange resin is collected, and the resulting fraction is adjusted to Brix60. Ethanol is added to a final concentration of 75% by volume, followed by centrifugal separation under the conditions of 8000 rpm and lOmin. A fraction insoluble in an organic solvent (ethanol) was collected, and the obtained fraction was freeze-dried to obtain 5.3 g of a lyophilized product.

Experiment 1 0

From the barley shochu distillation residue obtained in “Manufacturing barley shochu and barley shochu distillation residue”, a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm and lOmin to obtain a liquid component, and the resulting liquid component was placed in a column packed with Synthetic Adsorbent Amperai® XAD-16 manufactured by Organo Corporation. The fraction adsorbed on the synthetic adsorbent consisting of a flow-through liquid exhibiting non-adsorbability to the synthetic adsorbent of the column was fractionated by subjecting it to an adhesion separation treatment. The obtained fraction Adjust to BrixlO, and pass 1 L of this adjusted fraction to BrixlO through a column packed with 500 ml of Amberlite IRA67 (weakly acidic anion exchange resin) manufactured by Organo Corp. After the fraction was adjusted to Brix60, ethanol was added to a final concentration of 75% by volume, and the mixture was centrifuged at 8000 rpm and lOmin to obtain the organic solvent (ethanol). The insoluble fraction was collected, and the obtained fraction was freeze-dried to obtain 5.9 g of a freeze-dried product.

Experiment 1 1

The barley shochu distillation residue obtained in the above-mentioned “Production of barley shochu and barley shochu distillation residue” fractions insoluble in an organic solvent were collected by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 kg, lOmin to obtain a liquid component, and the obtained liquid component was applied to a column packed with a synthetic adsorbent Amberlite XAD-16 manufactured by Organo Corporation. Through the adsorption separation process, fractions that were not adsorbed to the synthetic adsorbent consisting of a flow-through solution exhibiting non-adsorbability to the synthetic adsorbent of the column were fractionated. The obtained fraction was adjusted to BrixlO, and 1 L of the fraction adjusted to BrixlO was passed through a column filled with 500 ml of Amberlite IR120B (strongly acidic cation exchange resin) manufactured by Organo Corporation. Fraction that is not adsorbed on the ion exchange resin is collected, and the resulting fraction is adjusted to Brix 60. Ethanol is added to a final concentration of 75% by volume, followed by centrifugation at 8000 rpm and lOmin. A fraction insoluble in an organic solvent (ethanol) was collected, and the obtained fraction was freeze-dried to obtain 5.6 g of a freeze-dried product.

Experiment 1 2

From the barley shochu distillation residue obtained in “Manufacturing barley shochu and barley shochu distillation residue”, a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm and lOmin to obtain a liquid component, and the resulting liquid component was applied to a column filled with synthetic adsorbent Amberlite XAD-16 manufactured by Organo Corporation. Through the adsorption separation process, fractions that were not adsorbed to the synthetic adsorbent consisting of a flow-through solution exhibiting non-adsorbability to the synthetic adsorbent of the column were fractionated. The obtained fraction The fraction 1 L adjusted to BrixlO was passed through a column filled with 500 ml of Amberlite 200CT (strongly acidic cation exchange resin) manufactured by Organo Corp., and the fraction not adsorbed to the ion exchange resin. After fractionation, the fraction obtained was adjusted to Brix 60, ethanol was added to a final concentration of 75% by volume, and the mixture was centrifuged at 8000 rpm and lOmin, and insoluble in the organic solvent (ethanol). The fraction was collected, and the obtained fraction was freeze-dried to obtain 4.1 g of a freeze-dried product.

Experiment 1 3

From the barley shochu distillation residue obtained in “Manufacturing barley shochu and barley shochu distillation residue”, a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue was centrifuged under the conditions of 8000 n> m and lOmin to obtain a liquid, and the resulting liquid was filled with a synthetic adsorbent Amberlite XAD-16 manufactured by Organo Corporation. By passing through a column and subjecting to an adsorption separation treatment, a fraction that was not adsorbed to the synthetic adsorbent consisting of a flow-through liquid that showed non-adsorbability to the synthetic adsorbent of the column was fractionated. The obtained fraction was adjusted to BrixlO, and 1 L of the fraction adjusted to BrixlO was passed through a column filled with 500 ml of Amberlite IRA402BL (strongest basic anion exchange resin) manufactured by Organo Corp. Fraction that is not adsorbed on the exchange resin, the resulting fraction is adjusted to Brix60, ethanol is added to a final concentration of 75% by volume, and the organic material is centrifuged at 8000 rpm and lOmin. The fraction insoluble in the solvent (ethanol) was collected, and the obtained fraction was freeze-dried to obtain 2.3 g of a freeze-dried product.

Experiment 1 4

From the barley shochu distillation residue obtained in “Manufacturing barley shochu and barley shochu distillation residue”, a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm and lOmin to obtain a liquid component, and the resulting liquid component was applied to a column filled with synthetic adsorbent Amberlite XAD-16 manufactured by Organo Corporation. Through the adsorption separation process, fractions that were not adsorbed to the synthetic adsorbent consisting of a flow-through solution exhibiting non-adsorbability to the synthetic adsorbent of the column were fractionated. The obtained fraction The fraction 1 L adjusted to Br ixlO was adjusted to 350 ml of Amberlite IRC76 (weakly acidic cation exchange resin) by Organo Co., Ltd. and 150 ml of Amberlite IRA67 (organo Co., Ltd.) A weakly basic anion exchange resin) is passed through a column packed with a mixed bed ion exchange resin, and a fraction not adsorbed on the mixed bed ion exchange resin is collected. After adjustment to Brix60, ethanol was added to a final concentration of 75 volumes, and the mixture was centrifuged under the conditions of 8000 rpiii, lOmin, and the fraction insoluble in the organic solvent (ethanol) was collected. By subjecting the portion to freeze-drying, 1.9 g of a freeze-dried product was obtained.

[Measurement of NK cell activity]

For each of the fractions (lyophilized product) insoluble in the organic solvent obtained in Experiments 8 to 14, the NK cell activity (ie, the activation action on M cells) was performed in the same manner as described in the Examples below. ) Was measured.

The following facts were found from the measurement results of NK cell activity exhibited by NK cells cultured with the insoluble fraction (lyophilized product) added to each organic solvent obtained in Experiment 8 to Experiment 14. That is, compared with the control in which no fraction insoluble in any organic solvent was added, each of the fractions (lyophilized product) insoluble in the organic solvent obtained in Experiment 8 to Experiment 14 was added and cultured. All NK cells had increased NK cell activity and increased cytotoxic activity against K562 cells. Among them, the NK cell activity exhibited by the NK cells cultured by adding the insoluble fraction (lyophilized product) to the organic solvent obtained in Experiment 12 was the highest. On the other hand, the NK cell activity exhibited by the NK cells cultured with the fraction (lyophilized product) insoluble in the organic solvent obtained in Experiment 8 was the lowest.

[Measurement of polysaccharide content]

(Measurement of total content of arabinose and xylose derived from polysaccharides)

For the fractions (lyophilized product) insoluble in each organic solvent obtained in Experiment 1 to Experiment 14, the polysaccharide content and the total content of arabinose and xylose derived from the polysaccharide were measured by the following methods. That is, the fraction insoluble in each organic solvent obtained in Experiment 1 to Experiment 14 (lyophilized product) was dissolved by adding 0.5 ml of ion-exchanged water to 0.05 g, and 200 l of concentrated hydrochloric acid was added thereto to obtain 95 ° C. C. Hydrolysis was performed for 4 hours, filtered through a 0.80 m membrane filter to obtain a filtrate, and the obtained filtrate was injected into a high performance liquid chromatograph, and obtained in Experiment 1 to Experiment 14. The content of the polysaccharide contained in the fraction insoluble in each organic solvent (lyophilized product) and the total content of arabinose and xylose derived from the polysaccharide were determined. For high-speed liquid chromatographic analysis, Waters600 manufactured by Waters is used, a differential refractometer RI-71 manufactured by Showa Denko KK is used as the detector, and the column is Aminex HPX-87H (300 mm X 7.8 mm) manufactured by BioRad. It was used. The column temperature was 60 ° C, the mobile phase was 5mM sulfuric acid, the flow rate was 0.5ml / min, and the sample injection volume was 20 ^ 1.

The following facts were found from the measurement results of the polysaccharide content of the fractions insoluble in each organic solvent obtained in Experiments 1 to 14 and the measurement results of the total content of arabinose and xylose derived from the polysaccharide. That is, the polysaccharide content of the fraction insoluble in the organic solvent (lyophilized product) obtained in Experiment 5 and Experiment 12 and the total content of arabinose and xylose derived from the polysaccharide showed particularly high values. On the other hand, the polysaccharide content of the fraction insoluble in the organic solvent (lyophilized product) obtained in Experiment 1 and Experiment 8 and the total content of alapinose and xylose derived from the polysaccharide showed the lowest values.

Furthermore, the M cell activity exhibited by NK cells cultured separately by adding each of the fractions (lyophilized product) insoluble in the organic solvent obtained in Experiment 1 to Experiment 14 is the fraction insoluble in the organic solvent. It was revealed that the content was increased in proportion to the total content of arabinose and xylose derived from the polysaccharide contained in the minute. The correlation coefficient between the NK cell activity and the total content of arabinose and xylose derived from polysaccharide was found to be 0 · 961. From the above results, the NK cell activation action of the fractions (lyophilized product) insoluble in each organic solvent obtained in Experiment 1 to Experiment 14 is contained in the fraction insoluble in the organic solvent, arabinose and It was found that the possibility of being derived from a polysaccharide mainly composed of xylose is very high. [Measurement of molecular weight distribution]

In order to clarify the molecular weight of polysaccharides mainly composed of alapinose and xylose contained in the fractions (lyophilized product) insoluble in each organic solvent obtained in Experiment 1 to Experiment 14, respectively. The molecular weight distribution of the fraction insoluble in the organic solvent was measured.

Specifically, Shodex standard P-82 (molecular weight 1300 to 1660000) and maltotriose (molecular weight 504) manufactured by Showa Denko Co., Ltd. were separately dissolved in O. lmol / L sodium nitrate solution. A standard solution of 0.05 W / V agricultural power was obtained, and the standard solution was injected into a high performance liquid chromatograph to prepare a calibration curve. Next, prepare 0.02 g of the fraction insoluble in each organic solvent (lyophilized product) obtained in Experiment 1 to Experiment 10, and add 10 ml of O. lmol / L sodium nitrate solution at room temperature.晚 After standing, filter through a 0.45 m membrane filter to obtain a filtrate, inject the filtrate into a high-speed liquid chromatograph, and make a 480-meter system manufactured by System Instruments Co., Ltd. The molecular weight distribution was determined using the GPC program. For high-performance liquid chromatographic analysis, Shodex GPC SYSTEM-21 made by Showa Denko Co., Ltd. is used, and a differential refractometer RI-71S made by Showa Denko Co., Ltd. is used as the detector. Two (φ7.8 mm × 300 mm) were connected and used. The column temperature was 40 ° C, O. lmol / L sodium nitrate solution was used as the mobile phase, the flow rate was 1.0 ml / min, and the sample injection volume was 100 1.

As a result of measuring the molecular weight distribution of the fractions insoluble in the organic solvents obtained in Experiments 1 to 14 by the above method, the arabinose and xylose derived from polysaccharides contained in the fractions insoluble in the organic solvents. It was found that the higher the total content of, the higher the fraction with a molecular weight distribution in the molecular weight range of 3000 to 100,000. For these reasons, the present inventors have the ability to activate NK rice field, comprising arabinose and xylose as the main constituents contained in the fraction insoluble in the organic solvent fractionated from the barley shochu distillation residue. Polysaccharides with a major range of 3000 to 100,000 Presuming that it has a molecular weight distribution, we examined concentration and purification using ultrafiltration.

[Concentration purification by ultrafiltration]

As a result of measuring the molecular weight distribution of the fractions insoluble in each organic solvent obtained in Experiment 1 to Experiment 14 by the above method, the fraction insoluble in each organic solvent obtained in Experiment 5 and Experiment 12 (lyophilized product). However, it was found that the fraction having a molecular weight distribution in the molecular weight range of 3000 to 100,000 was very high, and the total content of arabinose and xylose derived from polysaccharides was also very high. Therefore, concentration and purification through ultrafiltration was performed according to the following procedure.

That is, first, the barley shochu distillation residue obtained in the above-mentioned “Manufacture of barley shochu and barley shochu distillation residue” was centrifuged at 8000 rpm and lOmin to obtain a liquid component, and the liquid obtained After adjusting the volume to Brixl O, 1 L of the liquid volume adjusted to Brixl O was passed through a column packed with 500 ml of Amberlite 200CT (strongly acidic cation exchange resin) manufactured by Organo Corporation. Fraction that is not adsorbed on the exchange resin is obtained, and the obtained fraction is subjected to concentration treatment with an ultrafiltration membrane UFP-30-E-4MA (fraction molecular weight 30,000) manufactured by A / G Technology. After the concentrate is adjusted to Brix20, ethanol is added to a final concentration of 75% by volume, and the mixture is centrifuged at 8000 rpm and lOmin, and is insoluble in the organic solvent (ethanol). Fractionate the fraction and freeze-dry the resulting fraction to obtain 2.5 g of lyophilized product. .

For the obtained fraction insoluble in organic solvent (lyophilized product), the total content of polysaccharide-derived arabinose and xylose was measured by the method described in “Measurement of Polysaccharide Content” above. As a result, it was found that the total content of polysaccharide-derived arabinose and xylose increased to about 54.4 wt. Further, when the molecular weight distribution of the fraction insoluble in the organic solvent (lyophilized product) was measured by the method described in “Measurement of molecular weight distribution” above, the molecular weight distribution of the fraction insoluble in the organic solvent was 100,000 to 5%, 30,000 to 100,000 are 18%, 10,000 to 30,000 are 23%, 3,000 to 10,000 are 31%, 1,000 From 3,000 to 11%, 500 to 1,000 was found to be 3%, and 500 or less was found to be 9%. From the results of the chromatogram obtained, it was revealed that the chromatogram of the fraction insoluble in the organic solvent had the highest peak in the molecular weight range of 3,000 to 10,000.

Therefore, the NK cell activity of NK cells cultured by adding a fraction insoluble in the organic solvent was measured by the method described in the Examples below. As a result, the NK cell activation action of the fraction insoluble in the organic solvent was extremely strong, and was comparable to the positive control IL-2.

From the above experimental results, the NK cell activation action of the fraction insoluble in the organic solvent fractionated from the liquid fraction of the barley shochu distillation residue is contained in the fraction insoluble in the organic solvent, arabinose and xylose. A composition containing a significant amount of such polysaccharides mainly composed of arabinose and xylose is derived from a polysaccharide mainly composed of arabinose. The liquid is subjected to solid-liquid separation to obtain a liquid component, which is subjected to an ion exchange treatment using an ion exchange resin to fractionate a fraction not adsorbed on the ion exchange resin, and the resulting ion exchange is obtained. A fraction that is not adsorbed to the resin is subjected to a concentration treatment by ultrafiltration using an ultrafiltration membrane to obtain a concentrate, and an organic solvent is added to the concentrate to obtain a fraction insoluble in the organic solvent. It was found that it can be sorted as . As a result of component analysis, the fraction insoluble in the organic solvent contains about 84.1% by weight of polysaccharide, about 4.2% by weight of crude protein, and about 0.3% by weight of organic acid. About 1.2% by weight The fraction was found to contain polysaccharides as the main component. Next, the barley shochu distillation residue obtained in “Manufacture of barley shochu and barley shochu distillation residue” above was used. The liquid is centrifuged under the conditions of 8000 rpm and lOmin to obtain a liquid component, and the obtained liquid component is passed through a column filled with a synthetic adsorbent Amberlite XAD-16 manufactured by Organo Corporation for adsorption separation treatment. As a result, fractions that had not been adsorbed to the synthetic adsorbent consisting of a flow-through liquid exhibiting non-adsorbability to the synthetic adsorbent of the column were fractionated. Obtained fraction Was adjusted to Brixl O, and then 1 L of the fraction adjusted to BrixlO was passed through a column filled with 500 ml of Amberlite 200CT (strongly acidic cation exchange resin) manufactured by Organo Corporation. Fractionated non-adsorbed fraction is subjected to concentration treatment with ultrafiltration membrane UFP-30-E-4MA (fractional molecular weight 30,000) manufactured by A / G Technology. The resulting concentrate is adjusted to Brix20, ethanol is added to a final concentration of 75% by volume, and the mixture is centrifuged at 8000 n) m and lOmin, and the organic solvent (ethanol) is obtained. Fraction-insoluble fraction was collected, and the obtained fraction was freeze-dried to obtain 1.3 g of a freeze-dried product.

For the obtained fraction insoluble in organic solvent (lyophilized product), the total content of polysaccharide-derived arabinose and xylose was measured by the method described in “Measurement of Polysaccharide Content” above. As a result, it was found that the total content of arabinose and xylose derived from polysaccharides increased to about 58.6% by weight. In addition, when the molecular weight distribution of the fraction (lyophilized product) insoluble in the obtained organic solvent was measured by the method described in “Measurement of molecular weight distribution” above, the fraction insoluble in the organic solvent was obtained. The molecular weight distribution is 11% for 100,000 or more, 29% for 30,000 to 100,000, 24% for 10,000 to 30,000, 21% for 3,000 to 10,000, 1,000 to 3 , 000 was found to be 6%, 500 to 1,000 was 2%, and 500 or less was 7%. From the results of the obtained chromatogram, it was revealed that the chromatogram of the fraction insoluble in the organic solvent had the highest peak in the molecular weight range of 30,000 to 100,000. Therefore, the NK cell activity of NK cells cultured by adding a fraction insoluble in the organic solvent was measured by the method described in Examples below. As a result, the NK cell activation action of the fraction insoluble in the organic solvent was extremely remarkable, and was comparable to that of IL-2 as a positive control. From the above experimental results, the NK cell activation action of the fraction insoluble in the organic solvent fractionated from the liquid content of the barley shochu distillation residue by the above-described method is the arabinose contained in the fraction insoluble in the organic solvent In addition, it was found to be derived from a polysaccharide mainly composed of xylose. In addition, the main composition of arabinose and xylose in this way A composition containing a significant amount of polysaccharide as an element is obtained by solid-liquid separation of the barley shochu distillation residue produced as a by-product in the production of shochu using barley as a raw material, and this liquid is used as a synthetic adsorbent. A fraction that is non-adsorbed on the synthetic adsorbent is obtained by subjecting it to an adsorption separation treatment, and the obtained fraction is subjected to an ion exchange treatment using an ion exchange resin to obtain a fraction that is not adsorbed on the ion exchange resin. By subjecting the non-adsorbed fraction to the obtained ion exchange resin to a concentration treatment by ultrafiltration using an ultrafiltration membrane to obtain a concentrate, and adding an organic solvent to the concentrate It was found that it can be fractionated as a fraction insoluble in the organic solvent. As a result of component analysis, the fraction insoluble in the organic solvent is about 78.5% by weight of polysaccharide, about 3.3% by weight of crude protein, about 0.2% by weight of organic acid, and free. It was found that the saccharide contained about 1.5% by weight, and the fraction contained polysaccharide as a main component.

Incidentally, the hemicellulos described in Patent Document 1 is similar to the composition having the action of activating NK cells of the present invention in that the main constituents are xylose and arabinose. However, since the hemicellulose has an average molecular weight of 550,000 or 600,000, it is clearly different from the composition having an action of activating NK cells of the present invention.

The immunity enhancing substance described in Patent Document 2 is similar to the composition having an action of activating M cells of the present invention in that the main components are xylose and arabinose. Since the force-enhancing substance has an average molecular weight of 600,000 or 650,000, it is clearly different from the composition having the action of activating NK cells of the present invention.

Patent Document 3 describes that an aqueous extract of sake lees produced as a by-product in sake production produced using rice as a raw material only through fermentation has an NK cell activation effect. However, Patent Document 3 does not describe the component composition of the aqueous extract at all, and does not specifically describe the component involved in the NK cell activation action.

Patent Document 4 describes an aqueous extract of rice bran from a fraction having a molecular weight of about 30,000 to 40,000. It is described that the composition for immunostimulation comprising the extract as described above has mitogenic activity and excellent NK cell activation action. However, Patent Document 4 does not describe the component composition of the immunostimulatory composition at all, and does not specifically describe the component involved in the immunostimulatory action. Patent Document 5 discloses that hemicellulose obtained by alkali extraction of the residue obtained by removing starch and protein from corn hull, which is marketed under the name of “Cel Ace”, is treated with xylanase. In the co 1 on26 tumor-bearing mice administered with the partial degradation product of hemicellulose, NK cell activity was significantly increased, and cytokines (IL-2 and iNF) were obtained. -r) It is described that the production ability is also significantly increased. Non-Patent Document 1 includes “Celace” (partially decomposed hemicellulose obtained from corn hulls) described in Patent Document 5 containing xylose, arabinose, uronic acid, galactose, and glucose. It is described that it is. Therefore, Patent Document 5 and Non-Patent Document 1 relate to the same “partially decomposed product of hemicellulose obtained from corn hulls”, that is, commercially available “Cell Ace”. On the other hand, the composition having an action of activating NK cells of the present invention produces barley koji and steamed barley using barley as a raw material, and the starch contained in the obtained barley koji and steamed barley is added to the barley koji. Saccharified, and then subjected to alcoholic fermentation with yeast to obtain shochu-ripened mash, and the resulting shochu-ripened mash is subjected to distillation to produce shochu, that is, a by-product distillation residue, that is, barley shochu mash distillation residue It is obtained from the liquid. That is, the source for obtaining the composition having the effect of activating NK cells of the present invention is barley shochu distillation residue. On the other hand, the acquisition source of “cell ace”, a partial degradation product of hemicellulose described in Patent Document 5, is corn hull, that is, hull of corn kernel that is removed when corn kernel is refined. In this way, both sources are completely different. Moreover, the composition having the action of activating NK cells of the present invention is obtained by a production method completely different from the method for producing a partially decomposed product of hemicellulose described in Patent Document 5. And the NK cells of the present invention The composition having an activating effect contains arabinose, xylose and glucose, but does not substantially contain uronic acid. On the other hand, the partially decomposed product of hemicellulose described in Patent Document 5 contains a relatively large amount of uronic acid. In this respect both

, Completely different. Therefore, the composition having the effect of activating the M cells of the present invention is clearly distinguished from the partially decomposed product of hemicellulose (trade name Cellace) obtained from corn hull described in Patent Document 5. Obviously it is different. In Patent Document 5, the average molecular weight of the partially degraded hemicellulose is preferably 20,000 to 200,000, more preferably 20,000 to 100,000, and 20,000 to 40,000. However, the experimental data that led to the determination of the average molecular weight is not described at all, and a specific example of obtaining a partially decomposed product of hemicellulose having such an average molecular weight is as follows. It is not described at all. Therefore, the inventors described in Patent Document 5 that a partially decomposed product of hemicellulose is marketed under the trade name “Cel Ace” (manufactured by Nippon Shokuhin Kako Co., Ltd.). Cellace ”was obtained, and the molecular weight distribution of Cellace was measured by the method described in“ Measurement of molecular weight distribution ”described above. As a result, the molecular weight distribution of Cell Ace is 1% for 1 million or more, 9% for 300,000 to 1 million, 30% for 100,000 to 300,000, 30% for 30,000 to 100,000, and 10,000 to 30,000. 11%, 3000 to 10,000 is 4%, 1000 to 3000 is 2%, 1000 or less is 13%, and the highest peak observed in the chromatogram is in the molecular weight range of 100,000 to 300,000. The average molecular weight (Mw) was found to be 150,000. Therefore, it is understood that the partial degradation product of hemicellulose obtained from the corn hull described in Patent Document 5 falls under the category of “Cell Ace” marketed as described in Patent Document 5. .

On the other hand, as described above, the molecular weight distribution of the composition having the effect of activating the M cells of the present invention is only 5% or 11% of 100,000 or more, and has a molecular weight of 3,000 to 100,000. Components with molecular weight distribution in the range account for 72% or 74% of the total. In addition, the highest peaks observed in the chromatogram are molecular weights of 3,000 to 10,000, respectively. Is present in the molecular weight range of 30,000 to 100,000.

Accordingly, the molecular weight distribution of the composition having the action of stimulating NK cells of the present invention is “cell suspension”, that is, a composition comprising a partial degradation product of hemicellulose obtained from corn hulls. It is completely different from the molecular weight distribution. Therefore, it is clear that the composition having the effect of activating NK cells of the present invention is different from the partial degradation product of hemicellulose described in Patent Document 5.

Patent Document 6 describes a composition having an action to suppress fat dryness from a barley shochu distillation residue. The source of the composition having an action to suppress fatty liver is barley shochu distillate as in the case of the composition having an action to activate NK cells of the present invention. It is different from the action to become. The main component of the composition described in Patent Document 6 has a molecular weight of 3000 or less, and the micelle mouth contained in the composition contains xylose as a main component. It is a distinct distinction from a composition that has the effect of activating cells.

DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The composition having the effect of activating the M cells of the present invention is produced as follows. That is, Step A to obtain a liquid component by solid-liquid separation of the barley shochu distillation residue that is a by-product in the production of distilled liquor using barley, and the obtained liquid component is subjected to an ion exchange treatment using an ion exchange resin. Step B 1 for obtaining a non-adsorbed fraction on the ion exchange resin, and subjecting the obtained non-adsorbed fraction to the ion exchange resin to a concentration treatment by ultrafiltration using an ultrafiltration membrane. It is produced by sequentially performing Step C for obtaining a concentrated solution and Step D for fractionating a fraction insoluble in the organic solvent by adding an organic solvent to the obtained concentrated solution.

In addition, the composition having an action of activating NK cells of the present invention is produced as follows. That is, Step A to obtain a liquid component by solid-liquid separation of a barley shochu distillation residue, which is a by-product in the production of distilled liquor using barley, and the obtained liquid component using a synthetic adsorbent. Step E to obtain a fraction that is not adsorbed to the synthetic adsorbent by subjecting it to an adsorption separation treatment, and the fraction that is not adsorbed to the synthetic adsorbent thus obtained is subjected to an ion exchange treatment using an ion exchange resin. Step B 2 for obtaining a non-adsorbed fraction on the ion exchange resin, and subjecting the obtained non-adsorbed fraction to the ion exchange resin to a concentration treatment by ultrafiltration using an ultrafiltration membrane. Thus, the step C is obtained by sequentially performing the step C for obtaining a concentrated liquid and the step D for fractionating a fraction insoluble in the organic solvent by adding an organic solvent to the obtained concentrated liquid.

Below, the barley shochu distillation residual liquid by-produced in the production of shochu using barley as a raw material and the respective steps used in carrying out the production method of the present invention will be described in detail. The barley shochu distillation residue used in the present invention typically comprises barley koji and steamed barley using barley or polished barley as a raw material, and the starch contained in the barley koji and steamed barley obtained is When saccharifying with barley koji, subjecting them to alcoholic fermentation with yeast to obtain shochu-ripened mash, and distilling the obtained shochu-ripened mash with a single distillation apparatus such as vacuum distillation or atmospheric distillation Means a by-product as a distillation residue, that is, a distillation residue of barley shochu.

In the present invention, when the barley shochu distillation residue is obtained, the barley koji used for the production of barley shochu may be produced under the koji-making conditions used in normal barley shochu production.

As a koji mold to be used, the Aspergillus kawachi i generally used in the production of barley shochu is preferable. Alternatively, Aspergi lus genus strains such as Aspergillus awamori used in awamori manufacture and Aspergi lus oryzae used in sake production and the like can also be used. In addition, various yeasts for brewing shochu can be used as the yeast used in the production of barley shochu.

In the present invention, the step A for obtaining a liquid component by solid-liquid separation of the barley shochu distillation residue obtained in the distillation step in the production of barley shochu is made from raw barley or barley koji derived from the barley shochu distillation residue. The purpose is to remove SS components such as water-insoluble fermentation residues. The solid-liquid separation in step A can be performed by screw press or -Preliminary separation is performed by a solid-liquid separation method using the Ler press method or using a filter-press type solid-liquid separator, and then using a centrifuge, a diatomaceous earth filter, a ceramic filter, or a filter-press The solid-liquid separation process that can be performed according to the present invention is performed.

The step E of obtaining a non-adsorbed fraction on the synthetic adsorbent by subjecting the liquid content of the barley shochu distillation residue obtained in the step A to an adsorption separation process using a synthetic adsorbent is a barley shochu distillation The purpose is to remove components such as polyphenol contained in the liquid content of the residual liquid. As the synthetic adsorbent used in Step E, an aromatic, aromatic modified, or methacrylic synthetic adsorbent can be used. Preferable specific examples of the synthetic adsorbent used in Step E include Amberlay XAD-4, Amberlay XAD-16, Amberlite XAD-1180 and Amperlite XAD-2000 manufactured by Organo Corporation. Aromatic (or styrene) synthetic adsorbents such as Sepabeads SP850 and Diaion HP20 manufactured by Mitsubishi Chemical Co., Ltd. Amperlite XAD-7 manufactured by Organo Co., Ltd. and Diamond manufactured by Mitsubishi Chemical Co., Ltd. Examples include methacrylic (or acrylic) synthetic adsorbents such as Ion HP2MG. In addition to these, aromatic modified synthetic adsorbents such as Sepapies SP207 manufactured by Mitsubishi Chemical Corporation may be used in some cases.

The step B 1 of obtaining a non-adsorbed fraction on the ion exchange resin by subjecting the liquid content of the barley shochu distillation residue obtained in the step A to an ion exchange treatment using an ion exchange resin In step B2, in which the fraction adsorbed on the synthetic adsorbent obtained in E is subjected to an ion exchange treatment using an ion exchange resin to obtain a fraction not adsorbed on the ion exchange resin, the liquid fraction or Components other than the above-mentioned polysaccharides contained in the non-adsorbed fraction of the synthetic adsorbent, that is, amino acids, peptides, proteins, organic acids, or barley-derived polyphenols are removed using an ion exchange resin. This is done for the purpose. The ion exchange resin used in Step B1 and Step B2 includes a cation exchange resin, an anion exchange resin, or a mixed bed ion in which both are mixed. Exchange resins can be used. In the case of a cation exchange resin, either a strong acid cation exchange resin or a weak acid cation exchange resin can be used. In the case of an anion exchange resin, a strong base anion exchange resin and a weak base are used. Any sex anion exchange resin can be used. In the case of a mixed bed ion exchange resin, the above-described cation exchange resin and anion exchange resin can be freely combined and used at a predetermined ratio. Specific examples of such ion exchange resins include strong acid cation exchange resins such as Amberlite 200CT and Amberlite IR120B manufactured by Organo Corp., weak acid cation exchange resins such as Amberly MRC76, and amperite. Strongest basic anion exchange resin such as IRA402BL, weak basic anion exchange measurement such as Amberlite IRA67, or both the weak acid cation exchange resin and the weak basic anion exchange resin at a predetermined ratio A mixed bed type ion exchange resin obtained by mixing can be used. Among these, from the viewpoint of removing amino acids and peptides contained in the liquid or the fraction not adsorbed to the synthetic adsorbent, the weak acid cation exchange resin Amberlite IRC76 and the above-mentioned weak It is particularly preferable to use a mixed bed type ion exchange resin obtained by mixing both basic anion exchange resin ampere lye IRA67 at a predetermined ratio, or Amberlite 200CT, a strongly acidic cation exchange resin. preferable.

In the step C of obtaining a concentrated solution by subjecting the non-adsorbed fraction obtained in the step B 1 and the step B 2 to a concentration treatment by ultrafiltration using an ultrafiltration membrane, The purpose is to concentrate a polysaccharide mainly composed of arabinose and xylose, which are components involved in NK cell activation, using an ultrafiltration membrane. As the ultrafiltration membrane used in Step C, any membrane material and membrane module type can be used, and the molecular weight cut-off is preferably 3000 or more, particularly preferably 10,000 to 50,000. Can be used. Furthermore, by increasing the concentration rate of ultrafiltration to preferably 2 times or more, particularly preferably 5 times or more, a polysaccharide composed of arabinose and xylose. Thus, a composition having an effect of activating the M cells of the present invention containing a significant amount of can be obtained.

In step D, in which an organic solvent is added to the concentrate obtained in step C to fractionate a fraction insoluble in the organic solvent, an appropriate organic solvent is added until a predetermined final concentration is reached. In this case, ethanol is optimal as the organic solvent, but the organic solvent is not limited to this. The final concentration of the organic solvent affects the production efficiency of the components, and the optimum final concentration of the organic solvent is preferably 5 volume% or more, more preferably 30 to 75 volume%.

The composition having the action of activating NK cells of the present invention obtained as described above is obviously of course administered directly as it is, as is apparent from the results of test examples in the examples described later. The desired NK cell activation effect can also be obtained by dispersing the composition in a suitable carrier such as physiological saline and administering it to a patient by intravenous injection. The present invention also provides a composition containing NK cells activated with the composition having an action of activating NK cells (hereinafter referred to as an activated NK cell-containing composition). The activated NK cell-containing composition collects blood from an affected person such as leukemia, separates the obtained blood into lymphocytes and serum composed of immune cells such as NK cells, and the lymphocytes are separated into the NK cells. It is obtained by mixing a composition having an action of activating and subjecting it to culturing for a predetermined period. The activated NK cell-containing composition obtained in this way is washed with a drip physiology Can be suspended in saline and administered via intravenous infusion. Such activated NK cell-containing composition has the effect of necrotizing K562 cells. Therefore, the activated NK cell-containing composition can be used as a therapeutic agent for (myeloid) leukemia.

EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

Example 1

Barley shochu distillation residue obtained in “Manufacture of barley shochu and barley shochu distillation residue” above Centrifugation was performed at 8000 rpm and lOmin to obtain a liquid component. After the obtained liquid component was adjusted to Br ixlO, 1 L of the liquid component adjusted to Br ixlO was added to a 500 ml volume of Organo Corporation Pass through a column packed with Wright 200CT (strongly acidic cation exchange resin) to obtain a fraction that is not adsorbed to the ion exchange resin, and the fraction that is not adsorbed to the obtained ion exchange resin Concentrated with UFP-30-E-4MA (fractional molecular weight 30,000) manufactured by the company to obtain a concentrated solution. The resulting concentrated solution is adjusted to Brix20, and the final concentration is 75 volumes. Ethanol was added so that the concentration of the lyophilized product was 10%. Centrifugation was performed at 8000 rpm and lOmin, and a fraction insoluble in the organic solvent (ethanol) was collected. 2. 5g was obtained. The lyophilized product was powdered to obtain a grayish white, tasteless and odorless composition.

Example 2

The barley shochu distillation residue obtained in “Manufacture of barley shochu and barley shochu distillation residue” was centrifuged at 8000 rpm and lOmin to obtain a liquid content of the barley shochu distillation residue. By passing through a column filled with a synthetic adsorbent amperite XAD-16 manufactured by Co., Ltd. and subjecting it to an adsorption separation treatment, the above-mentioned flow-through liquid that exhibits non-adsorbability to the synthetic adsorbent of the column is used. The fraction not adsorbed on the synthetic adsorbent was collected. The fraction that was not adsorbed to the resulting synthetic adsorbent was adjusted to Br ixlO, and 1 L of the fraction adjusted to Br ixlO was added to 500 ml of Amberlite 200CT (strongly acidic cation exchange resin) manufactured by Organo Corporation. A fraction that is not adsorbed to the ion exchange resin is obtained, and the obtained fraction is filtered with an ultrafiltration membrane UFP-30-E-4MA manufactured by A / G Technology (fractionated molecular weight 3 The resulting concentrate is adjusted to Brix20, ethanol is added to a final concentration of 75% by volume, and the mixture is centrifuged at 8000 rpm and lOmin. A fraction insoluble in the organic solvent (ethanol) was collected, and the obtained fraction was freeze-dried to obtain 1.3 g of a lyophilized product. The freeze-dried product was powdered to obtain an off-white, tasteless and odorless composition.

Comparative Example 1 The barley shochu distillation residue obtained in “Manufacture of barley shochu and barley shochu distillation residue” was centrifuged at 8000 rpm and l Omin to obtain a liquid, and the final concentration was 75% by volume in 1 L of the liquid. Ethanol was added, and the mixture was centrifuged at 8000Π and l Omin to separate the fraction insoluble in the organic solvent (ethanol). The obtained fraction was freeze-dried using a vacuum freeze dryer. To give 11.8 g of lyophilized product. The freeze-dried product was powdered to obtain a grayish white and tasteless and odorless composition.

Test example 1

Examples In order to clarify whether the compositions obtained in Example 2 and Comparative Example 1 have an activating effect on NK cells, the following Test Example 1 was performed.

That is, heparin-added peripheral blood was collected from healthy humans, diluted twice with physiological saline, and the diluted blood was overlaid on Lymphosepar I (manufactured by Immune Biology Laboratories) at room temperature. After centrifugation at 1500ΠΜ for 30 minutes, the plasma was removed using a pipette to obtain a fraction of peripheral blood mononuclear cells (匪 C). Next, the PBMC fraction obtained to obtain a S t emSep NK cell fraction Ri by the subjecting (manufactured by Veritas), the NK cell fraction obtained 5% C0 ₂ atmosphere at 37 ° C for Culture was performed to obtain a NK cell culture solution. The obtained M cell culture solution was centrifuged at 1000 rpm for 5 minutes to collect NK cells. The number of NK cells collected was adjusted to IX cells / ml using RPMI-1640 medium supplemented with 10% FCS to obtain an NK cell suspension. On the other hand, K562 cells (chronic myeloid leukemia cells) obtained by subculture are centrifuged to separate the K562 cells, which are then washed with PBS (Phosphat e-bufered saline). The cells were washed twice and then further washed with RPMI-1640 medium supplemented with 10% FCS to obtain target cells consisting of K562 cells.

Next, for each of the three NK cell suspensions 100 1, the concentration of the fraction insoluble in each organic solvent (lyophilized product) obtained in Example 2 and Comparative Example 1 was previously determined. serial 10 ^ 3 added 11 ^ 11-1640 medium solution prepared use Ite 11 ^ / 1111 was added One not a respectively 11 1, 5¾C0 ₂ atmosphere, 72 hours of culture to NK cell culture medium at 37 ° C for Got. In addition, 10% FCS was added to another NK cell suspension 100 1 as a control. The same treatment was applied to the addition of 11 l of RPMI-1640 medium. Furthermore, as a positive control, 11 1 each of solutions prepared to 1 mg / ml of IL-2 concentration using RPMI-1640 medium with 10% FCS added to another NK cell suspension 100 1 The same process was applied to the above.

Each NK cell culture solution thus obtained was centrifuged at 1000 rpm for 5 minutes to collect NK cells. The number of NK cells collected was adjusted to IX cells / ml using RPMI-1640 medium supplemented with 10% FCS to obtain effect cells.

Next, the target cells adjusted to the optimum concentration were dispensed 100 1 into a 96-well V-bottom plate, and the effector cells were mixed separately. Plate mixing said target cell and said effector cells are, 800 rpm, centrifuged for 2 minutes, 5% C0 ₂ atmosphere, after 4 hours at 37 ° C, each culture 100 1 were taken, respectively, Measure the K562 cell viability using the following formula, using the CeilTiter-Glo Luminescent Cel 1 Vi ab i 1 i Assay Assay kit (Promega), and determine the NK cells of each sample. It was used as an index of activation ability.

K562 cell viability (%) = {(Luminescence of [NK cells + K562 cells treated with each sample]) 1 (Luminescence of NK cells only)} / (Luminescence of K562 cells treated with PBS) X 100

[Evaluation 1]

Table 1 shows the measurement results of NK cell activity against K562 cells. The following facts were found from the results shown in Table 1. That is, NK cells obtained by adding a predetermined amount of the composition obtained in Comparative Example 1 and subjecting it to culture are more than NK cells obtained by adding the predetermined amount of IL-2 and subjecting it to culture. Slightly low NK cell activity was shown. On the other hand, NK cells obtained by adding a predetermined amount of the composition obtained in Example 1 and Example 2 and subjecting them to culture are obtained by adding the predetermined amount of IL-2 and subjecting them to culture. It was revealed that the cells have extremely remarkable NK cell activation ability comparable to that of M cells.

Test example 2

By the way, in afflicted patients such as malignant tumors, the activity of NK cells is reduced. The cytotoxic activity against tumor cells caused by the NK cells is also extremely low. Therefore, the following tests were conducted for the purpose of more practically evaluating the NK cell activation by the fraction insoluble in the organic solvent and the resulting cytotoxic activity against tumor cells.

That is, heparin-added peripheral blood was collected from healthy humans, diluted twice with physiological saline, and the diluted blood was overlaid on Lymphosepar I (manufactured by Immune Biology Laboratories) at room temperature. After centrifugation at 1500 rpm for 30 minutes, plasma was removed using a pipette to obtain a fraction of peripheral blood mononuclear cells (IC). Then, the resulting: PBMC fraction to obtain a StemSep NK cell fraction Ri by the subjecting (manufactured by Veritas), and the NK cell fraction obtained 5% C0 ₂ atmosphere, the culture at 37 ° C for The NK cell culture solution was obtained. The obtained NK cell culture solution was centrifuged at 1000 rpm for 5 minutes to collect NK cells. The number of cells of each collected NK cell was adjusted to IX 10 ⁶ cells / ml using RPMI-1640 medium supplemented with 10% FCS to obtain an NK cell suspension. On the other hand, K562 cells (chronic myeloid leukemia cells) obtained by subculture are centrifuged to separate the K562 cells and washed twice with PBS (Phosphate-buf fered saline). Subsequently, the cells were further washed with RPMI-1640 medium supplemented with CS, to obtain target cells consisting of K562 cells.

Next, the NK cell suspension was dispensed into a 96-well V-bottom plate at a rate of 1001, and the target cells adjusted to the optimum concentration were added at a rate of 100 U. Add each of the solutions prepared to 1 mg / ml using RPMI-1640 medium with 10% FCS added to the concentration of the fraction insoluble in each organic solvent (lyophilized product) obtained in Comparative Example 1. Then, the cells were cultured for 4 hours at 37 ° C. in a 5% CO ₂ atmosphere. Separately, as a control, 100 L of the target cells adjusted to the optimal concentration was added to another M cell suspension 100 1, and then the RPMI-1640 medium with 10% FCS was added to 22 μ1. The same treatment was applied to the material to which the slag was added. Further, as a positive control, the target cell 100 χ 調整 adjusted to the optimal concentration was added to another ΝΚ cell suspension 100 1, and then the RPMI-1640 medium supplemented with 10% FCS was used. IL-2 water prepared to a concentration of lmg / ml The same treatment was applied to the solution to which 22 1 was added. Next, 100 1 of each culture was collected, and the amount of ATP was measured using CelTiter-Glo Luminescent Cell Viability Assay Kit (Promega), and the survival rate of K562 cells was calculated using the following formula. Was used as an index of NK cell activation ability of each sample.

K562 cell viability (%) = {([Luminescence of [NK cells + K562 cells treated in each sample])]

(Luminescence of NK cells only)} / (Luminescence of K562 cells treated with PBS) X 100

[Evaluation 2]

Table 2 shows the measurement results of the cytotoxic activity when the lyophilized product obtained in Comparative Example 1 was added to the mixture of M cells and K562 cells. The following facts were found from the results shown in Table 2. That is, when the lyophilized product obtained in Comparative Example 1 was added to the mixture of NK cells and K562 cells in the predetermined amount, it was clearer than the case where the predetermined amount of IL-2 was added to the mixture. Showed low cytotoxic activity. On the other hand, when the predetermined amount of the lyophilized product obtained in Example 1 and Example 2 was added to the mixture of NK cells and K562 cells, it was comparable to the case where the predetermined amount of IL-2 was added to the mixture. It was found to exhibit extremely strong cytotoxic activity.

That is, when the lyophilized product of Example 1 and Example 2 is added to a mixture of NK cells and K562 cells, it not only significantly activates NK cells but also suppresses the proliferation of K562 cells. From the above, it was revealed that there is an excellent leukemia treatment effect.

Test example 3

In order to clarify the in vivo NK cell activation action of the freeze-dried fraction insoluble in the organic solvent obtained in Example 1 and Example 2, the following test was performed. That is, C57BL / 6 mice (3 weeks old, 10 mice per group) were fed with a diet (control group) that did not contain the composition having the effect of activating NK cells of the present invention, and the organic obtained in Example 1 Diet containing 0.5% and 1.0% of lyophilized fraction insoluble in solvent (test group 1), and lyophilized fraction insoluble in organic solvent obtained in Example 2 of 0. 5% and 1.0% diet (Test group 2) was ingested, and after 5 weeks, the spleens were removed from both groups of mice, the number of cells was counted and NK fine Cell activity was measured as follows.

That is, spleen cells were suspended in C-RMI medium, and cell-labeled with fluorescein isothiocyanate (FITC) -conjugated anti-mouse IL-2 / 3 receptor antibody and phycoerythrin 0PE) -conjugated anti-mouse CD3 antibody, The NK cell fraction was measured by flow cytometry, and the CD3-IL-2 ιδ + fraction was identified as M cells. '

Mouse lymphoma cell line YAC-1 was labeled with ⁵¹ 0 ₄ as target cells, splenocytes: target cells = 100: 1, mixed and suspended for 6 hours, and the radioactivity of ⁵¹ Cr in the supernatant was determined. NK cell activity (%) was calculated from the following formula using a scintillation counter.

NK cell activity (%) = [(experimental release-spontaneous release) / (maximum release-spontaneous release)] X 100

[Evaluation 3]

Table 3 shows the results of measurement of the number of spleen cells, NK cell fraction, and spleen cell-derived NK cell activity. The following facts were found from the results shown in Table 3. That is, test group 1 ingesting a diet containing 0.5% and 1.0% of the composition having the effect of activating NK cells obtained in Example 1, and the organic solvent obtained in Example 2 above. The spleen cell-derived NK cell activity in test group 2 that received diets containing 0.5% and 1.0% lyophilized fractions insoluble in erythrocyte significantly increased compared to the control group, respectively. It has been found. On the other hand, regarding the number of spleen cells and NK cell fraction, there was no significant difference between the three groups consisting of test group 1, test group 2 and control group. From these results, it became clear that the composition having an action of activating NK cells of the present invention activates NK cells even in vivo.

As is clear from the results of Test Example 1 to Test Example 3 above, the M cells of the present invention, which are mainly composed of polysaccharides consisting of alapinose and xylose, obtained from the barley shochu distillation residue, are activated. It has been found that a composition having an action has an excellent activation effect on NK cells.

The composition having the effect of activating NK cells of the present invention has a very remarkable NK cell activation effect comparable to IL-2, which is a known NK cell activator, so leukemia is repelled. It is extremely suitable for the treatment of cancer.

table 1

Control IL-2 Example 1 Example 2 Comparative Example 1

K562 cell viability (¾) 84.7 38.7 48.4 42.6 68.3 Table 2 Control 1L-2 Example 1 Example 2 Comparative Example 1

K562 cell viability (%) 79.4 30.2 37.6 33.1 62.7 Table 3 Dried male g _jL fruit 1 Lyophilized product of Example 2

5 ^ ¹ 1 0.5¾ 1.0¾

NK cell activity (¾) 2.70 ± 0.25 18.8 ± 2.97 21.6 ± 5.18 20.2 ± 4.31 22.8 ± 3.75 fl Number of base cells (x10 ^a ) 1.17 ± 0.31 1.18 ± 0.16 1.08 + 0.61 1.22 ± 0.38 1.09 ± 0.45 NK cell fraction ( .81 ± 0.14 62 ± 0.71 4.43 ± 0.29 4,29 ± 0.87 4.58 ± 1.27

I

Claims

1. Solid-liquid separation of barley shochu distillation residue produced as a by-product in shochu production using barley

'To obtain a liquid fraction, and subject the liquid fraction to an ion exchange treatment using an ion exchange resin to fractionate a non-adsorbed fraction on the ion exchange resin, Leading ultrafiltration membranes

Concentrated by ultrafiltration to be used to obtain a concentrated solution, which was fractionated by adding an organic solvent to the concentrated solution, substantially free of uronic acid, from xylose, arabinose and glucose A composition having a function of activating natural killer cells, comprising a fraction insoluble in the organic solvent containing the polysaccharide.

Surrounding

2. The composition according to claim 1, wherein the fraction insoluble in the organic solvent has the following molecular weight distribution.

Molecular weight distribution:

100,000 or more 5%

30,000 to 100, 000 18%

10,000 to 30,000 23%

3,000 to 10,000 31%

1 000 to 3,000 11%

500 to 1,000 3%

500 or less 9%

3. The fraction insoluble in the organic solvent is about 84.1% by weight of polysaccharide, about 54.4% by weight of the total content of arabinose and xylose derived from polysaccharide, and about 4.2% by weight of crude protein. The composition according to claim 1, which has a component composition comprising about 0.3% by weight of organic acid and about 1.2% by weight of free sugars.

4. The barley shochu distillation residue obtained as a by-product in the production of shochu using barley as a raw material is subjected to solid-liquid separation to obtain a liquid, and the liquid is subjected to an adsorption separation process using a synthetic adsorbent to perform the synthetic adsorption. Fractionate the first fraction that is not adsorbed on the agent, and use the ion exchange resin for the first fraction. The second fraction that is not adsorbed on the ion exchange resin is fractionated, and the second fraction is subjected to a concentration treatment by ultrafiltration using an ultrafiltration membrane. Thus, a concentrated solution was obtained and fractionated by adding an organic solvent to the concentrated solution, which did not substantially contain uronic acid and was insoluble in the organic solvent containing a polysaccharide consisting of xylose, arabinose and glucose. A composition comprising a fraction and having an action of activating natural killer cells.

5. The composition according to claim 4, wherein the fraction insoluble in the organic solvent has the following molecular weight distribution.

Molecular weight distribution:

100,000 or more

30,000 to 100, 000

10,000 to 30,000

3,000 to 10,000

1, 000 to 3,000

500 to 1,000

500 or less

6. The fraction insoluble in the organic solvent is about 78.5% by weight of polysaccharide, about 58.6% by weight of the total content of arabinose and xylose derived from polysaccharide, and about 3.3% by weight of crude protein. 5. The composition according to claim 4, which has a component composition consisting of about 0.2% by weight of organic acid and about 1.5% by weight of free sugars.

7. The composition according to any one of claims 1 to 6, wherein the fraction insoluble in the organic solvent is in a powder form.

8. The composition according to any one of claims 1 to 6, which is used as a medicine.

9. The composition according to claim 4, wherein the synthetic adsorbent is an aromatic synthetic adsorbent or a methacrylic synthetic adsorbent.

10. The barley shochu distillation residue that is by-produced in the production of shochu using barley as a raw material Separating the liquid component, subjecting the liquid component to an ion exchange treatment using an ion exchange resin, and fractionating a non-adsorbed fraction on the ion exchange resin, and separating the ion exchange resin. The non-adsorbed fraction is subjected to a concentration treatment by ultrafiltration using an ultrafiltration membrane to obtain a concentrated solution, and an organic solvent is added to the concentrated solution to substantially contain uronic acid. Insoluble in the organic solvent having an action of activating natural killer cells, comprising a step of fractionating a fraction insoluble in the organic solvent containing a polysaccharide consisting of xylose, arabinose and glucose A method for producing a composition comprising the following fractions:

11. The method for producing a composition according to claim 10, wherein the fraction insoluble in the organic solvent has the following molecular weight distribution.

Molecular weight distribution:

100,000 or more 5%

30,000 to 100,000 18%

10,000 to 30,000 23%

3,000 to 10,000 31%

1,000 to 3,000 11%

500 to 1, 000 3%

500 or less 9%

12. The fraction insoluble in the organic solvent is about 84.1% by weight of polysaccharide, about 54.4% by weight of the total content of arapinose and xylose derived from polysaccharide, and about 4. 11. The method for producing a composition according to claim 10, wherein the composition comprises 2% by weight, organic acid is about 0.3% by weight, and free saccharide is about 1.2% by weight.

13. A step of obtaining a liquid component by separating a barley shochu distillation residue produced as a by-product in the production of shochu using barley as a raw material, and subjecting the liquid to an adsorption separation process using a synthetic adsorbent. A step of separating a non-adsorbed fraction into an agent, and subjecting the collected non-adsorbed fraction to an ion exchange treatment using an ion exchange resin to the ion A step of fractionating a non-adsorbed fraction on the exchange resin, and a step of subjecting the non-adsorbed fraction to the collected ion exchange resin to a concentration treatment by ultrafiltration using an ultrafiltration membrane to obtain a concentrated solution Adding an organic solvent to the concentrated solution to fractionate a fraction that is substantially free of uronic acid and insoluble in the organic solvent containing a polysaccharide consisting of xylose, arabinose, and glucose. A method for producing a composition comprising a fraction insoluble in the organic solvent having an action of activating natural killer cells, comprising:

14. The method for producing a composition according to claim 13, wherein the fraction insoluble in the organic solvent has the following molecular weight distribution.

Molecular weight distribution:

100,000 or more 11%

30,000 to 100, 000 29%

10,000 to 30,000 24%

3,000 to 10,000 21%

1,000 to 3,000 6%

500 to 1, 000 2%

500 or less 7%

15. The fraction insoluble in the organic solvent is about 78.5% by weight of polysaccharide, about 58.6% by weight of the total content of arabinose and xylose derived from polysaccharide, and about 3.3% of crude protein. 14. The method for producing a composition according to claim 13, wherein the composition has a component composition comprising about 0.2% by weight of organic acid, about 0.2% by weight of organic acid, and about 1.5% by weight of free sugars.

16. The method for producing a composition according to any one of claims 10 to 15, further comprising a step of freeze-drying a fraction insoluble in the organic solvent.

17. A barley shochu distillation residue produced as a by-product in the production of shochu using barley as a raw material is separated into solid and liquid to obtain a liquid, and the liquid is subjected to an ion exchange treatment using an ion exchange resin. The non-adsorbed fraction is fractionated into the ultrafiltration membrane. Concentrated by ultrafiltration using a solution to obtain a concentrated solution, and fractionated by adding an organic solvent to the concentrated solution, substantially free of uronic acid, xylose, arapinose And a natural killer cell activated using a fraction insoluble in the organic solvent, which contains a polysaccharide consisting of glucose and glucose and has an action of activating natural killer cells. Natural killer cell-containing composition.

18. The activated natural killer cell-containing composition according to claim 17, wherein the fraction insoluble in the organic solvent has the following molecular weight distribution.

Molecular weight distribution:

100, 0Q0 or more 5%

30,000 to 100, 000 18%

10,000 to 30,000 23%

3,000 to 10,000 31%

1,000 to 3,000 11%

500 to 1,000 3%

500 or less 9%

19. The fraction insoluble in the organic solvent is about 84.1% by weight of polysaccharide, about 54.4% by weight of the total content of arabinose and xylose derived from polysaccharide, and about 4.2% by weight of crude protein. 18. The activated natural killer cell-containing composition according to claim 17, which has a component composition consisting of about 0.3% by weight, about 0.3% by weight of organic acid, and about 1.2% by weight of free sugars.

20. The barley shochu distillation residue obtained as a by-product in the production of shochu using barley as a raw material is separated into solid and liquid to obtain a liquid, and the liquid is subjected to an adsorption separation process using a synthetic adsorbent. The non-adsorbed fraction is collected on the sample, and the collected fraction is subjected to a concentration treatment by ultrafiltration using an ultrafiltration membrane to obtain a concentrated solution, and an organic solvent is added to the concentrated solution. That is substantially free of uronic acid and contains polysaccharides consisting of xylose, arabinose and glucose, and activates natural killer cells. A natural killer cell-containing composition activated by using a natural killer cell activated using a fraction insoluble in the organic solvent.

21. The activated natural killer cell-containing composition according to claim 20, wherein the fraction insoluble in the organic solvent has the following molecular weight distribution.

Molecular weight distribution:

100, 000 or more 11%

30,000 to 100, 000 29%

10,000 to 30,000 24%

3,000 to 10,000 21¾

1, 000 to 3,000 6%

500 to 1,000 2%

500 or less 7%

22. The fraction insoluble in the organic solvent is approximately 78.5% by weight of polysaccharide, approximately 58.6% by weight of the total content of arapinose and xylose derived from polysaccharide, and approximately 3.3% by weight of crude protein. 21. The activated natural killer cell-containing composition according to claim 20, wherein the composition comprises an organic acid in an amount of about 0.2% by weight and a free saccharide in an amount of about 1.5% by weight.

23. The activated natural killer cell-containing composition according to any one of claims 17 to 22, which is used as a therapeutic agent for myeloid leukemia.

24. A step of obtaining a liquid component by separating a barley shochu distillation residue produced as a by-product in the production of shochu using barley as a raw material, and subjecting the liquid to an ion exchange treatment using an ion exchange resin. A step of fractionating a non-adsorbed fraction on the exchange resin, a step of subjecting the fractionated fraction to a concentration treatment by ultrafiltration using an ultrafiltration membrane to obtain a concentrate, and an organic solvent in the concentrate Is added to the organic solvent, which is substantially free of uronic acid, contains polysaccharides consisting of xylose, arabinose and glucose, and has the action of activating natural killer cells. The fractionation step, the fraction that is insoluble in the fractionated organic solvent and natural killer cells are mixed and cultured The manufacturing method of the activated natural killer cell containing composition characterized by including the process of activating the said natural killer cell by this.

25. The method for producing an activated natural killer cell-containing composition according to claim 24, wherein the fraction insoluble in the organic solvent has the following molecular weight distribution.

Molecular weight distribution:

100, 000 or more 5%

30,000 to 100, 000 18%

10,000 to 30,000 23%

3,000 to 10,000 31%

1,000 to 3,000 11%

500 to 1, 000 3%

500 or less 9%

26. The fraction insoluble in the organic solvent is about 84.1% by weight of polysaccharide, about 54.4% by weight of the total content of arapinose and xylose derived from polysaccharide, and about 4. 25. The production of an activated natural killer cell-containing composition according to claim 24, having a composition comprising 2 wt. ¾, an organic acid of about 0.3 wt.%, And a free saccharide of about 1.2 wt. Method.

• 27. A process for obtaining a liquid component by separating a barley shochu distillation residue produced as a by-product in the production of shochu using barley as a raw material, and subjecting the liquid to an adsorption separation process using a synthetic adsorbent. A step of fractionating a non-adsorbed fraction on the adsorbent, and subjecting the fractionated non-adsorbed fraction to the synthetic adsorbent to an ion exchange treatment using an ion exchange resin, A step of fractionating, a step of subjecting the fraction that has not been adsorbed to the ion exchange resin to a concentration treatment by ultrafiltration using an ultrafiltration membrane to obtain a concentrate, and an organic solvent in the concentrate Helping [? Thus, a fraction that is substantially free of uronic acid, contains a polysaccharide composed of xylose, arabinose, and glucose and has an action of activating natural killer cells is separated. Take Activated natural killer cells, characterized in that it comprises a step of activating the natural killer cells by mixing and culturing a fraction that is insoluble in the collected organic solvent and natural killer cells. The manufacturing method of a containing composition.

28. The method for producing an activated natural glitter single cell-containing composition according to claim 27, wherein the fraction insoluble in the organic solvent has the following molecular weight distribution.

Molecular weight distribution:

100,000 or more 11%

30,000 to 100, 000 29%

10,000 to 30,000 24%

3,000 to 10,000 21%

1,000 to 3,000 6%

500 to 1,000 2%

500 or less 7%

29. The fraction insoluble in the organic solvent is about 78.5% by weight of polysaccharide, about 58.6% by weight of the total content of arapinose and xylose derived from polysaccharide, and about 3. 28. The production of an activated natural killer cell-containing composition according to claim 27, having a composition comprising 3 wt. ¾, organic acid of about 0.2 wt%, and free saccharide of about 1.5 wt. Method.

30. A step of fermenting barley koji and shochu yeast produced from brown barley or polished barley as raw materials to produce ripened mash, and subjecting the ripened mash to distillation to produce barley shochu ( A), and the step (B) of treating the barley shochu distillation residual liquid as a distillation residue when producing the barley shochu in the step (A), and in the step (B), the barley shochu distillation A step of obtaining a liquid component by solid-liquid separation of the residual liquid, a step of subjecting the liquid component to an ion exchange treatment using an ion exchange resin, and fractionating a fraction not adsorbed on the ion exchange resin, Subjecting the non-adsorbed fraction to the ion exchange resin to a concentration treatment by ultrafiltration using an ultrafiltration membrane to obtain a concentrated solution; and By adding an organic solvent to the concentrate, it contains substantially no uronic acid, contains a polysaccharide consisting of xylose, arabinose, and glucose, and has the above-mentioned effect of stimulating natural killer cells. A composition comprising a fraction having an action of activating the natural killer cells is produced by sequentially performing a process of fractionating an insoluble fraction, and the process (A) and the process (B) are continuously performed. A method for continuously producing a composition comprising the barley shochu and a fraction having an action of activating the natural killer cells.

31. The method according to claim 30, wherein the fraction insoluble in the organic solvent has the following molecular weight distribution.

Molecular weight distribution:

100,000 or more

30,000 to 100,000

10,000 to 30,000

3,000 to 10,000

1,000 to 3,000

500 to 1,000

500 or less

32. The fraction insoluble in the organic solvent was about 84.1% by weight of polysaccharide, about 54.4% by weight of the total content of arapinose and xylose derived from polysaccharide, and about 4.2% of crude protein. 31. The method of claim 30, having a component composition comprising, by weight, about 0.3% by weight organic acid, and about 1.2% by weight free sugars.

33. A process for producing barley shochu by subjecting barley koji produced using brown barley or polished barley as a raw material and yeast for shochu to fermentation, and subjecting the maturation mash to distillation. A) and the step (B) of treating the barley shochu distillation residual liquid as a distillation residue when producing the barley shochu in the step (A), and in the step (B), the barley shochu A step of solid-liquid separating the distillation residue to obtain a liquid component, the liquid A step of subjecting the body fraction to an adsorption separation process using a synthetic adsorbent to fractionate a fraction that is not adsorbed on the synthetic adsorbent; A step of fractionating a non-adsorbed fraction on the ion-exchange resin by subjecting the ion-exchange resin to a fraction, and using an ultrafiltration membrane to fractionate the non-adsorbed fraction on the fractionated ion-exchange resin. A step of obtaining a concentrated solution by subjecting to a concentration treatment by the above, and by adding an organic solvent to the concentrated solution, containing a polysaccharide substantially free of uronic acid and comprising xylose, arabinose and glucose, A composition comprising a fraction having an action of activating natural killer cells is produced by sequentially performing a step of fractionating a fraction insoluble in the organic solvent having an action of activating natural killer cells. The process (A) And a method of continuously producing a composition comprising the barley shochu and a fraction having an action of activating the natural killer cell, wherein the step (B) is continuously performed.

34. The method according to claim 33, wherein the fraction insoluble in the organic solvent has the following molecular weight distribution.

Molecular weight distribution:

100,000 or more

30,000 to 100, 000

10,000 to 30,000

3,000 to 10,000

1,000 to 3,000

500 to 〖, 000

500 or less

35. The fraction insoluble in the organic solvent comprises about 78.5% by weight of polysaccharide, about 58.6% by weight of the total content of arabinose and xylose derived from polysaccharide, and about 3.3% of crude protein. 34. The method of claim 33, having a component composition consisting of about 0.2% organic acid, about 0.2% by weight organic acid, and about 1.5% by weight free sugars.

36. In the step (A), when obtaining the ripened mash, separately prepared brown barley or refined barley is subjected to fermentation together with the barley straw and the shochu yeast. The method according to 33.

37. The method according to claim 33, wherein the synthetic adsorbent is an aromatic synthetic adsorbent or a methacrylic synthetic adsorbent.