WO2007080962A1

WO2007080962A1 - Anti-inflammatory composition and method for production of anti-inflammatory composition

Info

Publication number: WO2007080962A1
Application number: PCT/JP2007/050317
Authority: WO
Inventors: Hidekazu Tonouchi; Hajime Sasaki; Hisae Kume; Yoshinobu Tuchiya
Original assignee: Meiji Dairies Corporation
Priority date: 2006-01-13
Filing date: 2007-01-12
Publication date: 2007-07-19
Also published as: JP5025491B2; JPWO2007080962A1

Abstract

[PROBLEMS] To discover an anti-inflammatory composition which comprises a casein digest or the like and has a higher activity. [MEANS FOR SOLVING PROBLEMS] Disclosed is an anti-inflammatory composition which is produced by subjecting a raw material such as a cheese or casein to lactic acid bacterium fermentation and/or enzymatic treatment. Preferably, a raw material such as a cheese or casein is fermented with a lactic acid bacterium belonging to the genus Lactococcus and then treated with two or more enzymes including a protease or a peptidase.

Description

Specification

Anti-inflammatory composition and method for producing anti-inflammatory composition

Technical field

[0001] The present invention relates to an anti-inflammatory composition and a method for producing the same, specifically, an anti-inflammatory composition having enhanced anti-inflammatory effects such as inhibition of TNF-a production and inhibition of IL-6 production, and the present anti-inflammatory composition. Concerning manufacturing methods.

Background art

[0002] Enzyme Modified Cheese (EMC) refers to cheese that has been modified by digestion with an enzyme such as protease lipase. Because cheese flavor is enhanced, it is used for the purpose of enhancing and improving the flavor by adding a small amount to cheese and other foods. Previously reported EMC functions include cheddar cheese-Utrease (derived from Bacillus amyloliqu efaciens: Novozyme Corp.), Lactobacillus casei enzyme, EMC force decomposed by combination of tebitrase, known ACE inhibition The ability to report several types of peptides containing active peptides in their partial sequences has been conducted until the study of the activity of these individual peptides (Non-patent Document 1).

[0003] TNF-a is an important factor in the regulation of the force immune system discovered as a factor inducing hemorrhagic necrosis in tumors. Not only stimulates T cell proliferation and differentiation, but also promotes B cell proliferation and antibody production, enhances macrophage histocompatibility antigen expression and chemotaxis, enhances neutrophil chemotaxis and phagocytosis Has many roles. TNF-α also induces inflammation. Inflammation is a biological defense reaction and at the same time a process of pathogenesis. The pathological conditions involving TNF-a include systemic inflammatory response syndrome (SIRS), Helicobacter pylori infection, allergic inflammation, rheumatoid arthritis, diffuse lung disease, liver disease, biliary tract disease, heart failure, miscarriage, pregnancy addiction, Examples include hemophagocytic syndrome, obesity, arteriosclerosis, neurological diseases, intestinal infections, AIDS, and aging (Non-patent Document 2).

[0004] Many food-derived physiologically active peptides have been reported so far, for example, a biopeptide (Non-patent document 3), a mineral absorption-promoting peptide (Non-patent document 4), and an antibacterial peptide (Non-patent document 5). ACE inhibitory peptide (Non-patent Document 6), etc. As food-derived protein degradation products, whey protein trypsin degradation product (Patent Document 1) and ratatofurin molecular weight 10,000 or less peptide (Patent Document 2) have been reported. However, no casein degradation product having anti-inflammatory activity has been reported.

[0005] Patent Document 1: Japanese Patent Application Laid-Open No. 2004-155751

Patent Document 2: JP-A-8-165248

Non-patent literature 1: Haueselassie Sb, Lee BH, Gibbs BF, Purmcation and Identification of Potentially Bioactive Peptides from Enzyme-Modified Cheese., J. Dairy Sci., 82, pp.1612-1617 (1999)

Non-Patent Document 2: Niro Imanishi, edited by "Separate Volume · History of Medicine, Site Power-in and Disease", published by Medical Dentistry, pp.3-116 ( ^July 10, 2000)

Non-patent document 3: Zioudrou C, Streaty RA, Klee WA, Opioid peptides derived from food proteins. ゝ J. Biol. Chem. ゝ 254 (7), pp.2446— 2449 (1979)

Non-Patent Document 4: Kitts DD, Bioactive substances in food: identification and potential us es. ゝ Can J Physiol Pharmacol, 72 (4), pp.423-434 (1994)

Non-Patent Document 5: Pellegrini A, Dettling C, Thomas U, Hunziker P, Isolation and characterization of four bactericidal domains in the bovine beta— lactoglobulin. ゝ Biochim. Biophys. Acta, 1526 (2), pp.131-140 (2001)

Non-Patent Document 6: Nakamura Y, Yamamoto N, Sakai K, Okubo A, Yamazaki S, Takano T., Purification and characterization of angiotensin I— converting enzyme inhibitors f so m milk milk. ゝ J. Dairy Sci. ゝ 78 (4) , Pp.777— 783 (1995)

Disclosure of the invention

Problems to be solved by the invention

[0006] An object of the present invention is to find an anti-inflammatory composition having higher activity, such as a degradation product of casein.

Means for solving the problem

[0007] The present inventors have found that EMC has an anti-inflammatory effect in an in vitro system using cultured cells and an in vivo system using mice. In addition, EMC can alleviate alcoholic liver damage caused by ethanol and lipopolysaccharide (hereinafter LPS). It has been confirmed by the team.

[0008] The anti-inflammatory composition of the present invention comprises cheese or casein, etc.

It is characterized by being treated with one or more proteases.

[0009] That is, the present invention provides

[I] an anti-inflammatory composition obtained by subjecting casein to lactic acid bacteria fermentation and Z or enzyme treatment,

[2] The anti-inflammatory composition according to [1], wherein the enzyme is one or a combination of protease and beptidase,

[3] The anti-inflammatory composition according to any one of [1] to [2], wherein the enzyme is one or a combination of Aspergillus oryzae-derived or Bacillus subtilis-derived,

1_4] Lactic acid power SLactococcus lactis subsp. The anti-inflammatory composition according to any one of [1] to [3], which is one or a combination of lactis, Lactococcus cremons, and Lactococcus diacetylactis,

[5] The anti-inflammatory composition according to any one of [1] to [4], which is used for suppressing hepatitis,

[6] Anti-inflammatory action is suppression of TNF-α production caused by Gram-negative bacteria or LPS, [

1)-[5] any one of the anti-inflammatory composition according to one,

[7] The anti-inflammatory composition according to any one of [1] to [5], wherein the anti-inflammatory action is suppression of hepatitis caused by autoimmune disease or concanaparin A (Con A),

[8] The anti-inflammatory composition according to any one of [1] to [5], wherein the anti-inflammatory action is suppression of alcoholic liver damage,

[9] An anti-inflammatory and Z or therapeutic drug comprising the anti-inflammatory composition according to any one of [1] to [8] as an active ingredient,

[10] Food and drink for inflammation prevention and Z or treatment containing the anti-inflammatory composition according to any one of [1] to [8],

[I I] The method for producing an anti-inflammatory composition according to any one of [1] to [8],

Consists of.

The invention's effect

[0010] The anti-inflammatory composition of the present invention is useful as a composition effective for the prevention and / or treatment of inflammation, particularly inflammation caused by Gram-negative bacteria, autoimmune diseases and alcohol disorders. Ma In addition, it is possible to efficiently prepare an anti-inflammatory composition with fewer steps. That is, there is an advantage that an anti-inflammatory composition excellent in safety that can be added to foods and drinks and medicines can be easily provided.

Brief Description of Drawings

[0011] FIG. 1 is a graph showing the TNF-a production inhibitory effect exhibited by EMC samples of various concentrations in Example 1 and Experiment 3. Represents the average value (n = 2).

FIG. 2 is a graph showing blood GOT activity of each group in Example 1 and Experiment 4. Mean value Indicates the standard error ( _n = 10). *: p <0.05 (Mann-Whitney U-test).

FIG. 3 is a graph showing blood GPT activity of each group in Example 1 and Experiment 4. Average value expresses standard error (n = 10). *: P <0.05 (Mann-Whitney U-test).

FIG. 4 is a graph showing blood IL-6 concentrations of each group in Example 1 and Experiment 4. Average value expresses standard error (n = 10). *: P <0.05 (Unpaired t-test).

[FIG. ⁵ ] Example 1 In Experiment 5, a dull showing the change (Δ ¾0) of ¹³ CO concentration in the breath of each group

2 2 Represents the mean value standard deviation (η = 5). *, **: positive vs. negative, * p 0.05, ** p 0.01 (Student's t-test). #: Positive vs. EMC, # p 〈0.05 (Student's t- test).

FIG. 6 is a graph showing the TNF-a production inhibitory effect exhibited by cheese enzyme-treated samples at various concentrations in Example 2. Represents the average value (n = 2).

FIG. 7 is a graph showing the TNF-a production inhibitory effect of each casein-treated sample in Example 3. Represents the average value (n = 2).

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely modified within the scope of the present invention.

[0013] The anti-inflammatory composition of the present invention is obtained by subjecting casein to lactic acid bacteria fermentation and Z or enzyme treatment. Examples of casein that can be used include a-casein, 13-casein, κ-casein, acid casein, and rennet casein. A readily available food material can also be used in place of casein, and as a food material containing a large amount of casein, power cheese that can be exemplified by cheese, skim milk, and skim milk powder is particularly preferable. Cheese is said to contain 95% of the protein contained in the protein zein (for example, “Science of Milk”, Shu Uenogawa, Asakura Shoten) Either Ral cheese or process cheese is preferably used. Moreover, defatted cheese, partial defatted cheese, and skim cheese may be sufficient. Casein can also obtain the milk power of mammals such as ushi, goat, hidge, buffalo, horse, pig and human. In addition, if the product of the present invention can be obtained, it is synthesized not only by the above method but also by a method such as organic synthesis which may be produced using other animal, plant, or microorganism-derived raw materials. May be manufactured. In practice, the above raw materials can be used alone or in combination.

[0014] Lactic acid bacteria fermentation is performed by adding a lactic acid bacteria starter, in which raw materials such as casein are dispersed and dissolved in water or hot water and cultivated in a skim milk medium. Further, it may be used in combination with an enzyme such as protease described later. The concentration of the raw material is not particularly limited, but if it is mentioned, the concentration range of 5 to 70% by weight is preferred, more preferably 25 to 65% by weight, and still more preferably 40 to 60% by weight. can do. The pH at the start of fermentation, fermentation time, and fermentation temperature are not particularly limited as long as the product of the present invention can be obtained, but the pH at the start of fermentation can be 3.0 to 7.5, preferably 3.5 to 6.5. The fermentation time can be 0.5 to 300 hours, preferably 100 to 200 hours, and the fermentation temperature can be 20 to 45 ° C, preferably 30 to 43 ° C.

[0015] The bacterium used as the lactic acid bacterium starter is not particularly limited as long as it is a lactic acid bacterium. Lactococcus lactis subsp. Lactis ^ Lactococcus lactis subsp. casei, Lactobacillus rhamnos us, Lactobacillus zeae, Lactobacillus reuteri, Lactobacillus delbruekii subsp.bulg aricus, Lactobacillus delbruekii subsp. lactis ^ Lactobacillus fermentum, Lactobacil lus murinus, Bifidobacterium animalif, Bifidobacterium animalis, Bifidobacterium um, Enterococcus faecium, Enterococcus fecalis, Streptococcus thermophilus can be preferably used. More preferably, the genus Lactococcus can be used. In the practice of the present invention, the lactic acid bacteria may be used alone or in combination of two or more. When combined, each fermentation may be performed simultaneously or separately. MM Culture (MM 100: Lactococcus lactis subsp. 1) The ability to include Dairy Connection Inc (Wisconsin, USA), including three types of bacteria, including actis, Lactococcus lactis subsp. cremoris, and Lactococcus lactis subsp. lactis biovar di acetylactis. The medium used for preparing the starter is not limited to the skim milk medium.

[0016] The enzyme treatment is performed by adding an enzyme to raw materials such as casein and the fermented product solution. The enzyme treatment may be performed before lactic acid bacteria fermentation, simultaneously with lactic acid bacteria fermentation, or at any time after lactic acid bacteria fermentation. In the present invention, only the enzyme treatment may be performed without performing lactic acid bacteria fermentation. As with lactic acid bacteria fermentation, the concentration of raw materials such as cheese or casein and the fermented product is not particularly limited, but if it is mentioned, the concentration range is preferably 5 to 70% by weight, more preferably 25 to 65%. %, More preferably 40-60% by weight. PH at the start of enzyme treatment, enzyme treatment time The enzyme treatment temperature is not particularly limited as long as the product of the present invention can be obtained, but the pH at the time of enzyme treatment can be 3.0 to 7.5, preferably 3.5 to 6.5. The enzyme treatment time can be 0.5 to 300 hours, preferably 100 to 200 hours. The enzyme treatment temperature can be 20 to 45 ° C, preferably 30 to 43 ° C.

[0017] The enzyme used in combination with lactic acid bacteria fermentation and the enzyme used in the enzyme treatment is one or a combination of protease or peptidase. The protease or beptidase to be used is not particularly limited. For example, food grade proteases include end protease, exo protease, exo peptidase / endo protease complex enzyme, and protease Z peptidase complex enzyme. . Endoproteases include, for example, chymosin (EC 3.4.23.4, Maxiren, modified yeast Kluyveromyces lac tis, GIST-BROCADES NV), AlcalaseR (Bacillus licheniformis, Novo), Esperase (B. lentus, Novo) ), NeutraseR (from B. subtilis, Novo), Protamex (from bacteria, Novo), PTN6.0S (porcine spleen trypsin, Novo), etc. For example, flavor zym (derived from Aspergillus oryzae, Novo) and the like. Other endo-type proteases include, for example, trypsin (CAS No. 9002-07-7, EC 3.4.21.4, derived from spleen, Produc t No. T8802, SIGMA), pepsin (CAS No. 9001-75-6). EC 3.4.4.1, derived from porcine gastric mucosa , SIGMA), Chymotrypsin (Novo, Boehringer), Protease N “Amano” G (from Bad llus subtilis; Amano Enzym), Biolase (from Bacillus subtilis; Nagase Sangyo), Papain W-40 (Amano Enzym), Exo type Examples of proteases include knee carboxypeptidase and small intestinal brush border aminopeptidase. In addition, as the protease Z peptidase complex enzyme, for example, hammazyme G (peptidase and peptidase, derived from Aspergillus oryzae, Enzyme Amano) can be used.

[0018] The origin of the enzyme is not limited to the above description and may be any of an animal, a plant, and a microorganism, but those derived from Aspergillus oryzae or Bacillus subtilis are preferable. These enzymes do not imply a limited name such as trade name, origin, manufacturer. In the practice of the present invention, the enzymes may be used alone or in combination. Preferred examples of enzyme combinations include Flavorzyme (from Aspergillus oryzae, Novo), Protease N “Amano” G (from Bacillus subtilis; Amano Enzyme), Ummamizyme G (peptidase and Protease, derived from Aspergillus oryzae, Amano Enzyme), but is not limited to this example. When combined, each enzyme reaction may be performed simultaneously or separately.

[0019] These lactic acid bacteria and Z or enzyme can be used singly or in combination of a plurality of types, but it is more preferable to use a combination of a plurality of types. Lactic acid bacteria fermentation and enzyme reaction can be stopped by sterilization or enzyme deactivation, that is, chemical treatment or heat treatment. As a preferred example of the present invention, a starter culture solution prepared from MM Culture and protease N “Amano” G and sodium chloride sodium are added to crushed cheese and decomposed for 2 days (pH at the start of fermentation is 5.5, fermentation (Temperature is 34 ° C), pH is adjusted to 4.1, humazyme G and flavorzyme are added, and the mixture is stirred and decomposed for 5 days (temperature is 34 ° C). A method of inactivating the enzyme by heating at 110 ° C. for 15 minutes can be mentioned, but the method is not limited to this.

[0020] Furthermore, the product of the present invention can be obtained by subjecting the above-mentioned lactic acid bacteria fermentation and Z or enzyme-treated product as it is, or as a soluble fraction or an insoluble fraction diluted with a solvent. As the solvent, water or a commonly used solvent, for example, alcohols, hydrocarbons, organic acids, organic bases, inorganic acids, inorganic bases, supercritical fluids, etc. may be used alone or in combination. it can.

[0021] The product of the present invention thus obtained can be used as it is, and, if necessary, can be used as a solution obtained by concentrating or diluting this solution by a known method. Furthermore, this concentrated liquid can also be used as a dried product by a known method. In addition, it is possible to use a combination of a plurality of materials derived from different raw materials or a material derived from a single material.

[0022] The anti-inflammatory composition of the present invention is useful as a composition effective for prevention and / or treatment of inflammation. In particular, effects on inflammation caused by Gram-negative bacteria, autoimmune diseases, and alcohol disorders have been confirmed in inflammation models induced by LPS, Con A, and alcohol.

[0023] LPS is a membrane component of Gram-negative bacteria, and is known to induce a strong heat-generating action on mammals and the like during bacterial infection. When RAW 264.7 cells (mouse macrophage-like cell line) were stimulated with LPS and cultured with the anti-inflammatory composition of the present invention added in advance, an increase in the amount of TNF-a in the culture supernatant was observed. It was possible to suppress it in a concentration-dependent manner. When using the amount of TNF-α in the above-mentioned system as an anti-inflammatory index, the production inhibition rate is preferably 13% or more, more preferably 30% or more, and more preferably 50% or more U, . As for TNF-a production-suppressing peptides reported so far in studies using cultured cells, (1) the peptide directly adsorbs to LPS and inhibits the adsorption of LPS to Toll like receptor (2 ) It has been confirmed that the activity of the peptide force κ 取り込ま incorporated into the cell is suppressed and the transcription level of the site force in is lowered, and the same mechanism is considered for the product of the present invention. The invention has been demonstrated to be applicable to Gram-negative bacterial infections and endotoxemia.

[0024] Con A is known as T cell mitogen. Because it has an effect of excessively stimulating immunocompetent cells such as T cells, it is used to create a model for autoimmune hepatitis. The mechanism of liver damage caused by Con A is due to the production of various cytokines by binding to the sugar chains on the surface of immunocompetent cells such as cells and macrophages and activating them. In the Atsy system in which Con A was pre-administered to mice, the anti-inflammatory composition of the present invention showed a significant increase inhibitory effect on blood IL-6, GOT and GPT. this thing Therefore, the product of the present invention may act on T cells and macrophages to exert an anti-inflammatory effect, and the product of the present invention was demonstrated to be applicable to autoimmune diseases such as rheumatoid arthritis.

[0025] It is known that excessive alcohol consumption causes liver damage. As its mechanism, ethanol metabolism (increase in NADH, production of acetoaldehyde, etc.), decrease in oxygen concentration in microcirculation, autoimmune reaction, etc. have been reported. Endotoxemia has also occurred in alcoholic cirrhosis, and macrophages activated by endotoxin have neutrophil force. Various site force ins (TNF, platelet activity factor, etc.) released cause organ damage. (Yuichi Yamamura, supervised by Kazutoshi Giri, “The Latest University of Internal Medicine 51”, Nakayama Shoten, pp.78-84 (issued April 30, 1992)). Enomoto et al. Also reported that the sensitivity of liver Kupper cells to LPS increased after oral administration of ethanol. The mechanism involves the increase in CD14 (LPS receptor) protein and LPS binding protein (LPB). Furthermore, since the permeability of the intestinal tract is increased by ethanol, endotoxins (including LPS) derived from gram-negative bacteria in the intestine are easily transferred into the blood. Kupper cells activated in this way produce more TNF-α and promote the induction and progression of liver damage. Based on this phenomenon, an attempt was made to create an animal model of alcoholic liver injury that can be administered orally with ethanol and intravenously with LPS (Nobuyuki Enomoto et al., 7, Alcohol and Medical Biology, Vol. 20, ρρ 121-126 (2000), Enomoto N et al, Hepatology, 29 (6), pp.1680-1689 (1999), Erika Hashizume and others 5 (2002, pp.155 (2002)) o The anti-inflammatory composition of the present invention shows the effect of suppressing the decrease in liver function in the Atsy system where ethanol is administered orally and LPS is administered intravenously to rats. It was. From this, it was demonstrated that the peptide present in the product of the present invention is useful for reducing liver function associated with liver damage.

[0026] Liver function was based on the metabolic capacity of aminovirin. When an animal is administered amaminopyrine, it is absorbed and then metabolized in the liver. At this time, liver cytochrome P450 2D1 was metabolized to 4-methylaminoantipyrine, which was further metabolized by the same enzyme to give 4-aminoantipyrine. The formaldehyde produced by these two stages of metabolism is further metabolized to produce CO. When the liver is damaged, aminopyrine to 4-methylaminoantipy Excretion of CO derived from aminovirin due to stagnant metabolism of phosphorus to 4-aminoantipyrine

2 will decrease. Therefore, administration of ¹³ c labeled Aminopirin, ¹³ CO excretion in breath

By measuring 2, the residual metabolic capacity when the liver is damaged can be measured.

[0027] The anti-inflammatory composition of the present invention is an inflammatory disease (chronic rheumatoid arthritis, osteoarthritis, rheumatoid myelitis, ventilatory arthritis, periosteitis) as an effective composition for preventing and / or treating inflammation. Arthritis, low back pain, surgery, inflammation after trauma, swelling relief, neuralgia, stomatitis, pharyngitis, cystitis, hepatitis (viral, alcoholic, etc.), cirrhosis, gastritis, splenitis, rhinitis, atopic Inflammatory bowel diseases (IBD) such as dermatitis, Crohn's disease, autoimmune disease (systemic lupus erythematosus, myasthenia gravis, pernicious anemia, type 1 diabetes, Hashimoto's thyroiditis, Graves' disease, infertility, etc.) ), Meningitis, inflammatory eye disease, inflammatory lung disease, diabetic complications, etc.), cardiovascular diseases (eg, angina pectoris, myocardial infarction, congestive heart failure, generalized intravascular coagulation syndrome), asthma allergy Disease, systemic lupus erythematosus, autoimmune hemolysis Anemia, psoriasis, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, AIDS encephalopathy, etc.), central neuropathy (cerebral vascular disorders, head trauma, spinal injury, brain edema, multiple sclerosis, etc.), toxemia (Sepsis, septic shock, endotoxin shock, gram-negative bacterial sepsis, toxin shock syndrome, etc.), Addison disease, Creutzfeldt-Jakob disease, viral infection, transplant rejection, dialysis hypotension, osteoporosis, etc. Application is possible. In addition, it is possible to efficiently prepare an anti-inflammatory composition with fewer steps. That is, there is an advantage that an anti-inflammatory composition excellent in safety that can be added to foods and drinks and pharmaceuticals can be easily provided.

[0028] The anti-inflammatory composition of the present invention can be used in the form of pharmaceuticals or foods and drinks. For example, it is expected to treat and / or prevent various inflammations by direct administration as pharmaceuticals, or by direct intake as special-purpose foods such as foods for specified health use or nutritional functional foods. In addition, various foods (milk, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crusts, prepared milk powder, liquid foods, sick people, regardless of the form of liquid, paste, solid, powder, etc. Foods, nutritional foods, frozen foods, processed foods and other commercial foods). The intake varies depending on the target symptom, age, weight, etc., and can be determined as appropriate depending on its effectiveness (effect). [0029] The food containing the anti-inflammatory composition of the present invention uses water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors, and the like as main components. Can. Examples of proteins include whole milk powder, skim milk powder, partially skim milk powder, force zein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, j8-lacto Globulin, α-ratatorebumin, ratatofurin, soy protein, egg protein, meat protein and other animal and vegetable proteins, and their degradation products; butter, milk minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipid, Examples include various milk-derived components such as lactose. It may contain peptides and amino acids such as casein phosphopeptides, arginine and lysine. Examples of the saccharide include saccharides, processed starch (in addition to text phosphorus, soluble starch, pre-taste starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Examples of fats include animal oils such as lard, fish oil, etc., fractionated oils, hydrogenated oils, transesterified oils, etc .; palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractions thereof Examples thereof include vegetable oils such as oil, hydrogenated oil and transesterified oil. Examples of vitamins include vitamin ビタミン, carotene, vitamin Β, vitamin C, vitamin D, vitamin E, vitamin K, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, Examples include inositol, choline, and folic acid. Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, and selenium. Examples of the organic acid include malic acid, citrate, lactic acid, tartaric acid, and the like. These components can be used in combination of two or more, and synthetic products and Z or foods rich in these may be used. The food form may be solid or liquid. It may be in the form of a gel.

[0030] When the anti-inflammatory composition of the present invention is used as a pharmaceutical, it can be administered in various forms. Examples of the form include oral administration by tablets, capsules, granules, powders, syrups and the like. These various preparations can be generally used in the pharmaceutical formulation technical field such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, and coating agents as main ingredients according to conventional methods. It can be formulated using known adjuvants. Further, it may contain an appropriate amount of calcium. In addition, an appropriate amount of vitamin Minerals, minerals, organic acids, sugars, amino acids, peptides and the like may be added. Example 1

[0031] Hereinafter, the present invention will be described by way of examples. The present invention is not limited thereby.

[0032] [Experiment 1] (Preparation of EMC)

(Preparation of starter culture)

20 g of skim milk powder (for biochemistry, Wako Pure Chemical Industries) was dissolved in 200 ml of distilled water and sterilized to prepare a 10 w / v% nonfat dry milk medium. This includes about 3 O.lg MM Culture (MM 100: Lact ococcus lactis subsp. Lactis, Lactococcus lactis subsp. Cremons, Lactococcus lactis subsp. Lactis Diovar diacetylactis, Dairy Connection Inc (Wisco nsin, USA) ) And cultured at 37 ° C for 16 hours.

[0033] (Preparation method)

Distilled water is added to Danish skim cheese (ripening period 6 months) crushed with meat chopper, starter culture solution, sodium chloride and protease N “Amano” G (protease derived from Bacillus subtilis, Amano Enzym) prepared by the above method Added. The pH at the start of the fermentation was 5.5. The mixture was stirred and decomposed at a fermentation temperature of 34 ° C for 2 days. The pH was adjusted to 4.1 with quenic acid, and Ummamizyme G (peptidase and protease, from Aspergillus oryzae, Amano Enzyme) and Flavorzyme (endoprotease and exopeptidase, from Aspergillus oryzae, Novozymes A / S ) Decomposed by stirring at 34 ° C for 5 days. After the completion of decomposition, the pH was adjusted to 5.0 with sodium hydroxide and heated at 110 ° C for 15 minutes to deactivate the enzyme, and EMC was obtained. Table 1 shows the recipe.

[0034] [Table 1] Danish skim cheese (ripening period 6 months) 150g Starter culture solution (MM100) 27.00g Protease N "Amano JG 0.90g Ummamizyme G 0.45g Flavored potato 0.45g Sodium chloride 0.51g Quenic acid (100g / 100ml) 13.50g Hydroxide Sodium (4N) 15.00 g Distilled water 105.00 g Total 312.81 g

[0035] [Experiment 2] (Preparation of free peptide contained in EMC)

An equal volume of water was added to the EMC produced in Experiment 1 and centrifuged at 10,000 g for 40 minutes to collect the water-soluble fraction. This water-soluble fraction was lyophilized to obtain a free peptide fraction.

[0036] [Experiment 3] (EMC TNF-a production suppression effect using cultured cells)

(Method)

RAW 264.7 cells (mouse macrophage-like cell line) are cultured at 37 ° C (prepared with RPMI1 640 (Sigma) supplemented with 10% fetal bovine serum (Mitsubishi Chemical) and 1% kanamycin sulfate (GIBCO). ), 1 x 10 ⁵ cells / ml was seeded in a 24-well plate at 400 μl. Subsequently, the free peptide prepared in Experiment 2 (hereinafter also referred to as EMC-derived peptide) was added to final concentrations of 100, 500, and 1000 g / ml, and then cultured. Subsequently, LPS was added at a final concentration of 0.1 ng / ml for stimulation, and the culture supernatant was collected 4 hours after addition of LPS, and the amount of TNF-a in the culture supernatant was measured by ELISA.

[0037] (Result)

The results are shown in Figure 1. EMC-derived peptides suppressed the production of TNF-o, an inflammatory site force-in, in a concentration-dependent manner. The TNF-α production inhibitory peptides reported in studies using cultured cells to date are (1) peptides that directly adsorb to LPS, and LPS Toll like receptor (2) Peptide force taken up into cells κB activity is suppressed, transcription level of site force-in is reduced, etc. Various mechanisms can be considered.

[0038] [Experiment 4] (Hepatitis suppression effect using Con A-induced liver injury model mice)

Con A has the effect of excessively stimulating immunocompetent cells such as T cells, and is therefore used to create a model for autoimmune hepatitis.

(Method)

As experimental animals, male 6-week-old C57BL / 6 mice were purchased from Japan SLC Co., Ltd., and after acclimatization for 7 days, 1 group was divided into 10 groups of casein group and EMC group according to body weight. The EMC-derived peptide was the free peptide prepared according to Experiment 2. After grouping, the casein group was bred with the control diet of Table 2, and the EMC group was bred with the EMC diet of Table 2. On the 14th day of breeding, Con A was intravenously administered at 12 mg / kg. Blood was collected after 2, 4, 8 and 24 hours, and blood GOT, GPT (after 8 and 24 hours), and IL-6 levels (after 2, 4, 8 and 24 hours) were measured.

[0039] [Table 2]

[0040] The feed shown in Table 2 was prepared based on AIN-93M purified feed (Oriental Yeast Co., Ltd.) . The control feed was prepared with casein (ARACID 720, NZMP) as protein and corn starch as j8 corn starch (Oriental Yeast Co., Ltd.). The EMC feed was prepared by replacing half of the control feed casein with the EMC-derived peptide prepared in Experiment 2.

[0041] The results are shown in FIG. 2, FIG. 3, and FIG. Administration of the EMC-derived peptide showed a significant inhibitory effect on blood IL-6, GOT, and GPT in comparison with the control group. The mechanism of liver damage caused by Con A is due to the occurrence of Con A force ^ various cyto force in production forces S by binding to sugar chains on the surface of immunocompetent cells such as cells and macrophages and activating them. . As with cultured cells, it may be possible that they exerted anti-inflammatory effects by acting on peptide-powered cells and macrophages present in EMC.

[0042] [Experiment 5] (EMC effect on alcoholic liver injury induced by combined administration of ethanol and LPS)

When an animal is administered amaminopyrine, it is absorbed and then metabolized in the liver. At this time, it is metabolized by cytochrome P450 2D1 in the liver to give 4-methylaminoantipyrine, which is further metabolized by the same enzyme to give 4-aminoaminopyrine. The formaldehyde generated by this two-stage metabolism is further metabolized to produce CO. When the liver is damaged,

2

Aminopyrine to 4-methylaminoantipyrine to 4-amaminoantipyrine metabolism stagnation, which reduces the excretion of CO derived from aminovirin. Therefore, ¹³ c labeled amino

2

Nopiline and ¹³ CO in exhaled breath

2 By measuring excretion, the residual metabolic capacity when the liver is damaged can be measured. In Example 1, this system was used to show the effect of EMC on alcoholic liver damage.

[0043] (1) Materials and methods

[Animal experimentation]

As experimental animals, female 4-week-old SD rats were purchased from Japan SLC Co., Ltd., acclimated for 7 days, and then divided into 5 groups per group by weight, negative control group, positive control group and EMC group. After grouping, the negative control group and the positive control group were reared for 2 weeks with the control sample shown in Table 2 and the EMC group receiving the EMC sample shown in Table 2 freely. The EMC-derived peptide was a free peptide prepared according to Experiment 2. On the 15th day, 4 g / kg of ethanol was orally administered to each group (administered as 50 v / v% ethanol). Six hours later, LPS (derived from E. coli 055: B5, Sig ma-Aldrich Co.) was administered at 1.66 mg / kg tail vein (dosage volume 2 ml / kg), and the same volume of physiological saline was administered to the negative control group via the tail vein. 20 hours after administration of LPS or physiological saline, each group was orally administered ¹³ C-labeled aminovirin (hereinafter ¹³ C-aminovirin) with 20 mg / kg (dose volume 10 ml / kg). Rats were placed in a desiccator for each individual, and thereafter, expired breaths were collected over time after 10 minutes, 10, 20, 30, 40, 50, 60, 70, 80, 90, 120, 150, 180, 210, 240 minutes. The exhaled breath excreted in the desiccator was sucked at a rate of 150 ml / min, and the breath was collected in a breath collection bag 20 (Otsuka Pharmaceutical Co., Ltd.) for UBiT · PO Cone. Specifically, exhalation was collected by the method described below, and 1.5 minutes of exhalation was collected at each time point.

[0044] [Exhalation collection device]

Place a plate insole with many small holes of several millimeters in a 2L volume desiccator, place the rat on it, close the lid, and exhale exhaled from parts other than the breath collection port or air intake port! /, The lid was fixed as follows. A silicone tube was passed through the side opening (intake air) of the desiccator, and one end of the tube opening was fixed to the bottom of the desiccator to make the breath collection port. Use a silicon tube that is smaller in diameter than the air inlet! The exhaled breath will also be transferred to a breath collecting loca provided under the insole. Connect the other end of the tube opening to the UBiT POCone dedicated breath collection bag 20, and connect a peristaltic pump (Master Flex L / S; Cole-Parmer Instrument Company) between the desiccator and the UBiT POCone dedicated breath collection bag 20. The exhaled air excreted in the desiccator was continuously aspirated at a constant rate of 150 ml / min and collected in the UBiT · POCone dedicated breath collection bag 20

[0045] (2) Evaluation

Connect the UBiT · POCone dedicated breath collection bag 20 that collects exhaled breath to the UBiT-IR300 autosampler UBiT-AS10 (Otsuka Pharmaceutical Co., Ltd.) connected to the UBiT-IR300 and mix gas (0: 95%, CO: 5%) was used as a control to measure A ¹³ CO in exhaled breath. Evaluation is individual over time

2 2 2

Specific delta ¹³ CO measurements, Cmax (maximum delta ¹³ CO measurements), Tmax (maximum delta ¹³ CO measurements expression

2 2 2

Time). In addition, the dose of ¹³ C-aminopyrine was also 0 to 240 minutes AUC (A ¹³ CO measurement-

The area under the 2-hour curve) was calculated by the trapezoidal area method.

[0046] (3) Results and discussion Figure 3 shows the measured A ¹³ CO over time, and Table 3 shows Cmax, Tmax, and AUC. Compared to the negative group

2

In the positive group, ¹³ CO emissions decreased significantly 20-210 minutes after administration of aminovirin,

2

The combined use of ethanol and LPS reduced liver metabolic capacity.

In contrast, in the EMC group, ¹³ CO emissions increased significantly compared to the positive group 30 to 80 minutes after administration of aaminopyrine, and the liver metastases decreased by the combined administration of ethanol and LPS.

2

It showed the effect of significantly suppressing the ability of appreciation.

Regarding Cmax, the positive group had a significantly lower value compared to the negative group. The EMC group showed a significantly higher value than the positive group. Tmax was strong with no significant difference between the groups of deviations. Even with AUC, the positive group decreased significantly compared to the negative group. However, there was no significant difference between the EMC group and the positive group.

[0047] [Table 3] Negative (n = 5) Positive (n = 5) EMC (n = 5)

Cmax 29.40 ± 7.30 16.90 26.80 ± 5.50

(△: ' ³ C:% o)

Tmax 59.0 ± 18.2 70.0 53.0 Sat 14.8

(min)

AUC (0-240min) 5168 ± 937 3030 4383 ± 965 (min,% o)

[0048] Table 3 above shows Cmax, Tmax, AU in the change of ¹³ CO concentration in the breath (A ¹³ CO) of each group.

twenty two

C is shown. Represents the mean value standard deviation (n = 5). *: Positive vs. negative, * p 0.05 (Student's t — test). #: Positive vs. EMC, # p 〈0.05 (Student's t- test).

Example 2

[0049] Next,

The TNF-a production inhibitory effect was confirmed.

[Experiment 1] (Preparation of processed cheese protease)

(Preparation method)

Danish skim cheese crushed with meat chopper (ripening period 6 months) with distilled water In addition, sodium chloride and protease N “Amano” G (protease, derived from Bacillus subtilis, Amano Enzyme) were added. After agitation and digestion at 34 ° C for 2 days, the pH was adjusted to 4.1 with citrate, and humamizyme G (peptidase and protease, from Aspergillus oryzae, Amano Enzyme) and flavor zyme (endoprotease and exopeptidase, Aspergillus oryzae origin, Novozymes A / S) was added. Decomposition with stirring at 34 ° C for 5 days. After the decomposition, the pH was adjusted to 5.0 with sodium hydroxide and heated at 110 ° C for 15 minutes to deactivate the enzyme. The formulation is the same as Table 1 except for the starter culture.

[Experiment 2] (Preparation of free peptide contained in protease-treated product)

An equal amount of water was added to the protease-treated product produced in Experiment 1 and centrifuged at 10,000 g for 40 minutes to collect a water-soluble fraction. This water-soluble fraction was lyophilized to obtain a free peptide fraction.

[0050] [Experiment 3] (EMC TNF-a production suppression effect using cultured cells)

With respect to this preparation, the TNF-o; production inhibitory effect was examined in the same manner as in Experiment 3 of Example 1. The free peptide prepared in Experiment 2 was added to a final concentration of 500 and 1000 / z g / ml. The result is shown in Fig. 6. Peptides derived from cheese enzyme-treated products suppress the production of TNF-a, which is a inflammatory site force-in, in a concentration-dependent manner, and are thought to have anti-inflammatory effects similar to EMC.

Example 3

[0051] Next, TNF-a production inhibitory effect was confirmed for the protease-treated product of casein.

[Experiment 1] (Preparation of casein protease product)

(Preparation method)

Distilled water was added to Na Casein (manufactured by NZMP), and sodium chloride and protease N “Amano” G (protease, derived from Bacillus subtilis, Enzyme Amano) were added. After stirring and digesting at 34 ° C for 2 days, the pH was adjusted to 4.1 with citrate, and humamizyme G (peptidase and protease, from Aspergillus oryzae, Amano Enzyme) and flavor zyme (end protease and exo). Peptidase, derived from Aspergillus oryzae, Novozymes A / S) was added. Decomposition with stirring at 34 ° C for 5 days. After the decomposition, adjust the pH with sodium hydroxide 5. The enzyme was inactivated by adjusting to 0 and heating at 110 ° C. for 15 minutes. The recipe is shown in Table 4.

[Table 4]

[0052] [Experiment 2] (Preparation of free peptide contained in protease-treated product)

[0053] With respect to this preparation, the TNF-a production inhibitory effect of peptides present in casein protease treated products was examined in the same manner as in Experiments 2 and 3 of Example 2. The results are shown in Fig. 7. Peptide strength derived from casein enzyme-treated product It suppresses the production of TNF-a, an inflammatory site force-in, in a concentration-dependent manner, and is considered to have anti-inflammatory effects similar to EMC.

[0054] From the results of Examples 1 to 3 described above, it is possible to obtain a composition having excellent anti-inflammatory action by enzymatic treatment of not only EMC but also casein or cheese in which casein occupies most of the contained protein. Indicated.

Industrial applicability

[0055] Since it has an anti-inflammatory action, it can be applied as a medicine or food or drink having the same function.

Claims

The scope of the claims

[I] An anti-inflammatory composition obtained by subjecting casein to lactic acid bacteria fermentation and Z or enzyme treatment.

[2] The anti-inflammatory composition according to claim 1, wherein the enzyme is one or a combination of protease and beptidase.

[3] The anti-inflammatory composition according to any one of claims 1 and 2, wherein the enzyme is one or a combination of Aspergillus oryzae or Bacillus subtilis.

[4] The anti-inflammatory composition according to any one of claims 1 to 3, which is one or a combination of Lactococcus metis subsp. Lactis ^ Lactococcus cremons and Lactococcus diacetylactis.

[5] The anti-inflammatory composition according to any one of claims 1 to 4, which is used for suppressing hepatitis.

6. The anti-inflammatory composition according to any one of claims 1 to 5, wherein the anti-inflammatory action is suppression of TNF-a production caused by gram-negative bacteria or lipopolysaccharide (LPS).

7. The anti-inflammatory composition according to any one of claims 1 to 5, wherein the anti-inflammatory action is suppression of hepatitis caused by autoimmune disease or concanaparin A (Con A).

[8] The anti-inflammatory composition according to any one of claims 1 to 5, wherein the anti-inflammatory action is suppression of alcoholic liver injury.

[9] A pharmaceutical product for inflammation prevention and / or treatment containing the anti-inflammatory composition according to any one of claims 1 to 8 as an active ingredient.

[10] A food and drink for inflammation prevention and / or treatment containing the anti-inflammatory composition according to any one of claims 1 to 8.

[I I] The method for producing an anti-inflammatory composition according to any one of claims 1 to 10.