WO2013073648A1 - Anti-inflammatory functional agent for oral application - Google Patents
Anti-inflammatory functional agent for oral application Download PDFInfo
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- WO2013073648A1 WO2013073648A1 PCT/JP2012/079734 JP2012079734W WO2013073648A1 WO 2013073648 A1 WO2013073648 A1 WO 2013073648A1 JP 2012079734 W JP2012079734 W JP 2012079734W WO 2013073648 A1 WO2013073648 A1 WO 2013073648A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/14—Vegetable proteins
- A23J3/16—Vegetable proteins from soybean
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/185—Vegetable proteins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C19/00—Cheese; Cheese preparations; Making thereof
- A23C19/02—Making cheese curd
- A23C19/05—Treating milk before coagulation; Separating whey from curd
- A23C19/054—Treating milk before coagulation; Separating whey from curd using additives other than acidifying agents, NaCl, CaCl2, dairy products, proteins, fats, enzymes or microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
- A23C9/1322—Inorganic compounds; Minerals, including organic salts thereof, oligo-elements; Amino-acids, peptides, protein-hydrolysates or derivatives; Nucleic acids or derivatives; Yeast extract or autolysate; Vitamins; Antibiotics; Bacteriocins
Definitions
- the present invention relates to a peptide having an anti-inflammatory function.
- Inflammation is a proper defense reaction of the living body, but an excessive inflammatory reaction causes damage to the self-organization of the living body.
- histamine and serotonin are first released from mast cells and platelets. Histamine and serotonin cause temporary vasoconstriction in a short time, dilate blood vessels in the inflamed area (arterioles, venules, and capillaries), increase blood flow, and cause heat and redness.
- vascular permeability is increased (actin of vascular endothelial cells contracts), the interval between vascular endothelial cells is expanded, leukocytes are leached locally from the whole body (in blood), and protective factors such as plasma leak locally Causes swelling (edema).
- factor 12 of blood coagulation is activated and bradykinin is produced in the kinin / caniclein system, causing pain. These reactions occur in the tissue, resulting in tissue dysfunction. These “fever, redness, swelling, pain, dysfunction” are called the five major signs of inflammation.
- neutrophils treat bacteria, viruses, dead cells, and other foreign substances in the early stages of inflammation, but macrophages gather and treat dead cells and bacteria by phagocytosis at the later stage of inflammation.
- Macrophages that encounter bacteria and dead cells are activated and release cytokines such as TNF- ⁇ , IL-1, and IL-6, and chemokines such as IL-8.
- cytokines cause fever, redness, and swelling due to increased vascular permeability
- chemokines increase leukocyte chemotaxis and cause swelling.
- macrophages play an important role in maintaining homeostasis, but macrophage dysfunction such as excessive activation is involved in many diseases of the immune system and excessive inflammation.
- the development of a drug that suppresses the reaction has been intensively studied.
- Patent Document 1 describes many di- and tripeptides as peptides having an anti-inflammatory effect.
- Patent Document 2 discloses a technique for obtaining an effect on gastrointestinal inflammation with a food-derived component, although it does not directly have an anti-inflammatory function.
- this product is a hydrolyzate of cheese, its palatability is high due to its unique flavor.
- Patent Document 3 discloses the anti-inflammatory function of soybean peptides, particularly Phe-Leu-Val and Val-Pro-Tyr, which are peptides derived from the 11S protein.
- soybean peptides particularly Phe-Leu-Val and Val-Pro-Tyr
- Patent Document 4 describes the anti-inflammatory function of pea protein, which is not a major food allergen, but there is no specific description as an example, and it is intended for skin application. Is not intended.
- proteins contained in peas are composed of 11S (legmin), 7S (bicillin), combicillin and globulin from the sedimentation coefficient by ultracentrifugation analysis.
- Pea 11S (Legmin) forms a hexamer with a molecular weight of 320,000 to 380,000, and like other legumins of seeds, acidic subunits and basic subunits are accumulated in a disulfide bond.
- pea 7S (bicillin) forms a trimer with a molecular weight of 170,000 and is composed of subunits with molecular weights of 47,000, 50,000, 34,000 and 30,000.
- Combicillin has a molecular weight of 290,000 and is composed of subunits with a molecular weight of 71,000.
- combicilin is 9-12% in the isolated pea protein
- 7S (bicillin) is 30-38%
- 11S (legumin) is 28%- 33% included.
- proteins contained in soybean are classified into 2S, 7S, 11S and 15S globulin fractions based on the sedimentation coefficient by ultracentrifugation analysis.
- 7S and 11S are the main constituent proteins, 7S ( ⁇ -conglycinin) accounts for about 1/3, and 11S (glycinin) accounts for about 2/3.
- the object of the present invention was to obtain an anti-inflammatory functional agent from a food material that does not contain major food allergens.
- the present inventor diligently studied the pea protein, which has about 11% of the 11S component, which is less than 30% of the soy protein containing 1/3 of the 11S component, which is considered the main body of anti-inflammation.
- the present inventors have found that a peptide obtained by hydrolyzing a protein composition containing pea protein as a main raw material has an anti-inflammatory effect equivalent to or higher than that of soybean protein, and completed the present invention.
- the present invention is (1) an oral anti-inflammatory functional agent comprising pea protein hydrolyzate as an active ingredient.
- the anti-inflammatory functional agent according to (1), wherein the di-tripeptide content in the hydrolyzate is 40% by weight or more.
- an oral anti-inflammatory functional agent having an anti-inflammatory effect is obtained from pea, which is a food material that does not contain major food allergens, and is added to a form of a drug effective for inflammatory diseases, or to food or feed.
- pea which is a food material that does not contain major food allergens
- pea protein means each of the aforementioned pea protein subunits, or an aggregate thereof.
- the pea protein material is a material derived from pea seeds and containing a large amount of pea protein, preferably contains 50% by weight or more of pea protein, more preferably contains 65% by weight or more, More preferably, it is contained by weight% or more.
- Methods for preparing pea protein material from pea seeds are well known. Extracting and concentrating protein components from peas with a general composition using water, and pea seed material by pulverizing and classifying pea seeds. Methods for concentrating are known. Moreover, what mixed other components with the pea protein raw material with high protein contents, such as isolation
- Pea protein hydrolyzate is a peptide mixture prepared by hydrolyzing a pea protein material.
- the pea protein hydrolyzate preferably has a higher degree of degradation, and in particular, the proportion of dipeptides and tripeptides in the total amount of peptides and free amino acids in the hydrolyzate is preferably high.
- the proportion of dipeptides and tripeptides in the total amount of peptides and free amino acids is preferably 40% by weight or more. It is more preferably 60% by weight or more, and most preferably 64% by weight or more.
- dipeptides and tripeptides are defined as a fraction obtained by removing free amino acids from a fraction having a molecular weight of 500 or less. Therefore, the proportion of dipeptides and tripeptides in the total amount of peptides and free amino acids was measured by amino acid analysis after measuring the proportion of peptide fractions with a molecular weight of 500 or less in the hydrolyzate by gel filtration chromatography for peptides. It can be calculated by subtracting the free amino acid content in the hydrolyzate.
- the content of free amino acids in the hydrolyzate is preferably 10% by weight or less, and more preferably 5% by weight or less.
- the proportion of the fraction having a molecular weight of 500 or more in the total amount of peptide and free amino acid in the hydrolyzate is preferably 40% by weight or less, More preferably, it is not more than wt%.
- protease Hydrolysis is preferably performed by protease treatment.
- Protease treatment is carried out using the above-mentioned pea protein material slurry or aqueous solution as a substrate and the following proteases.
- the protease used here is of animal origin, plant origin or microbial origin, and is classified into “metal protease”, “acidic protease”, “thiol protease”, “serine protease” in the classification of protease, preferably “metal” It can be appropriately selected from proteases classified as “protease”, “thiol protease”, and “serine protease”.
- a degradation method in which enzymes belonging to two or more kinds or three or more kinds of different classes are acted on sequentially or simultaneously can increase the proportion of relatively low molecular weight peptides such as dipeptides and tripeptides.
- protease is a classification method based on the type of amino acid at the active center, which is usually performed in the field of enzyme science.
- Methodal Protease includes Bacillus Neutral Protease, Streptomyces Neutral Protease, Aspergillus Neutral Protease, “Samoase”, etc.
- Protease includes bromelain, papain and the like
- serine protease includes trypsin, chymotrypsin, subtilisin, Streptomyces alkaline protease, “Alcalase”, “Biolase” and the like.
- the classification of other enzymes can also be confirmed by their pH and reactivity with inhibitors.
- enzymes having different active centers differ greatly in the site of action on the substrate, it is possible to reduce the “uncut residue” and efficiently obtain an enzyme degradation product.
- enzymatic degradation products can be more efficiently produced by using enzymes from different origins (origin organisms). Even in the same classification, if the origin is different, the site of action of the substrate protein is also different, and as a result, the proportion of dipeptides and tripeptides can be increased. These proteases preferably have low exo activity.
- the reaction pH and reaction temperature of the protease treatment may be set in accordance with the characteristics of the protease to be used, and the reaction pH is usually carried out near the optimum pH, and the reaction temperature may be carried out around the optimum temperature. In general, the reaction can be carried out at a reaction temperature of 20 to 80 ° C, preferably 40 to 60 ° C. After the reaction, the enzyme is heated at a temperature sufficient to inactivate the enzyme (about 60 to 170 ° C.) to inactivate the remaining enzyme activity.
- the reaction solution after the protease treatment can be used as it is or after being concentrated, but it can usually be used in the form of a dry powder by sterilization, spray drying, freeze drying or the like.
- Sterilization is preferably heat sterilization, and the heating temperature is preferably 110 to 170 ° C, more preferably 130 to 170 ° C.
- the heating time is preferably 3 to 20 seconds.
- the reaction solution can be adjusted to an arbitrary pH, and precipitates and suspensions generated during pH adjustment can be removed by centrifugation, filtration, or the like. Furthermore, it can also refine
- the hydrolyzate of the protein composition mainly composed of the pea protein hydrolyzate of the present invention can be widely used as a pharmaceutical form.
- the oral form ie, food or As a form added to the feed, it can be appropriately mixed with other raw materials as necessary.
- it can be used in various forms such as liquid, powder, tablet, capsule and the like.
- When used as a mixed form in foods it can be used by mixing with solid foods such as biscuits, cakes, breads, etc., or it can be used for fluid or semi-solid foods such as bavaria, mousse or pudding. There is no problem with mixing. It can also be taken as a beverage by dissolving in water or the like.
- the oral anti-inflammatory functional agent of the present invention can be used in combination with other foods and drinks and ingredients exhibiting an anti-inflammatory effect.
- lactic acid bacteria fermentation for example, lactic acid bacteria drinks, yogurts, and cheeses
- a particularly strong effect can be obtained.
- other anti-inflammatory effects include polyphenols (tea catechin, anthocyanin, cyanidin, chlorogenic acid, etc.), carotenoids (astaxanthin, lycopene, lutein, etc.), glucosamine, chondroitin, curcumin, capsaicin, etc. Can be illustrated.
- the peptide of the present invention may be denatured by decomposition or the like due to heat at the time of sterilization of food.
- carbohydrates, vitamins, minerals and the like are mixed and used as a supplement.
- it can be used by mixing with known feed without being limited to land and fishery.
- lactic acid bacteria used for lactic acid bacteria fermented foods such as lactic acid bacteria beverages
- lactic acid bacteria generally used as foods can be used.
- Lactobacillus genus bacteria L.casei, L.plantarum, L.brevis, l.acidophilus, L.pentous etc.
- Lactococcus genus bacteria L.lactis, L.cremoris etc.
- Bifidobacterium genus bacteria B.bididum, B.adolescentis etc.
- Example 1 Preparation of Isolated Pea Protein
- a separated pea protein as a pea protein material was prepared as follows. That is, 50 g of pea seed pulverized product was dissolved and dispersed in 500 ml of water, and extracted at 50 ° C. for 1 hr under the condition of pH 9.0. The fiber fraction and pea soymilk were separated using a centrifuge. The pH of the obtained soymilk was adjusted to 4.5, and the precipitated curd fraction was collected with a centrifuge. This fraction was neutralized and sterilized by heating at 120 ° C. for 10 seconds to prepare a separated pea protein.
- Example 2 Preparation of pea protein hydrolyzate
- an enzymatic degradation product by protease was prepared as follows. To a 3% by weight separated pea protein solution, “Samoase” (origin; Bacillus thermoproteolyticus, metalloprotease, Daiwa Kasei) was added at 2% by weight per protein and allowed to act at pH 9.0, 58 ° C. for 60 minutes. Next, “Biolase” (origin; Bacillus sp., Serine protease, Nagase Chemtech) was added at 1% by weight per protein and allowed to act at pH 7.5 for 60 minutes at 58 ° C.
- “Samoase” origin; Bacillus thermoproteolyticus, metalloprotease, Daiwa Kasei
- Biolase origin; Bacillus sp., Serine protease, Nagase Chemtech
- “Sumitum FP” (source: Aspergillus sp., Metalloprotease, Shin Nippon Chemical Industry) was added at 1% by weight per protein and allowed to act at pH 7.5, 58 ° C. for 60 minutes. After the above treatment, the reaction was stopped at 90 ° C. for 20 minutes, and then freeze-dried to obtain a pea protein hydrolyzate sample.
- Example 3 Preparation of isolated soybean protein Isolated soybean protein was prepared from low-denatured defatted soybean as follows. A 1 kg kg of low-denatured defatted soybean was extracted with a hot water slurry to obtain defatted soymilk by removing the okara fraction with a centrifuge. Next, the pH of the obtained defatted soymilk was adjusted to 4.5 and subjected to isoelectric precipitation, and an acid precipitation card was obtained and neutralized with a centrifuge. Furthermore, each obtained fraction was neutralized and sterilized by heating at 120 ° C. for 10 seconds.
- Example 4 Preparation of Soy Protein Hydrolyzate A sample obtained by treating the isolated soy protein obtained in Production Example 3 in the same manner as in Production Example 2 was used as a soy protein hydrolysate.
- Example 5 Other food-derived protein hydrolysates For comparison with other food-derived protein hydrolysates, commercially available products were obtained. Casein hydrolyzate CE90M and Peptopro (manufactured by DMV) as whey protein hydrolysates, HW-3 (manufactured by Snow Brand Milk Products) and Thermox690 (manufactured by Glanbia), collagen hydrolysates as whey whey hydrolysates FSP-AS-L (Nippi) and Chicken collagen (Nippon Ham Health Create) were obtained as samples.
- CE90M and Peptopro manufactured by DMV
- HW-3 manufactured by Snow Brand Milk Products
- Thermox690 manufactured by Glanbia
- IL-6 concentration by ELISA In order to measure the IL-6 concentration in the culture medium by ELISA, Purified rat anti-mouse IL-6 (primary antibody), Biotinylated rat anti-mouse IL-6 (secondary antibody), Recombinant mouse IL-6 ( Standard IL-6) and Streptavidin-Alkalin Phosphate were all purchased from BD Pharmingen and used.
- the plate was washed 3 times with PBS-Tween20 (PBS-T) and thoroughly drained. Thereafter, 3% BSA in PBS-T was added in an amount of 100 ⁇ l / well, and allowed to stand at room temperature for 1 hr, and then washed 3 times with PBS-T to thoroughly drain water. Samples diluted appropriately with PBS-T were added in an amount of 50 ⁇ l / well, and allowed to stand at 4 ° C. for 24 hours, and then washed 3 times with PBS-T to drain thoroughly.
- PBS-Tween20 PBS-Tween20
- the molecular weight distribution of each food-derived protein hydrolyzate was measured by the HPLC method using the following gel filtration column.
- An HPLC system using a gel filtration column for peptides was assembled, a known peptide serving as a molecular weight marker was charged, and a calibration curve was obtained in relation to the molecular weight and the retention time.
- the molecular weight markers are [ ⁇ -Asp] -Angiotensin II ⁇ -Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (molecular weight 1046) as octapeptide and Angiotensin IV Val-Tyr- as hexapeptide.
- the ratio (%) of the peptide fraction with a molecular weight of 500 or less to the total amount of peptide and free amino acid in the hydrolyzate is determined by the ratio of the area with a molecular weight of 500 or less (time range) to the total absorbance chart area. Determined (column used: Superdex Peptide 7.5 / 300GL (manufactured by GE Healthcare Japan). Solvent: 1% SDS / 10 mM phosphate buffer, pH 8.0, column temperature 25 ° C, flow rate 0.25 ml / min, detection Wavelength: 220 nm).
- each food-derived protein hydrolyzate was measured by amino acid analysis.
- Hydrolyzate (4 mg / ml) was added to an equal volume of 3% sulfosalicylic acid and shaken at room temperature for 15 minutes. Centrifugation was carried out at 10,000 rpm for 10 minutes, and the resulting supernatant was filtered through a 0.45 ⁇ m filter, and free amino acids were measured with an amino acid analyzer (JLC500V manufactured by JEOL Ltd.).
- the free amino acid content in the protein was calculated as a ratio to the protein content obtained by the Kjeldahl method.
- the value obtained by subtracting the “free amino acid content” from the “ratio of the peptide fraction having a molecular weight of 500 or less” obtained above was defined as the “dipeptide / tripeptide content” in the hydrolyzate.
- Dr.SPSSII manufactured by SPSS was used for the significant difference test, and one-way analysis of variance between multiple groups was evaluated using the Turkey-Kramer method.
- Example 6 Anti-inflammatory effect of each food-derived protein hydrolyzate on mouse macrophage cells Pea protein hydrolyzate obtained in Example 2, soybean protein hydrolyzate obtained in Example 4, and each food-derived In order to evaluate the anti-inflammatory effect of the protein hydrolyzate, mouse macrophage cell culture described in the measurement method and IL-6 concentration were measured by ELISA.
- the anti-inflammatory effect by the cheese of patent document 2 has only a low effect compared with a pea, or originates in the metabolite of the lactic acid bacteria used at the time of cheese production.
- the pea protein has less anti-inflammatory function than non-patent document 1 and non-patent document 2 because 11S type protein (legmin type) that exhibits anti-inflammatory function as described in patent document 3 is less than soybean protein.
- the anti-inflammatory function exhibited by pea protein hydrolyzate was equivalent to or better than that of soybean protein hydrolyzate.
- Example 7 Combined effect with lactic acid bacteria Regarding the anti-inflammatory effect on mouse macrophage cells, the combined effect of pea protein hydrolyzate and lactic acid bacteria was verified. Lactobacillus casei was used as the lactic acid bacterium, and mouse macrophage cell culture described in the measurement method and IL-6 concentration were measured by ELISA. Lactic acid bacteria were added at a concentration of 1 ⁇ 10 5 cells when CpG was added, and a system in which pea protein hydrolyzate was added and a system in which pea protein hydrolyzate was not added were set, and IL-6 concentration was measured. As a result, it was observed that IL-6 secreted by mouse macrophage cells was reduced even in the lactic acid bacteria addition system, but it was confirmed that the effect was increased by using it together with pea protein hydrolysate.
- Example 8 Preparation of lactic acid bacteria beverage and anti-inflammatory effect
- the above-mentioned pea protein hydrolysis was applied to the commercially available lactic acid bacteria beverage "Yakult" (manufactured by Yakult Honsha Co., Ltd.).
- the sample was dissolved to a final concentration of 1% and used as a sample.
- IL-6 secreted by mouse macrophage cells was reduced.
Abstract
The purpose of the present invention is to produce an anti-inflammatory functional agent from a raw material that contains no major food allergen and can be used widely as a food material. An anti-inflammatory functional agent for oral application which contains no allergen and has a potent effect can be produced using, as an active ingredient, a pea protein hydrolysate that is a food material. The product of the present invention can be used widely in the form of a medicinal agent or in such a form contained in a food or a feed.
Description
本発明は、抗炎症機能を有するペプチドに関する。
The present invention relates to a peptide having an anti-inflammatory function.
炎症とは、生体の合目的な防御反応であるが、過剰な炎症反応は生体の自己組織の損傷をもたらす。炎症の初期段階ではまず、ヒスタミンやセロトニンが肥満細胞と血小板から放出される。ヒスタミンやセロトニンは短時間に一過的な血管収縮を起こし、炎症局所（細動脈、細静脈、及び毛細血管）の血管を拡張させ、血流を増加させ熱感や発赤が生じる。続いて、血管透過性を亢進させ（血管内皮細胞のアクチンが収縮）、血管内皮細胞の間隔が広がって、全身（血液中）から、白血球を局所に浸出させ血漿などの防御因子を局所に漏出させることにより腫脹（浮腫）が生じる。血管内皮細胞の破壊に伴い、血液凝固の第12因子が活性化されキニン・カニクレイン系でブラジキニンが産生されることにより疼痛が生じる。これらの反応が組織内で起こることにより組織の機能障害が生じる。これら「発熱、発赤、腫脹、疼痛、機能障害」を炎症の５大兆候と呼ばれている。
Inflammation is a proper defense reaction of the living body, but an excessive inflammatory reaction causes damage to the self-organization of the living body. In the early stages of inflammation, histamine and serotonin are first released from mast cells and platelets. Histamine and serotonin cause temporary vasoconstriction in a short time, dilate blood vessels in the inflamed area (arterioles, venules, and capillaries), increase blood flow, and cause heat and redness. Subsequently, vascular permeability is increased (actin of vascular endothelial cells contracts), the interval between vascular endothelial cells is expanded, leukocytes are leached locally from the whole body (in blood), and protective factors such as plasma leak locally Causes swelling (edema). With the destruction of vascular endothelial cells, factor 12 of blood coagulation is activated and bradykinin is produced in the kinin / caniclein system, causing pain. These reactions occur in the tissue, resulting in tissue dysfunction. These “fever, redness, swelling, pain, dysfunction” are called the five major signs of inflammation.
これら炎症反応の中で、炎症の初期には好中球が細菌，ウイルス，死細胞等の異物の処理を行うが、炎症後期になるとマクロファージが集まり死んだ細胞や細菌を食作用により処理を行っている。細菌や死細胞と遭遇したマクロファージは、活性化しTNF-α，IL-1，IL-6などのサイトカイン、及びIL-8などのケモカインを放出する。これらのサイトカインにより血管透過性の亢進による発熱，発赤，腫脹がおこり、ケモカインにより白血球の走性を亢進し腫脹が引き起こされる。上記のように、マクロファージは生体の恒常性維持のために重要な役割を担っているが、過剰な活性化などのマクロファージの機能異常は、免疫システムの多くの病気に関わっており、過剰な炎症反応を抑制する医薬品の開発が鋭意検討されている。
Among these inflammatory reactions, neutrophils treat bacteria, viruses, dead cells, and other foreign substances in the early stages of inflammation, but macrophages gather and treat dead cells and bacteria by phagocytosis at the later stage of inflammation. ing. Macrophages that encounter bacteria and dead cells are activated and release cytokines such as TNF-α, IL-1, and IL-6, and chemokines such as IL-8. These cytokines cause fever, redness, and swelling due to increased vascular permeability, and chemokines increase leukocyte chemotaxis and cause swelling. As mentioned above, macrophages play an important role in maintaining homeostasis, but macrophage dysfunction such as excessive activation is involved in many diseases of the immune system and excessive inflammation. The development of a drug that suppresses the reaction has been intensively studied.
その中で、食品由来ペプチドを用いた研究もなされており、例えば特許文献１では多くのジ，トリペプチドが抗炎症効果を持つペプチドとして記載されている。しかし、合成による製造を前提としており、安全性が十分に確立されたものとは言い難い。特許文献２には、直接抗炎症機能を謳うものではないが、食品由来の成分で、消化管炎症への効果を謳う技術が開示されている。しかし、本品はチーズの加水分解物であるため、その独特な風味から嗜好性が高い。
Among them, research using food-derived peptides has also been conducted. For example, Patent Document 1 describes many di- and tripeptides as peptides having an anti-inflammatory effect. However, since it is premised on production by synthesis, it cannot be said that safety is sufficiently established. Patent Document 2 discloses a technique for obtaining an effect on gastrointestinal inflammation with a food-derived component, although it does not directly have an anti-inflammatory function. However, since this product is a hydrolyzate of cheese, its palatability is high due to its unique flavor.
特許文献３では、大豆ペプチド、特に11S蛋白質に由来するペプチドであるPhe-Leu-ValおよびVal-Pro-Tyrの抗炎症機能が開示されている。しかし、大豆は主要な食物アレルゲンである事から、大豆アレルギーを発症している患者への応用が出来ないなど、利用範囲が限定される。特許文献４において、主要な食物アレルゲンでは無いエンドウ蛋白質の抗炎症機能について記載が見られるが、実施例としての具体的記載はない上に、皮膚塗布を目的としているものであり、経口摂取による効果は目的としていない。
Patent Document 3 discloses the anti-inflammatory function of soybean peptides, particularly Phe-Leu-Val and Val-Pro-Tyr, which are peptides derived from the 11S protein. However, since soy is a major food allergen, the range of use is limited, such as being unable to be applied to patients with soy allergy. Patent Document 4 describes the anti-inflammatory function of pea protein, which is not a major food allergen, but there is no specific description as an example, and it is intended for skin application. Is not intended.
ところで、エンドウに含まれる蛋白質は、超遠心分析による沈降係数から、11S（レグミン），7S（ビシリン），コンビシリン及びグロブリンから構成されている。エンドウ11S（レグミン）は分子量320,000～380,000の６量体を形成しており、他の種子のレグミンと同様に酸性サブユニットと塩基性サブユニットがジスルフィド結合でつながった状態で集積している。一方エンドウ7S（ビシリン）は分子量170,000の三量体を形成しており、分子量47,000，50,000，34,000及び30,000のサブユニットから構成されている。コンビシリンは290,000の分子量を持ち、分子量71,000のサブユニットから構成されている。非特許文献１より多くのエンドウ品種について、構成タンパク質の含量を比較した結果、コンビシリンは分離エンドウ蛋白質中９～12%、7S（ビシリン）は30～38%、11S（レグミン）は28%～33%含まれる。
一方、大豆に含まれる蛋白質は、超遠心分析による沈降係数から、2S，7S，11S及び15Sの各グロブリン画分に分類される。分離大豆蛋白質中では、7Sおよび11Sが主要構成蛋白質であり、7S（β-コングリシニン）が約1/3を、11S（グリシニン）が約2/3を占めている。 By the way, proteins contained in peas are composed of 11S (legmin), 7S (bicillin), combicillin and globulin from the sedimentation coefficient by ultracentrifugation analysis. Pea 11S (Legmin) forms a hexamer with a molecular weight of 320,000 to 380,000, and like other legumins of seeds, acidic subunits and basic subunits are accumulated in a disulfide bond. On the other hand, pea 7S (bicillin) forms a trimer with a molecular weight of 170,000 and is composed of subunits with molecular weights of 47,000, 50,000, 34,000 and 30,000. Combicillin has a molecular weight of 290,000 and is composed of subunits with a molecular weight of 71,000. As a result of comparing the content of constituent proteins in more pea varieties than Non-Patent Document 1, combicilin is 9-12% in the isolated pea protein, 7S (bicillin) is 30-38%, 11S (legumin) is 28%- 33% included.
On the other hand, proteins contained in soybean are classified into 2S, 7S, 11S and 15S globulin fractions based on the sedimentation coefficient by ultracentrifugation analysis. Among the isolated soybean proteins, 7S and 11S are the main constituent proteins, 7S (β-conglycinin) accounts for about 1/3, and 11S (glycinin) accounts for about 2/3.
一方、大豆に含まれる蛋白質は、超遠心分析による沈降係数から、2S，7S，11S及び15Sの各グロブリン画分に分類される。分離大豆蛋白質中では、7Sおよび11Sが主要構成蛋白質であり、7S（β-コングリシニン）が約1/3を、11S（グリシニン）が約2/3を占めている。 By the way, proteins contained in peas are composed of 11S (legmin), 7S (bicillin), combicillin and globulin from the sedimentation coefficient by ultracentrifugation analysis. Pea 11S (Legmin) forms a hexamer with a molecular weight of 320,000 to 380,000, and like other legumins of seeds, acidic subunits and basic subunits are accumulated in a disulfide bond. On the other hand, pea 7S (bicillin) forms a trimer with a molecular weight of 170,000 and is composed of subunits with molecular weights of 47,000, 50,000, 34,000 and 30,000. Combicillin has a molecular weight of 290,000 and is composed of subunits with a molecular weight of 71,000. As a result of comparing the content of constituent proteins in more pea varieties than Non-Patent Document 1, combicilin is 9-12% in the isolated pea protein, 7S (bicillin) is 30-38%, 11S (legumin) is 28%- 33% included.
On the other hand, proteins contained in soybean are classified into 2S, 7S, 11S and 15S globulin fractions based on the sedimentation coefficient by ultracentrifugation analysis. Among the isolated soybean proteins, 7S and 11S are the main constituent proteins, 7S (β-conglycinin) accounts for about 1/3, and 11S (glycinin) accounts for about 2/3.
本発明は、主要な食物アレルゲンを含有しない食品素材から、抗炎症機能剤を得ることを目的とした。
The object of the present invention was to obtain an anti-inflammatory functional agent from a food material that does not contain major food allergens.
本発明者は本課題について鋭意検討する中で、抗炎症の本体と考えられる11S成分を、蛋白質中役1/3含む大豆蛋白質に比べ、11S成分が30%程度と少ないエンドウ蛋白質について、敢えてその抗炎症効果を確認したところ、エンドウ蛋白質を主原料とする蛋白質組成物を加水分解したペプチドが、大豆蛋白質と同等以上の抗炎症効果を有することを見出し、本発明を完成させた。
即ち本発明は
（１）エンドウ蛋白質加水分解物を有効成分とする、経口性抗炎症機能剤。
（２）加水分解物中のジ・トリペプチド含量が40重量%以上である、（１）記載の抗炎症機能剤。
（３）エンドウ蛋白質加水分解物の、経口性抗炎症機能剤への使用。
（４）ジ・トリペプチド含量が40重量%以上である、エンドウ蛋白質加水分解物の、経口性抗炎症機能剤への使用。
（５）エンドウ蛋白質加水分解物を有効成分とする、経口性抗炎症機能剤の製造方法。（６）ジ・トリペプチド含量が40重量%以上である、エンドウ蛋白質加水分解物を有効成分とする、経口性抗炎症機能剤の製造方法。
（７）（１）記載の抗炎症機能剤を含有した飼料。
である。 The present inventor diligently studied the pea protein, which has about 11% of the 11S component, which is less than 30% of the soy protein containing 1/3 of the 11S component, which is considered the main body of anti-inflammation. As a result of confirming the anti-inflammatory effect, the present inventors have found that a peptide obtained by hydrolyzing a protein composition containing pea protein as a main raw material has an anti-inflammatory effect equivalent to or higher than that of soybean protein, and completed the present invention.
That is, the present invention is (1) an oral anti-inflammatory functional agent comprising pea protein hydrolyzate as an active ingredient.
(2) The anti-inflammatory functional agent according to (1), wherein the di-tripeptide content in the hydrolyzate is 40% by weight or more.
(3) Use of pea protein hydrolyzate as an oral anti-inflammatory functional agent.
(4) Use of a pea protein hydrolyzate having a di-tripeptide content of 40% by weight or more as an oral anti-inflammatory functional agent.
(5) A method for producing an oral anti-inflammatory functional agent comprising pea protein hydrolyzate as an active ingredient. (6) A method for producing an oral anti-inflammatory functional agent comprising a pea protein hydrolyzate having a di-tripeptide content of 40% by weight or more as an active ingredient.
(7) A feed containing the anti-inflammatory function agent according to (1).
It is.
即ち本発明は
（１）エンドウ蛋白質加水分解物を有効成分とする、経口性抗炎症機能剤。
（２）加水分解物中のジ・トリペプチド含量が40重量%以上である、（１）記載の抗炎症機能剤。
（３）エンドウ蛋白質加水分解物の、経口性抗炎症機能剤への使用。
（４）ジ・トリペプチド含量が40重量%以上である、エンドウ蛋白質加水分解物の、経口性抗炎症機能剤への使用。
（５）エンドウ蛋白質加水分解物を有効成分とする、経口性抗炎症機能剤の製造方法。（６）ジ・トリペプチド含量が40重量%以上である、エンドウ蛋白質加水分解物を有効成分とする、経口性抗炎症機能剤の製造方法。
（７）（１）記載の抗炎症機能剤を含有した飼料。
である。 The present inventor diligently studied the pea protein, which has about 11% of the 11S component, which is less than 30% of the soy protein containing 1/3 of the 11S component, which is considered the main body of anti-inflammation. As a result of confirming the anti-inflammatory effect, the present inventors have found that a peptide obtained by hydrolyzing a protein composition containing pea protein as a main raw material has an anti-inflammatory effect equivalent to or higher than that of soybean protein, and completed the present invention.
That is, the present invention is (1) an oral anti-inflammatory functional agent comprising pea protein hydrolyzate as an active ingredient.
(2) The anti-inflammatory functional agent according to (1), wherein the di-tripeptide content in the hydrolyzate is 40% by weight or more.
(3) Use of pea protein hydrolyzate as an oral anti-inflammatory functional agent.
(4) Use of a pea protein hydrolyzate having a di-tripeptide content of 40% by weight or more as an oral anti-inflammatory functional agent.
(5) A method for producing an oral anti-inflammatory functional agent comprising pea protein hydrolyzate as an active ingredient. (6) A method for producing an oral anti-inflammatory functional agent comprising a pea protein hydrolyzate having a di-tripeptide content of 40% by weight or more as an active ingredient.
(7) A feed containing the anti-inflammatory function agent according to (1).
It is.
本発明により、主要な食物アレルゲンを含有しない食品素材であるエンドウから、抗炎症効果を持つ経口性抗炎症機能剤を得、炎症性疾患に効果がある医薬品の形態あるいは、食品または飼料に添加された形態で使用することができる。
According to the present invention, an oral anti-inflammatory functional agent having an anti-inflammatory effect is obtained from pea, which is a food material that does not contain major food allergens, and is added to a form of a drug effective for inflammatory diseases, or to food or feed. Can be used in different forms.
以下、本発明をより詳細に説明する。
Hereinafter, the present invention will be described in more detail.
（エンドウ蛋白質）
本発明でいうエンドウ蛋白質とは、前述したエンドウ蛋白質のサブユニットの各々、またはその集合物を意味する。 (Pea protein)
The term “pea protein” as used in the present invention means each of the aforementioned pea protein subunits, or an aggregate thereof.
本発明でいうエンドウ蛋白質とは、前述したエンドウ蛋白質のサブユニットの各々、またはその集合物を意味する。 (Pea protein)
The term “pea protein” as used in the present invention means each of the aforementioned pea protein subunits, or an aggregate thereof.
（エンドウ蛋白質素材）
エンドウ蛋白質素材とは、エンドウ種子に由来し、エンドウ蛋白質を多く含む素材であり、エンドウ蛋白質が50重量%以上含まれていることが好ましく、65重量%以上含まれていることがより好ましく、90重量%以上含まれていることがさらに好ましい。エンドウ種子からエンドウ蛋白質素材を調製する方法は公知であり、一般的な組成のエンドウから蛋白質成分を水を用いて抽出及び濃縮する方法と、エンドウ種子を粉砕し、分級する事でエンドウ蛋白質素材を濃縮する方法が知られている。また、分離エンドウ蛋白質等の蛋白質含量の高いエンドウ蛋白質素材に他成分を混合したものも、後述する加水分解の原料として扱うことができる。 (Pea protein material)
The pea protein material is a material derived from pea seeds and containing a large amount of pea protein, preferably contains 50% by weight or more of pea protein, more preferably contains 65% by weight or more, More preferably, it is contained by weight% or more. Methods for preparing pea protein material from pea seeds are well known. Extracting and concentrating protein components from peas with a general composition using water, and pea seed material by pulverizing and classifying pea seeds. Methods for concentrating are known. Moreover, what mixed other components with the pea protein raw material with high protein contents, such as isolation | separation pea protein, can also be handled as a raw material of a hydrolysis mentioned later.
エンドウ蛋白質素材とは、エンドウ種子に由来し、エンドウ蛋白質を多く含む素材であり、エンドウ蛋白質が50重量%以上含まれていることが好ましく、65重量%以上含まれていることがより好ましく、90重量%以上含まれていることがさらに好ましい。エンドウ種子からエンドウ蛋白質素材を調製する方法は公知であり、一般的な組成のエンドウから蛋白質成分を水を用いて抽出及び濃縮する方法と、エンドウ種子を粉砕し、分級する事でエンドウ蛋白質素材を濃縮する方法が知られている。また、分離エンドウ蛋白質等の蛋白質含量の高いエンドウ蛋白質素材に他成分を混合したものも、後述する加水分解の原料として扱うことができる。 (Pea protein material)
The pea protein material is a material derived from pea seeds and containing a large amount of pea protein, preferably contains 50% by weight or more of pea protein, more preferably contains 65% by weight or more, More preferably, it is contained by weight% or more. Methods for preparing pea protein material from pea seeds are well known. Extracting and concentrating protein components from peas with a general composition using water, and pea seed material by pulverizing and classifying pea seeds. Methods for concentrating are known. Moreover, what mixed other components with the pea protein raw material with high protein contents, such as isolation | separation pea protein, can also be handled as a raw material of a hydrolysis mentioned later.
（エンドウ蛋白質加水分解物）
エンドウ蛋白質加水分解物とは、エンドウ蛋白質素材を加水分解することで調製されるペプチド混合物である。エンドウ蛋白質加水分解物は分解度がより高いことが好ましく、特に加水分解物中におけるペプチド及び遊離アミノ酸の合計量に占める、ジペプチド及びトリペプチドの割合が高いことが好ましい。具体的には、ペプチド及び遊離アミノ酸の合計量に占めるジペプチド及びトリペプチドの割合が40重量%以上であることが好ましい。60重量%以上であることが更に好ましく、64重量%以上であることが最も好ましい。
なお本願では、ジペプチド及びトリペプチドを、分子量500以下の画分から遊離アミノ酸を除いた画分と規定した。したがって、ペプチド及び遊離アミノ酸の合計量に占めるジペプチド及びトリペプチドの割合は、ペプチド用ゲルろ過クロマトグラフィーにより加水分解物中の分子量500以下のペプチド画分の割合を測定した後、アミノ酸分析により測定した加水分解物中の遊離アミノ酸含量を差し引くことにより算出することが可能である。加水分解物中の遊離アミノ酸含量は10重量%以下であることが好ましく、５重量%以下であることがより好ましい。さらに、ペプチド体はより低分子であることが望ましいことから、加水分解物中のペプチド及び遊離アミノ酸の合計量に占める分子量500以上の画分の割合は40重量%以下であることが好ましく、35重量%以下であることがより好ましい。 (Pea protein hydrolyzate)
Pea protein hydrolyzate is a peptide mixture prepared by hydrolyzing a pea protein material. The pea protein hydrolyzate preferably has a higher degree of degradation, and in particular, the proportion of dipeptides and tripeptides in the total amount of peptides and free amino acids in the hydrolyzate is preferably high. Specifically, the proportion of dipeptides and tripeptides in the total amount of peptides and free amino acids is preferably 40% by weight or more. It is more preferably 60% by weight or more, and most preferably 64% by weight or more.
In the present application, dipeptides and tripeptides are defined as a fraction obtained by removing free amino acids from a fraction having a molecular weight of 500 or less. Therefore, the proportion of dipeptides and tripeptides in the total amount of peptides and free amino acids was measured by amino acid analysis after measuring the proportion of peptide fractions with a molecular weight of 500 or less in the hydrolyzate by gel filtration chromatography for peptides. It can be calculated by subtracting the free amino acid content in the hydrolyzate. The content of free amino acids in the hydrolyzate is preferably 10% by weight or less, and more preferably 5% by weight or less. Furthermore, since it is desirable that the peptide body has a lower molecular weight, the proportion of the fraction having a molecular weight of 500 or more in the total amount of peptide and free amino acid in the hydrolyzate is preferably 40% by weight or less, More preferably, it is not more than wt%.
エンドウ蛋白質加水分解物とは、エンドウ蛋白質素材を加水分解することで調製されるペプチド混合物である。エンドウ蛋白質加水分解物は分解度がより高いことが好ましく、特に加水分解物中におけるペプチド及び遊離アミノ酸の合計量に占める、ジペプチド及びトリペプチドの割合が高いことが好ましい。具体的には、ペプチド及び遊離アミノ酸の合計量に占めるジペプチド及びトリペプチドの割合が40重量%以上であることが好ましい。60重量%以上であることが更に好ましく、64重量%以上であることが最も好ましい。
なお本願では、ジペプチド及びトリペプチドを、分子量500以下の画分から遊離アミノ酸を除いた画分と規定した。したがって、ペプチド及び遊離アミノ酸の合計量に占めるジペプチド及びトリペプチドの割合は、ペプチド用ゲルろ過クロマトグラフィーにより加水分解物中の分子量500以下のペプチド画分の割合を測定した後、アミノ酸分析により測定した加水分解物中の遊離アミノ酸含量を差し引くことにより算出することが可能である。加水分解物中の遊離アミノ酸含量は10重量%以下であることが好ましく、５重量%以下であることがより好ましい。さらに、ペプチド体はより低分子であることが望ましいことから、加水分解物中のペプチド及び遊離アミノ酸の合計量に占める分子量500以上の画分の割合は40重量%以下であることが好ましく、35重量%以下であることがより好ましい。 (Pea protein hydrolyzate)
Pea protein hydrolyzate is a peptide mixture prepared by hydrolyzing a pea protein material. The pea protein hydrolyzate preferably has a higher degree of degradation, and in particular, the proportion of dipeptides and tripeptides in the total amount of peptides and free amino acids in the hydrolyzate is preferably high. Specifically, the proportion of dipeptides and tripeptides in the total amount of peptides and free amino acids is preferably 40% by weight or more. It is more preferably 60% by weight or more, and most preferably 64% by weight or more.
In the present application, dipeptides and tripeptides are defined as a fraction obtained by removing free amino acids from a fraction having a molecular weight of 500 or less. Therefore, the proportion of dipeptides and tripeptides in the total amount of peptides and free amino acids was measured by amino acid analysis after measuring the proportion of peptide fractions with a molecular weight of 500 or less in the hydrolyzate by gel filtration chromatography for peptides. It can be calculated by subtracting the free amino acid content in the hydrolyzate. The content of free amino acids in the hydrolyzate is preferably 10% by weight or less, and more preferably 5% by weight or less. Furthermore, since it is desirable that the peptide body has a lower molecular weight, the proportion of the fraction having a molecular weight of 500 or more in the total amount of peptide and free amino acid in the hydrolyzate is preferably 40% by weight or less, More preferably, it is not more than wt%.
（プロテアーゼ）
加水分解はプロテアーゼ処理により行われることが好ましい。プロテアーゼ処理は上記エンドウ蛋白質素材のスラリー又は水溶液を基質とし、以下に挙げるプロテアーゼで処理を行なう。ここで用いるプロテアーゼは動物起源，植物起源あるいは微生物起源は問わず、プロテアーゼの分類において「金属プロテアーゼ」、「酸性プロテアーゼ」、「チオールプロテアーゼ」、「セリンプロテアーゼ」に分類されるプロテアーゼ、好ましくは「金属プロテアーゼ」、「チオールプロテアーゼ」、「セリンプロテアーゼ」に分類されるプロテアーゼの中から適宜選択する事ができる。特に２種類以上、あるいは３種類以上の異なった分類に属する酵素を、順次若しくは同時に作用させる分解方法がジペプチドやトリペプチド等の比較的分子量の低いペプチドの割合を増加させる事ができ好ましい。 (Protease)
Hydrolysis is preferably performed by protease treatment. Protease treatment is carried out using the above-mentioned pea protein material slurry or aqueous solution as a substrate and the following proteases. The protease used here is of animal origin, plant origin or microbial origin, and is classified into “metal protease”, “acidic protease”, “thiol protease”, “serine protease” in the classification of protease, preferably “metal” It can be appropriately selected from proteases classified as “protease”, “thiol protease”, and “serine protease”. Particularly, a degradation method in which enzymes belonging to two or more kinds or three or more kinds of different classes are acted on sequentially or simultaneously can increase the proportion of relatively low molecular weight peptides such as dipeptides and tripeptides.
加水分解はプロテアーゼ処理により行われることが好ましい。プロテアーゼ処理は上記エンドウ蛋白質素材のスラリー又は水溶液を基質とし、以下に挙げるプロテアーゼで処理を行なう。ここで用いるプロテアーゼは動物起源，植物起源あるいは微生物起源は問わず、プロテアーゼの分類において「金属プロテアーゼ」、「酸性プロテアーゼ」、「チオールプロテアーゼ」、「セリンプロテアーゼ」に分類されるプロテアーゼ、好ましくは「金属プロテアーゼ」、「チオールプロテアーゼ」、「セリンプロテアーゼ」に分類されるプロテアーゼの中から適宜選択する事ができる。特に２種類以上、あるいは３種類以上の異なった分類に属する酵素を、順次若しくは同時に作用させる分解方法がジペプチドやトリペプチド等の比較的分子量の低いペプチドの割合を増加させる事ができ好ましい。 (Protease)
Hydrolysis is preferably performed by protease treatment. Protease treatment is carried out using the above-mentioned pea protein material slurry or aqueous solution as a substrate and the following proteases. The protease used here is of animal origin, plant origin or microbial origin, and is classified into “metal protease”, “acidic protease”, “thiol protease”, “serine protease” in the classification of protease, preferably “metal” It can be appropriately selected from proteases classified as “protease”, “thiol protease”, and “serine protease”. Particularly, a degradation method in which enzymes belonging to two or more kinds or three or more kinds of different classes are acted on sequentially or simultaneously can increase the proportion of relatively low molecular weight peptides such as dipeptides and tripeptides.
このプロテアーゼの分類は、酵素科学の分野において通常行なわれている活性中心のアミノ酸の種類による分類方法である。各々の代表として「金属プロテアーゼ」にはBacillus中性プロテアーゼ，Streptomyces中性プロテアーゼ，Aspergillus中性プロテアーゼ，『サモアーゼ』等、「酸性プロテアーゼ」にはペプシン，Aspergillus酸性プロテアーゼ，『スミチュームAP』等、「チオールプロテアーゼ」にはブロメライン，パパイン等、「セリンプロテアーゼ」にはトリプシン，キモトリプシン，ズブチリシン，Streptomycesアルカリプロテアーゼ，『アルカラーゼ』，『ビオプラーゼ』等が挙げられる。これら以外の酵素でも作用pHや阻害剤との反応性により、その分類を確認する事ができる。活性中心が異なる酵素間では、基質への作用部位が大きく異なるため、「切れ残り」を減らし、効率よく酵素分解物を得る事ができる。あるいは異なった起源の（起源生物） の酵素を併用する事で、更に効率よく酵素分解物を製造する事ができる。同分類でも起源が異なれば、基質である蛋白質への作用部位も異なり、結果としてジペプチドやトリペプチドの割合を増やす事ができる。これらプロテアーゼはエキソ活性が少ない事が好ましい。
This classification of protease is a classification method based on the type of amino acid at the active center, which is usually performed in the field of enzyme science. As representatives of each, “Metal Protease” includes Bacillus Neutral Protease, Streptomyces Neutral Protease, Aspergillus Neutral Protease, “Samoase”, etc. “Protease” includes bromelain, papain and the like, and “serine protease” includes trypsin, chymotrypsin, subtilisin, Streptomyces alkaline protease, “Alcalase”, “Biolase” and the like. The classification of other enzymes can also be confirmed by their pH and reactivity with inhibitors. Since enzymes having different active centers differ greatly in the site of action on the substrate, it is possible to reduce the “uncut residue” and efficiently obtain an enzyme degradation product. Alternatively, enzymatic degradation products can be more efficiently produced by using enzymes from different origins (origin organisms). Even in the same classification, if the origin is different, the site of action of the substrate protein is also different, and as a result, the proportion of dipeptides and tripeptides can be increased. These proteases preferably have low exo activity.
プロテアーゼ処理の反応pHや反応温度は、用いるプロテアーゼの特性に合わせて設定すれば良く、通常反応pHは至適pH付近で行ない、反応温度は至適温度付近で行なえば良い。概ね反応温度は20～80℃、好ましくは40～60℃で反応を行なう事ができる。反応後は酵素を失活するに十分な温度（60～170℃程度）で加熱し、残存酵素活性を失活させる。
The reaction pH and reaction temperature of the protease treatment may be set in accordance with the characteristics of the protease to be used, and the reaction pH is usually carried out near the optimum pH, and the reaction temperature may be carried out around the optimum temperature. In general, the reaction can be carried out at a reaction temperature of 20 to 80 ° C, preferably 40 to 60 ° C. After the reaction, the enzyme is heated at a temperature sufficient to inactivate the enzyme (about 60 to 170 ° C.) to inactivate the remaining enzyme activity.
プロテアーゼ処理後の反応液は、そのまま又は濃縮して用いることもできるが、通常、殺菌して噴霧乾燥、凍結乾燥等して乾燥粉末の状態で利用する事ができる。殺菌は、加熱殺菌が好ましく、加熱温度は110～170℃が好ましく、130～170℃が更に好ましい。加熱時間は３～20秒間が好ましい。また反応液を任意のpHに調整しておくこともでき、pH調整時に発生する沈殿物や懸濁物を遠心分離や濾過等により除去することもできる。また更に活性炭や吸着樹脂により精製することもできる。
The reaction solution after the protease treatment can be used as it is or after being concentrated, but it can usually be used in the form of a dry powder by sterilization, spray drying, freeze drying or the like. Sterilization is preferably heat sterilization, and the heating temperature is preferably 110 to 170 ° C, more preferably 130 to 170 ° C. The heating time is preferably 3 to 20 seconds. In addition, the reaction solution can be adjusted to an arbitrary pH, and precipitates and suspensions generated during pH adjustment can be removed by centrifugation, filtration, or the like. Furthermore, it can also refine | purify with activated carbon or adsorption resin.
（利用）
本発明のエンドウ蛋白質加水分解物を主成分とする蛋白質組成物の加水分解物は、医薬品の形態として広く使用できるが、食品素材に由来する特長を生かす意味でも、経口性の形態、すなわち食品または飼料に添加された形態として、必要により適宜その他の原材料と混合して使用することができる。医薬品の形態として供する場合は、液体，散薬，錠剤，カプセル等の種々の形態で使用することができる。食品に混合された形態として供する場合は、ビスケット，ケーキ，パン等の固形状食品に混合して使用しても差し支えなく、または、ババロア、ムースまたはプリンのような流動状，半固形状食品に混合しても問題ない。また、水等に溶解して飲料として、摂取することもできる。 (Use)
The hydrolyzate of the protein composition mainly composed of the pea protein hydrolyzate of the present invention can be widely used as a pharmaceutical form. However, the oral form, ie, food or As a form added to the feed, it can be appropriately mixed with other raw materials as necessary. When used as a pharmaceutical form, it can be used in various forms such as liquid, powder, tablet, capsule and the like. When used as a mixed form in foods, it can be used by mixing with solid foods such as biscuits, cakes, breads, etc., or it can be used for fluid or semi-solid foods such as bavaria, mousse or pudding. There is no problem with mixing. It can also be taken as a beverage by dissolving in water or the like.
本発明のエンドウ蛋白質加水分解物を主成分とする蛋白質組成物の加水分解物は、医薬品の形態として広く使用できるが、食品素材に由来する特長を生かす意味でも、経口性の形態、すなわち食品または飼料に添加された形態として、必要により適宜その他の原材料と混合して使用することができる。医薬品の形態として供する場合は、液体，散薬，錠剤，カプセル等の種々の形態で使用することができる。食品に混合された形態として供する場合は、ビスケット，ケーキ，パン等の固形状食品に混合して使用しても差し支えなく、または、ババロア、ムースまたはプリンのような流動状，半固形状食品に混合しても問題ない。また、水等に溶解して飲料として、摂取することもできる。 (Use)
The hydrolyzate of the protein composition mainly composed of the pea protein hydrolyzate of the present invention can be widely used as a pharmaceutical form. However, the oral form, ie, food or As a form added to the feed, it can be appropriately mixed with other raw materials as necessary. When used as a pharmaceutical form, it can be used in various forms such as liquid, powder, tablet, capsule and the like. When used as a mixed form in foods, it can be used by mixing with solid foods such as biscuits, cakes, breads, etc., or it can be used for fluid or semi-solid foods such as bavaria, mousse or pudding. There is no problem with mixing. It can also be taken as a beverage by dissolving in water or the like.
本発明の経口性抗炎症機能剤は、抗炎症効果を示す他の飲食品や成分と併用して使用することが出来る。中でも、乳酸菌発酵を利用した食品や飲料、例えば乳酸菌飲料，ヨーグルト類，チーズ類に使用する場合、特に強い効果を得ることができる。また、併用できる他の抗炎症効果を示す成分として、例えば、ポリフェノール類（茶カテキン、アントシアニン、シアニジン、クロロゲン酸など）、カロテノイド類（アスタキサンチン、リコピン、ルテインなど）、グルコサミン、コンドロイチン、クルクミン、カプサイシンなどを例示することが出来る。ただし、食品の殺菌の際の熱により本発明のペプチドが分解等の変性する可能性もあり、好ましくは、糖質，ビタミン，ミネラル等を混合してサプリメントとして利用するのがよい。飼料に混合された形態として供する場合は、陸産，水産に限定されることなく、既知の飼料と混合して使用することができる。
The oral anti-inflammatory functional agent of the present invention can be used in combination with other foods and drinks and ingredients exhibiting an anti-inflammatory effect. Especially, when using for foods and drinks using lactic acid bacteria fermentation, for example, lactic acid bacteria drinks, yogurts, and cheeses, a particularly strong effect can be obtained. Examples of other anti-inflammatory effects that can be used in combination include polyphenols (tea catechin, anthocyanin, cyanidin, chlorogenic acid, etc.), carotenoids (astaxanthin, lycopene, lutein, etc.), glucosamine, chondroitin, curcumin, capsaicin, etc. Can be illustrated. However, there is a possibility that the peptide of the present invention may be denatured by decomposition or the like due to heat at the time of sterilization of food. Preferably, carbohydrates, vitamins, minerals and the like are mixed and used as a supplement. When provided as a form mixed with feed, it can be used by mixing with known feed without being limited to land and fishery.
乳酸菌飲料等の乳酸菌発酵食品に使用する乳酸菌としては、食品として一般に使用される乳酸菌を使用することが出来る。例えば、Lactobacillus属菌（L.casei, L.plantarum, L. brevis, l.acidophilus, L.pentousなど）、Lactococcus属菌（L.lactis, L.cremorisなど）、Bifidobacterium属菌（B.bididum, B.adolescentisなど）を例示使用することが出来る。
As lactic acid bacteria used for lactic acid bacteria fermented foods such as lactic acid bacteria beverages, lactic acid bacteria generally used as foods can be used. For example, Lactobacillus genus bacteria (L.casei, L.plantarum, L.brevis, l.acidophilus, L.pentous etc.), Lactococcus genus bacteria (L.lactis, L.cremoris etc.), Bifidobacterium genus bacteria (B.bididum, B.adolescentis etc.) can be used as an example.
As lactic acid bacteria used for lactic acid bacteria fermented foods such as lactic acid bacteria beverages, lactic acid bacteria generally used as foods can be used. For example, Lactobacillus genus bacteria (L.casei, L.plantarum, L.brevis, l.acidophilus, L.pentous etc.), Lactococcus genus bacteria (L.lactis, L.cremoris etc.), Bifidobacterium genus bacteria (B.bididum, B.adolescentis etc.) can be used as an example.
以下、実施例により本発明の実施態様を具体的に説明する。
Hereinafter, embodiments of the present invention will be described specifically by way of examples.
（実施例１）分離エンドウ蛋白質の調製
非特許文献１に記載の方法に準じて以下の通り、エンドウ蛋白質素材である分離エンドウ蛋白質を調製した。つまり、50gのエンドウ種子粉砕物を500mlの水に溶解分散させ、pH9.0の条件で50℃、1hr抽出した。遠心分離機にて繊維画分とエンドウ豆乳に分離した。得られた豆乳のpHを4.5に調整し、遠心分離機にて沈殿カード画分を回収した。この画分を中和し、120℃で10秒間加熱殺菌を行い、分離エンドウ蛋白質を調製した。 Example 1 Preparation of Isolated Pea Protein According to the method described in Non-Patent Document 1, a separated pea protein as a pea protein material was prepared as follows. That is, 50 g of pea seed pulverized product was dissolved and dispersed in 500 ml of water, and extracted at 50 ° C. for 1 hr under the condition of pH 9.0. The fiber fraction and pea soymilk were separated using a centrifuge. The pH of the obtained soymilk was adjusted to 4.5, and the precipitated curd fraction was collected with a centrifuge. This fraction was neutralized and sterilized by heating at 120 ° C. for 10 seconds to prepare a separated pea protein.
非特許文献１に記載の方法に準じて以下の通り、エンドウ蛋白質素材である分離エンドウ蛋白質を調製した。つまり、50gのエンドウ種子粉砕物を500mlの水に溶解分散させ、pH9.0の条件で50℃、1hr抽出した。遠心分離機にて繊維画分とエンドウ豆乳に分離した。得られた豆乳のpHを4.5に調整し、遠心分離機にて沈殿カード画分を回収した。この画分を中和し、120℃で10秒間加熱殺菌を行い、分離エンドウ蛋白質を調製した。 Example 1 Preparation of Isolated Pea Protein According to the method described in Non-Patent Document 1, a separated pea protein as a pea protein material was prepared as follows. That is, 50 g of pea seed pulverized product was dissolved and dispersed in 500 ml of water, and extracted at 50 ° C. for 1 hr under the condition of pH 9.0. The fiber fraction and pea soymilk were separated using a centrifuge. The pH of the obtained soymilk was adjusted to 4.5, and the precipitated curd fraction was collected with a centrifuge. This fraction was neutralized and sterilized by heating at 120 ° C. for 10 seconds to prepare a separated pea protein.
（実施例２）エンドウ蛋白質加水分解物の調製
実施例１で得られた分離エンドウ蛋白質について、以下のようにプロテアーゼによる酵素分解物を調製した。
３重量%の分離エンドウ蛋白質の溶液に対して、『サモアーゼ』（起源;Bacillus thermoproteolyticus、金属プロテアーゼ、大和化成）を対蛋白質当たり２重量%加え、pH9.0，58℃で60分間作用させた。次に『ビオプラーゼ』（起源;Bacillus sp., セリンプロテアーゼ、ナガセケムテック）を対蛋白質当たり１重量%加え、pH7.5に、58℃で60分間作用させた。次に『スミチュームFP』（起源;Aspergillus sp., 金属プロテアーゼ，新日本化学工業）を対蛋白質当たり１重量%加え、pH7.5, 58℃で60分間作用させた。以上の処理の後、90℃, 20分で反応を停止した後、凍結乾燥し、エンドウ蛋白質加水分解物試料とした。 (Example 2) Preparation of pea protein hydrolyzate For the isolated pea protein obtained in Example 1, an enzymatic degradation product by protease was prepared as follows.
To a 3% by weight separated pea protein solution, “Samoase” (origin; Bacillus thermoproteolyticus, metalloprotease, Daiwa Kasei) was added at 2% by weight per protein and allowed to act at pH 9.0, 58 ° C. for 60 minutes. Next, “Biolase” (origin; Bacillus sp., Serine protease, Nagase Chemtech) was added at 1% by weight per protein and allowed to act at pH 7.5 for 60 minutes at 58 ° C. Next, “Sumitum FP” (source: Aspergillus sp., Metalloprotease, Shin Nippon Chemical Industry) was added at 1% by weight per protein and allowed to act at pH 7.5, 58 ° C. for 60 minutes. After the above treatment, the reaction was stopped at 90 ° C. for 20 minutes, and then freeze-dried to obtain a pea protein hydrolyzate sample.
実施例１で得られた分離エンドウ蛋白質について、以下のようにプロテアーゼによる酵素分解物を調製した。
３重量%の分離エンドウ蛋白質の溶液に対して、『サモアーゼ』（起源;Bacillus thermoproteolyticus、金属プロテアーゼ、大和化成）を対蛋白質当たり２重量%加え、pH9.0，58℃で60分間作用させた。次に『ビオプラーゼ』（起源;Bacillus sp., セリンプロテアーゼ、ナガセケムテック）を対蛋白質当たり１重量%加え、pH7.5に、58℃で60分間作用させた。次に『スミチュームFP』（起源;Aspergillus sp., 金属プロテアーゼ，新日本化学工業）を対蛋白質当たり１重量%加え、pH7.5, 58℃で60分間作用させた。以上の処理の後、90℃, 20分で反応を停止した後、凍結乾燥し、エンドウ蛋白質加水分解物試料とした。 (Example 2) Preparation of pea protein hydrolyzate For the isolated pea protein obtained in Example 1, an enzymatic degradation product by protease was prepared as follows.
To a 3% by weight separated pea protein solution, “Samoase” (origin; Bacillus thermoproteolyticus, metalloprotease, Daiwa Kasei) was added at 2% by weight per protein and allowed to act at pH 9.0, 58 ° C. for 60 minutes. Next, “Biolase” (origin; Bacillus sp., Serine protease, Nagase Chemtech) was added at 1% by weight per protein and allowed to act at pH 7.5 for 60 minutes at 58 ° C. Next, “Sumitum FP” (source: Aspergillus sp., Metalloprotease, Shin Nippon Chemical Industry) was added at 1% by weight per protein and allowed to act at pH 7.5, 58 ° C. for 60 minutes. After the above treatment, the reaction was stopped at 90 ° C. for 20 minutes, and then freeze-dried to obtain a pea protein hydrolyzate sample.
（実施例３）分離大豆蛋白質の調製
低変性脱脂大豆から以下のように分離大豆蛋白質を調製した。低変性脱脂大豆１kgの温水抽出スラリーを遠心分離機にてオカラ画分を除き脱脂豆乳とした。次に、得られた脱脂豆乳のpHを4.5に調整して等電点沈殿し、遠心分離機にて酸沈殿カードを得て中和した。更に、得られた各画分を中和して、120℃で10秒間加熱殺菌を行った。 (Example 3) Preparation of isolated soybean protein Isolated soybean protein was prepared from low-denatured defatted soybean as follows. A 1 kg kg of low-denatured defatted soybean was extracted with a hot water slurry to obtain defatted soymilk by removing the okara fraction with a centrifuge. Next, the pH of the obtained defatted soymilk was adjusted to 4.5 and subjected to isoelectric precipitation, and an acid precipitation card was obtained and neutralized with a centrifuge. Furthermore, each obtained fraction was neutralized and sterilized by heating at 120 ° C. for 10 seconds.
低変性脱脂大豆から以下のように分離大豆蛋白質を調製した。低変性脱脂大豆１kgの温水抽出スラリーを遠心分離機にてオカラ画分を除き脱脂豆乳とした。次に、得られた脱脂豆乳のpHを4.5に調整して等電点沈殿し、遠心分離機にて酸沈殿カードを得て中和した。更に、得られた各画分を中和して、120℃で10秒間加熱殺菌を行った。 (Example 3) Preparation of isolated soybean protein Isolated soybean protein was prepared from low-denatured defatted soybean as follows. A 1 kg kg of low-denatured defatted soybean was extracted with a hot water slurry to obtain defatted soymilk by removing the okara fraction with a centrifuge. Next, the pH of the obtained defatted soymilk was adjusted to 4.5 and subjected to isoelectric precipitation, and an acid precipitation card was obtained and neutralized with a centrifuge. Furthermore, each obtained fraction was neutralized and sterilized by heating at 120 ° C. for 10 seconds.
（実施例４）大豆蛋白質加水分解物の調製
製造例３で得られた分離大豆蛋白質を製造例２と同様に処理した試料を大豆蛋白質加水分解物とした。 Example 4 Preparation of Soy Protein Hydrolyzate A sample obtained by treating the isolated soy protein obtained in Production Example 3 in the same manner as in Production Example 2 was used as a soy protein hydrolysate.
製造例３で得られた分離大豆蛋白質を製造例２と同様に処理した試料を大豆蛋白質加水分解物とした。 Example 4 Preparation of Soy Protein Hydrolyzate A sample obtained by treating the isolated soy protein obtained in Production Example 3 in the same manner as in Production Example 2 was used as a soy protein hydrolysate.
（実施例５）他の食品由来蛋白質加水分解物
他の食品由来蛋白質の加水分解物と比較するために、市販品を入手した。乳蛋白の加水分解物として、カゼイン加水分解物のCE90M及びPeptopro（DMV社製）、乳清ホエー加水分解物としてHW-3（雪印乳業社製）及びThermax690（Glanbia社製）、コラーゲン加水分解物としてFSP-AS-L(ニッピ社製)及びChicken collagen(日本ハムヘルスクリエイト社製）を入手し、試料とした。 (Example 5) Other food-derived protein hydrolysates For comparison with other food-derived protein hydrolysates, commercially available products were obtained. Casein hydrolyzate CE90M and Peptopro (manufactured by DMV) as whey protein hydrolysates, HW-3 (manufactured by Snow Brand Milk Products) and Thermox690 (manufactured by Glanbia), collagen hydrolysates as whey whey hydrolysates FSP-AS-L (Nippi) and Chicken collagen (Nippon Ham Health Create) were obtained as samples.
他の食品由来蛋白質の加水分解物と比較するために、市販品を入手した。乳蛋白の加水分解物として、カゼイン加水分解物のCE90M及びPeptopro（DMV社製）、乳清ホエー加水分解物としてHW-3（雪印乳業社製）及びThermax690（Glanbia社製）、コラーゲン加水分解物としてFSP-AS-L(ニッピ社製)及びChicken collagen(日本ハムヘルスクリエイト社製）を入手し、試料とした。 (Example 5) Other food-derived protein hydrolysates For comparison with other food-derived protein hydrolysates, commercially available products were obtained. Casein hydrolyzate CE90M and Peptopro (manufactured by DMV) as whey protein hydrolysates, HW-3 (manufactured by Snow Brand Milk Products) and Thermox690 (manufactured by Glanbia), collagen hydrolysates as whey whey hydrolysates FSP-AS-L (Nippi) and Chicken collagen (Nippon Ham Health Create) were obtained as samples.
（蛋白質含量（CP）の測定）
105℃,12時間乾燥した各種蛋白質素材の乾燥重量に対して、ケルダール法により測定した粗蛋白質量の重量を、重量%で表した。尚、窒素係数は6.25とした。 (Measurement of protein content (CP))
The weight of the crude protein mass measured by the Kjeldahl method with respect to the dry weight of various protein materials dried at 105 ° C. for 12 hours was expressed in wt%. The nitrogen coefficient was 6.25.
105℃,12時間乾燥した各種蛋白質素材の乾燥重量に対して、ケルダール法により測定した粗蛋白質量の重量を、重量%で表した。尚、窒素係数は6.25とした。 (Measurement of protein content (CP))
The weight of the crude protein mass measured by the Kjeldahl method with respect to the dry weight of various protein materials dried at 105 ° C. for 12 hours was expressed in wt%. The nitrogen coefficient was 6.25.
（抗炎症効果評価のための細胞培養）
各食品由来蛋白質加水分解物のマクロファージに対する抗炎症効果を評価するために下記の手法により評価を行なった。細胞はマウスマクロファージ細胞J774.1（RIKEN Bioresource center）を使用し、基本培地としてRPMI-1690（和光純薬社製）を選択し、これに終濃度10%となるようにウシ血清（FBS）を添加し、抗生物質として終濃度100IU/mL Penicillin及び100μg/ ml Streptomycinを添加し細胞培養用培地とした。
J774.1細胞を細胞培養用培地にて37℃, ５%CO2条件下で培養し、10cmシャーレでコンフルエントになるまで培養を行った。コンフルエントに達した細胞を回収し、細胞数をカウントした上で１×105個/mlになるように希釈し、24穴細胞培養用プレートに１mlずつ添加した。添加後２日間37℃, ５%CO2濃度で培養し、培地をアスピレーターにて除去した。除去後、各規定濃度に調製した大豆蛋白質加水分解物を350ml、及び炎症誘導物質としてCpGを終濃度200nmol/Lになるように添加し24時間、37℃、５%CO2濃度で培養した。 (Cell culture for anti-inflammatory effect evaluation)
In order to evaluate the anti-inflammatory effect of each food-derived protein hydrolyzate on macrophages, the following methods were used for evaluation. Use mouse macrophage cell J774.1 (RIKEN Bioresource center) as the cell, select RPMI-1690 (manufactured by Wako Pure Chemical Industries, Ltd.) as the basic medium, and add bovine serum (FBS) to a final concentration of 10%. Then, final concentrations of 100 IU / mL Penicillin and 100 μg / ml Streptomycin were added as antibiotics to obtain a cell culture medium.
J774.1 cells were cultured in a cell culture medium under conditions of 37 ° C. and 5% CO 2 and cultured in a 10 cm petri dish until confluent. The cells that reached confluence were collected, the number of cells was counted, diluted to 1 × 10 5 cells / ml, and 1 ml each was added to a 24-well cell culture plate. After the addition, the cells were cultured for 2 days at 37 ° C. and 5% CO 2 concentration, and the medium was removed with an aspirator. After the removal, 350 ml of soy protein hydrolyzate prepared to each specified concentration and CpG as an inflammation inducer to a final concentration of 200 nmol / L were added and cultured for 24 hours at 37 ° C., 5% CO 2 concentration.
各食品由来蛋白質加水分解物のマクロファージに対する抗炎症効果を評価するために下記の手法により評価を行なった。細胞はマウスマクロファージ細胞J774.1（RIKEN Bioresource center）を使用し、基本培地としてRPMI-1690（和光純薬社製）を選択し、これに終濃度10%となるようにウシ血清（FBS）を添加し、抗生物質として終濃度100IU/mL Penicillin及び100μg/ ml Streptomycinを添加し細胞培養用培地とした。
J774.1細胞を細胞培養用培地にて37℃, ５%CO2条件下で培養し、10cmシャーレでコンフルエントになるまで培養を行った。コンフルエントに達した細胞を回収し、細胞数をカウントした上で１×105個/mlになるように希釈し、24穴細胞培養用プレートに１mlずつ添加した。添加後２日間37℃, ５%CO2濃度で培養し、培地をアスピレーターにて除去した。除去後、各規定濃度に調製した大豆蛋白質加水分解物を350ml、及び炎症誘導物質としてCpGを終濃度200nmol/Lになるように添加し24時間、37℃、５%CO2濃度で培養した。 (Cell culture for anti-inflammatory effect evaluation)
In order to evaluate the anti-inflammatory effect of each food-derived protein hydrolyzate on macrophages, the following methods were used for evaluation. Use mouse macrophage cell J774.1 (RIKEN Bioresource center) as the cell, select RPMI-1690 (manufactured by Wako Pure Chemical Industries, Ltd.) as the basic medium, and add bovine serum (FBS) to a final concentration of 10%. Then, final concentrations of 100 IU / mL Penicillin and 100 μg / ml Streptomycin were added as antibiotics to obtain a cell culture medium.
J774.1 cells were cultured in a cell culture medium under conditions of 37 ° C. and 5% CO 2 and cultured in a 10 cm petri dish until confluent. The cells that reached confluence were collected, the number of cells was counted, diluted to 1 × 10 5 cells / ml, and 1 ml each was added to a 24-well cell culture plate. After the addition, the cells were cultured for 2 days at 37 ° C. and 5% CO 2 concentration, and the medium was removed with an aspirator. After the removal, 350 ml of soy protein hydrolyzate prepared to each specified concentration and CpG as an inflammation inducer to a final concentration of 200 nmol / L were added and cultured for 24 hours at 37 ° C., 5% CO 2 concentration.
（ELISAによるIL-6濃度の測定）
培地中のIL-6濃度をELISA法により測定するために、Purified rat anti-mouse IL-6（１次抗体）、Biotinylated rat anti-mouse IL-6（２次抗体）、Recombinant mouse IL-6（標準IL-6）及びStreptavidin-Alkalin PhosphateはすべてBD Pharmingenより購入し使用した。
１次抗体を0.1M Na2HPO4 buffer（pH9.0）で0.5μg/mlになるように調製したものを96ウェルマイクロプレートに50μl/wellずつ添加し室温で2hr静置した。PBS-Tween20（PBS-T）で３回washし十分に水切りを行った。その後、3%BSA in PBS-Tを100μl/wellずつ添加し室温で１hr静置後、PBS-Tで３回washし十分に水切りを行った。PBS-Tで適宜希釈したサンプルを50μl/wellずつ添加し、4℃で24時間放置後、PBS-Tで３回washし十分に水切りを行った。PBS-Tで0.5μg/mlに調製した２次抗体を50μl/wellずつ添加し、アルミホイルで遮光して室温で1hr静置後、PBS-Tで3回washし十分に水切りを行った。Streptavidin-Alkaline Phosphate in PBS-Tを50μl/wellずつ添加し、アルミホイルで遮光して室温で1hr静置後PBS-Tで３回washし十分に水切りを行った。0.2mg/ml 4-ニトロフェニルリン酸2ナトリウム in 1M ジエタノールアミン bufferを50μl/wellずつ入れて、アルミホイルで遮光して10分間静置後、マイクロプレートリーダーにて405nm（参考波長492nm）の吸光度を２回測定し、平均値を算出した。 (Measurement of IL-6 concentration by ELISA)
In order to measure the IL-6 concentration in the culture medium by ELISA, Purified rat anti-mouse IL-6 (primary antibody), Biotinylated rat anti-mouse IL-6 (secondary antibody), Recombinant mouse IL-6 ( Standard IL-6) and Streptavidin-Alkalin Phosphate were all purchased from BD Pharmingen and used.
The primary antibody prepared with 0.1 M Na2HPO4 buffer (pH 9.0) to 0.5 μg / ml was added to a 96-well microplate at 50 μl / well and allowed to stand at room temperature for 2 hr. The plate was washed 3 times with PBS-Tween20 (PBS-T) and thoroughly drained. Thereafter, 3% BSA in PBS-T was added in an amount of 100 μl / well, and allowed to stand at room temperature for 1 hr, and then washed 3 times with PBS-T to thoroughly drain water. Samples diluted appropriately with PBS-T were added in an amount of 50 μl / well, and allowed to stand at 4 ° C. for 24 hours, and then washed 3 times with PBS-T to drain thoroughly. Secondary antibody prepared to 0.5 μg / ml with PBS-T was added at 50 μl / well, and after light-shielding with aluminum foil and allowed to stand at room temperature for 1 hr, it was washed 3 times with PBS-T and drained thoroughly. Streptavidin-Alkaline Phosphate in PBS-T was added in an amount of 50 μl / well, light-shielded with aluminum foil, allowed to stand at room temperature for 1 hr, and then washed 3 times with PBS-T to thoroughly drain water. Add 0.2 mg / ml of disodium 4-nitrophenyl phosphate in 1 M diethanolamine buffer at 50 μl / well, leave light-shielded with aluminum foil for 10 minutes, and then absorb the absorbance at 405 nm (reference wavelength 492 nm) with a microplate reader. Measurement was performed twice and the average value was calculated.
培地中のIL-6濃度をELISA法により測定するために、Purified rat anti-mouse IL-6（１次抗体）、Biotinylated rat anti-mouse IL-6（２次抗体）、Recombinant mouse IL-6（標準IL-6）及びStreptavidin-Alkalin PhosphateはすべてBD Pharmingenより購入し使用した。
１次抗体を0.1M Na2HPO4 buffer（pH9.0）で0.5μg/mlになるように調製したものを96ウェルマイクロプレートに50μl/wellずつ添加し室温で2hr静置した。PBS-Tween20（PBS-T）で３回washし十分に水切りを行った。その後、3%BSA in PBS-Tを100μl/wellずつ添加し室温で１hr静置後、PBS-Tで３回washし十分に水切りを行った。PBS-Tで適宜希釈したサンプルを50μl/wellずつ添加し、4℃で24時間放置後、PBS-Tで３回washし十分に水切りを行った。PBS-Tで0.5μg/mlに調製した２次抗体を50μl/wellずつ添加し、アルミホイルで遮光して室温で1hr静置後、PBS-Tで3回washし十分に水切りを行った。Streptavidin-Alkaline Phosphate in PBS-Tを50μl/wellずつ添加し、アルミホイルで遮光して室温で1hr静置後PBS-Tで３回washし十分に水切りを行った。0.2mg/ml 4-ニトロフェニルリン酸2ナトリウム in 1M ジエタノールアミン bufferを50μl/wellずつ入れて、アルミホイルで遮光して10分間静置後、マイクロプレートリーダーにて405nm（参考波長492nm）の吸光度を２回測定し、平均値を算出した。 (Measurement of IL-6 concentration by ELISA)
In order to measure the IL-6 concentration in the culture medium by ELISA, Purified rat anti-mouse IL-6 (primary antibody), Biotinylated rat anti-mouse IL-6 (secondary antibody), Recombinant mouse IL-6 ( Standard IL-6) and Streptavidin-Alkalin Phosphate were all purchased from BD Pharmingen and used.
The primary antibody prepared with 0.1 M Na2HPO4 buffer (pH 9.0) to 0.5 μg / ml was added to a 96-well microplate at 50 μl / well and allowed to stand at room temperature for 2 hr. The plate was washed 3 times with PBS-Tween20 (PBS-T) and thoroughly drained. Thereafter, 3% BSA in PBS-T was added in an amount of 100 μl / well, and allowed to stand at room temperature for 1 hr, and then washed 3 times with PBS-T to thoroughly drain water. Samples diluted appropriately with PBS-T were added in an amount of 50 μl / well, and allowed to stand at 4 ° C. for 24 hours, and then washed 3 times with PBS-T to drain thoroughly. Secondary antibody prepared to 0.5 μg / ml with PBS-T was added at 50 μl / well, and after light-shielding with aluminum foil and allowed to stand at room temperature for 1 hr, it was washed 3 times with PBS-T and drained thoroughly. Streptavidin-Alkaline Phosphate in PBS-T was added in an amount of 50 μl / well, light-shielded with aluminum foil, allowed to stand at room temperature for 1 hr, and then washed 3 times with PBS-T to thoroughly drain water. Add 0.2 mg / ml of disodium 4-nitrophenyl phosphate in 1 M diethanolamine buffer at 50 μl / well, leave light-shielded with aluminum foil for 10 minutes, and then absorb the absorbance at 405 nm (reference wavelength 492 nm) with a microplate reader. Measurement was performed twice and the average value was calculated.
（ジ・トリペプチド含量）
各食品由来蛋白質加水分解物の分子量分布を、以下のゲルろ過カラムを用いたHPLC法により測定した。ペプチド用ゲルろ過カラムを用いたHPLCシステムを組み、分子量マーカーとなる既知のペプチドをチャージし、分子量と保持時間の関係において検量線を求めた。なお、分子量マーカーは、オクタペプチドとして[β-Asp]-Angiotensin IIのβ-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe（分子量1046）、ヘキサペプチドとしてAngiotensin IVのVal-Tyr-Ile-His-Pro-Phe（分子量775）、ペンタペプチドとしてLeu-EnkephalinのTyr-Gly-Gly-Phe-Leu（分子量555）、トリペプチドとしてGlu-Glu-Glu（分子量405）、遊離アミノ酸としてPro（分子量115）を用いた。加水分解物（１%）を10,000rpm、10分で遠心分離した上清を、ゲル濾過用溶媒で２倍に希釈し、その５μlをHPLCにアプライした。加水分解物中のペプチド及び遊離アミノ酸の合計量に占める分子量500以下のペプチド画分の割合（%）を、全体の吸光度のチャート面積に対する、分子量500以下の範囲（時間範囲）の面積の割合によって求めた（使用カラム：Superdex Peptide 7.5/300GL（GEヘルスケア・ジャパン株式会社製）。溶媒：１%SDS/10mMリン酸緩衝液, pH8.0, カラム温度25℃, 流速0.25ml/min, 検出波長：220nm）。 (Di-tripeptide content)
The molecular weight distribution of each food-derived protein hydrolyzate was measured by the HPLC method using the following gel filtration column. An HPLC system using a gel filtration column for peptides was assembled, a known peptide serving as a molecular weight marker was charged, and a calibration curve was obtained in relation to the molecular weight and the retention time. The molecular weight markers are [β-Asp] -Angiotensin II β-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (molecular weight 1046) as octapeptide and Angiotensin IV Val-Tyr- as hexapeptide. Ile-His-Pro-Phe (molecular weight 775), Leu-Enkephalin Tyr-Gly-Gly-Phe-Leu (molecular weight 555) as pentapeptide, Glu-Glu-Glu (molecular weight 405) as tripeptide, Pro as free amino acid (Molecular weight 115) was used. The supernatant obtained by centrifuging the hydrolyzate (1%) at 10,000 rpm for 10 minutes was diluted 2-fold with a solvent for gel filtration, and 5 μl thereof was applied to HPLC. The ratio (%) of the peptide fraction with a molecular weight of 500 or less to the total amount of peptide and free amino acid in the hydrolyzate is determined by the ratio of the area with a molecular weight of 500 or less (time range) to the total absorbance chart area. Determined (column used: Superdex Peptide 7.5 / 300GL (manufactured by GE Healthcare Japan). Solvent: 1% SDS / 10 mM phosphate buffer, pH 8.0, column temperature 25 ° C, flow rate 0.25 ml / min, detection Wavelength: 220 nm).
各食品由来蛋白質加水分解物の分子量分布を、以下のゲルろ過カラムを用いたHPLC法により測定した。ペプチド用ゲルろ過カラムを用いたHPLCシステムを組み、分子量マーカーとなる既知のペプチドをチャージし、分子量と保持時間の関係において検量線を求めた。なお、分子量マーカーは、オクタペプチドとして[β-Asp]-Angiotensin IIのβ-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe（分子量1046）、ヘキサペプチドとしてAngiotensin IVのVal-Tyr-Ile-His-Pro-Phe（分子量775）、ペンタペプチドとしてLeu-EnkephalinのTyr-Gly-Gly-Phe-Leu（分子量555）、トリペプチドとしてGlu-Glu-Glu（分子量405）、遊離アミノ酸としてPro（分子量115）を用いた。加水分解物（１%）を10,000rpm、10分で遠心分離した上清を、ゲル濾過用溶媒で２倍に希釈し、その５μlをHPLCにアプライした。加水分解物中のペプチド及び遊離アミノ酸の合計量に占める分子量500以下のペプチド画分の割合（%）を、全体の吸光度のチャート面積に対する、分子量500以下の範囲（時間範囲）の面積の割合によって求めた（使用カラム：Superdex Peptide 7.5/300GL（GEヘルスケア・ジャパン株式会社製）。溶媒：１%SDS/10mMリン酸緩衝液, pH8.0, カラム温度25℃, 流速0.25ml/min, 検出波長：220nm）。 (Di-tripeptide content)
The molecular weight distribution of each food-derived protein hydrolyzate was measured by the HPLC method using the following gel filtration column. An HPLC system using a gel filtration column for peptides was assembled, a known peptide serving as a molecular weight marker was charged, and a calibration curve was obtained in relation to the molecular weight and the retention time. The molecular weight markers are [β-Asp] -Angiotensin II β-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (molecular weight 1046) as octapeptide and Angiotensin IV Val-Tyr- as hexapeptide. Ile-His-Pro-Phe (molecular weight 775), Leu-Enkephalin Tyr-Gly-Gly-Phe-Leu (molecular weight 555) as pentapeptide, Glu-Glu-Glu (molecular weight 405) as tripeptide, Pro as free amino acid (Molecular weight 115) was used. The supernatant obtained by centrifuging the hydrolyzate (1%) at 10,000 rpm for 10 minutes was diluted 2-fold with a solvent for gel filtration, and 5 μl thereof was applied to HPLC. The ratio (%) of the peptide fraction with a molecular weight of 500 or less to the total amount of peptide and free amino acid in the hydrolyzate is determined by the ratio of the area with a molecular weight of 500 or less (time range) to the total absorbance chart area. Determined (column used: Superdex Peptide 7.5 / 300GL (manufactured by GE Healthcare Japan). Solvent: 1% SDS / 10 mM phosphate buffer, pH 8.0, column temperature 25 ° C, flow rate 0.25 ml / min, detection Wavelength: 220 nm).
次に、アミノ酸分析により各食品由来蛋白質加水分解物の遊離アミノ酸含量の測定を行った。加水分解物（4mg/ml）を等量の３%スルホサリチル酸に加え、室温で15分間振とうした。10,000rpmで10分間遠心分離し、得られた上澄みを0.45μmフィルターでろ過し、アミノ酸分析器（日本電子製 JLC500V）にて、遊離アミノ酸を測定した。蛋白質中の遊離アミノ酸含量はケルダール法にて得られた蛋白質含量に対する割合として算出した。
以上より得られた、「分子量500以下のペプチド画分の割合」から「遊離アミノ酸含量」を差し引いた値を、加水分解物中の「ジペプチド・トリペプチド含量」とした。 Next, the free amino acid content of each food-derived protein hydrolyzate was measured by amino acid analysis. Hydrolyzate (4 mg / ml) was added to an equal volume of 3% sulfosalicylic acid and shaken at room temperature for 15 minutes. Centrifugation was carried out at 10,000 rpm for 10 minutes, and the resulting supernatant was filtered through a 0.45 μm filter, and free amino acids were measured with an amino acid analyzer (JLC500V manufactured by JEOL Ltd.). The free amino acid content in the protein was calculated as a ratio to the protein content obtained by the Kjeldahl method.
The value obtained by subtracting the “free amino acid content” from the “ratio of the peptide fraction having a molecular weight of 500 or less” obtained above was defined as the “dipeptide / tripeptide content” in the hydrolyzate.
以上より得られた、「分子量500以下のペプチド画分の割合」から「遊離アミノ酸含量」を差し引いた値を、加水分解物中の「ジペプチド・トリペプチド含量」とした。 Next, the free amino acid content of each food-derived protein hydrolyzate was measured by amino acid analysis. Hydrolyzate (4 mg / ml) was added to an equal volume of 3% sulfosalicylic acid and shaken at room temperature for 15 minutes. Centrifugation was carried out at 10,000 rpm for 10 minutes, and the resulting supernatant was filtered through a 0.45 μm filter, and free amino acids were measured with an amino acid analyzer (JLC500V manufactured by JEOL Ltd.). The free amino acid content in the protein was calculated as a ratio to the protein content obtained by the Kjeldahl method.
The value obtained by subtracting the “free amino acid content” from the “ratio of the peptide fraction having a molecular weight of 500 or less” obtained above was defined as the “dipeptide / tripeptide content” in the hydrolyzate.
（統計処理）
有意差検定はDr.SPSSII（SPSS社製）を使用し、多群間の１元配置分散分析はTurkey-Kramer法を用いて評価を行った。 (Statistical processing)
Dr.SPSSII (manufactured by SPSS) was used for the significant difference test, and one-way analysis of variance between multiple groups was evaluated using the Turkey-Kramer method.
有意差検定はDr.SPSSII（SPSS社製）を使用し、多群間の１元配置分散分析はTurkey-Kramer法を用いて評価を行った。 (Statistical processing)
Dr.SPSSII (manufactured by SPSS) was used for the significant difference test, and one-way analysis of variance between multiple groups was evaluated using the Turkey-Kramer method.
（表１） 各種食品由来蛋白質加水分解物の分析値
(Table 1) Analytical values of various food-derived protein hydrolysates
（実施例６）マウスマクロファージ細胞に対する各食品由来蛋白質加水分解物の抗炎症効果
実施例２で得られたエンドウ蛋白質加水分解物及び実施例４で得られた大豆蛋白質加水分解物、及び各食品由来蛋白質加水分解物の抗炎症効果を評価するために、測定方法記載のマウスマクロファージ細胞培養及びELISA法によるIL-6濃度の測定を行った。 (Example 6) Anti-inflammatory effect of each food-derived protein hydrolyzate on mouse macrophage cells Pea protein hydrolyzate obtained in Example 2, soybean protein hydrolyzate obtained in Example 4, and each food-derived In order to evaluate the anti-inflammatory effect of the protein hydrolyzate, mouse macrophage cell culture described in the measurement method and IL-6 concentration were measured by ELISA.
実施例２で得られたエンドウ蛋白質加水分解物及び実施例４で得られた大豆蛋白質加水分解物、及び各食品由来蛋白質加水分解物の抗炎症効果を評価するために、測定方法記載のマウスマクロファージ細胞培養及びELISA法によるIL-6濃度の測定を行った。 (Example 6) Anti-inflammatory effect of each food-derived protein hydrolyzate on mouse macrophage cells Pea protein hydrolyzate obtained in Example 2, soybean protein hydrolyzate obtained in Example 4, and each food-derived In order to evaluate the anti-inflammatory effect of the protein hydrolyzate, mouse macrophage cell culture described in the measurement method and IL-6 concentration were measured by ELISA.
各蛋白質加水分解物の抗炎症効果を検証した結果、図１に示すようにエンドウ蛋白質加水分解物及び大豆蛋白質加水分解物において、Positive controlと比較してマクロファージの出すIL-6の量がポジティブコントロールと比較して有意に低下している様子が観察された。乳蛋白由来の加水分解物では全く抗炎症効果が認められず、中でも、CE90M，Peptopro及びHW-3は大豆蛋白質加水分解物１，２と同程度のジ・トリペプチド含量にも関わらず、抗炎症効果を示さなかった（表１）。これらのことから、特許文献２記載のチーズによる抗炎症効果は、エンドウと比較して低い効果しか持っていないか、チーズ生産時に使用する乳酸菌の代謝物に由来するものであると示唆された。エンドウ蛋白質は非特許文献１及び非特許文献２から、特許文献３に記載されているような抗炎症機能を発揮する11Sタイプのタンパク質(レグミンタイプ)が大豆蛋白質と比較して少ないため、抗炎症機能については低いと考えられたが、エンドウ蛋白質加水分解物が発揮した抗炎症機能は、大豆蛋白質加水分解物と同等以上のものだった。
As a result of verifying the anti-inflammatory effect of each protein hydrolyzate, the amount of IL-6 produced by macrophages is positive control in pea protein hydrolyzate and soybean protein hydrolyzate as compared with Positive control as shown in FIG. It was observed that there was a significant decrease compared to. Milk protein-derived hydrolysates have no anti-inflammatory effect, and CE90M, Peptopro and HW-3 have anti-anti-drug content comparable to that of soybean protein hydrolysates 1 and 2, despite the anti-inflammatory effect. There was no inflammatory effect (Table 1). From these, it was suggested that the anti-inflammatory effect by the cheese of patent document 2 has only a low effect compared with a pea, or originates in the metabolite of the lactic acid bacteria used at the time of cheese production. The pea protein has less anti-inflammatory function than non-patent document 1 and non-patent document 2 because 11S type protein (legmin type) that exhibits anti-inflammatory function as described in patent document 3 is less than soybean protein. However, the anti-inflammatory function exhibited by pea protein hydrolyzate was equivalent to or better than that of soybean protein hydrolyzate.
（実施例７）乳酸菌との併用効果
マウスマクロファージ細胞に対する抗炎症効果に関して、エンドウ蛋白質加水分解物と乳酸菌の併用効果を検証した。乳酸菌はLactobacillus caseiを使用し、測定方法記載のマウスマクロファージ細胞培養及びELISA法によるIL-6濃度の測定を行った。乳酸菌はCpG添加時に細胞数１×105個の濃度で添加し、これにエンドウ蛋白質加水分解物を添加する系と添加しない系を設定し、IL-6の濃度測定を行った。その結果、乳酸菌添加系でもマウスマクロファージ細胞が分泌するIL-6を低下させる様子が観察されたが、エンドウ蛋白質加水分解物と併用することによりその効果が増大することが確認された。 (Example 7) Combined effect with lactic acid bacteria Regarding the anti-inflammatory effect on mouse macrophage cells, the combined effect of pea protein hydrolyzate and lactic acid bacteria was verified. Lactobacillus casei was used as the lactic acid bacterium, and mouse macrophage cell culture described in the measurement method and IL-6 concentration were measured by ELISA. Lactic acid bacteria were added at a concentration of 1 × 10 5 cells when CpG was added, and a system in which pea protein hydrolyzate was added and a system in which pea protein hydrolyzate was not added were set, and IL-6 concentration was measured. As a result, it was observed that IL-6 secreted by mouse macrophage cells was reduced even in the lactic acid bacteria addition system, but it was confirmed that the effect was increased by using it together with pea protein hydrolysate.
マウスマクロファージ細胞に対する抗炎症効果に関して、エンドウ蛋白質加水分解物と乳酸菌の併用効果を検証した。乳酸菌はLactobacillus caseiを使用し、測定方法記載のマウスマクロファージ細胞培養及びELISA法によるIL-6濃度の測定を行った。乳酸菌はCpG添加時に細胞数１×105個の濃度で添加し、これにエンドウ蛋白質加水分解物を添加する系と添加しない系を設定し、IL-6の濃度測定を行った。その結果、乳酸菌添加系でもマウスマクロファージ細胞が分泌するIL-6を低下させる様子が観察されたが、エンドウ蛋白質加水分解物と併用することによりその効果が増大することが確認された。 (Example 7) Combined effect with lactic acid bacteria Regarding the anti-inflammatory effect on mouse macrophage cells, the combined effect of pea protein hydrolyzate and lactic acid bacteria was verified. Lactobacillus casei was used as the lactic acid bacterium, and mouse macrophage cell culture described in the measurement method and IL-6 concentration were measured by ELISA. Lactic acid bacteria were added at a concentration of 1 × 10 5 cells when CpG was added, and a system in which pea protein hydrolyzate was added and a system in which pea protein hydrolyzate was not added were set, and IL-6 concentration was measured. As a result, it was observed that IL-6 secreted by mouse macrophage cells was reduced even in the lactic acid bacteria addition system, but it was confirmed that the effect was increased by using it together with pea protein hydrolysate.
（実施例８）乳酸菌飲料の作成と抗炎症効果
エンドウ蛋白質加水分解物を含有する乳酸菌飲料を作成するために、市販乳酸菌飲料「ヤクルト」(株式会社ヤクルト本社製)に、上述のエンドウ蛋白質加水分解物を終濃度１%となるように溶解させ試料とした。この乳酸菌飲料のマウスマクロファージ細胞に対する抗炎症効果を検証した結果、マウスマクロファージ細胞が分泌するIL-6を低下させることが確認された。 (Example 8) Preparation of lactic acid bacteria beverage and anti-inflammatory effect In order to prepare a lactic acid bacteria beverage containing pea protein hydrolyzate, the above-mentioned pea protein hydrolysis was applied to the commercially available lactic acid bacteria beverage "Yakult" (manufactured by Yakult Honsha Co., Ltd.). The sample was dissolved to a final concentration of 1% and used as a sample. As a result of examining the anti-inflammatory effect of this lactic acid bacteria beverage on mouse macrophage cells, it was confirmed that IL-6 secreted by mouse macrophage cells was reduced.
エンドウ蛋白質加水分解物を含有する乳酸菌飲料を作成するために、市販乳酸菌飲料「ヤクルト」(株式会社ヤクルト本社製)に、上述のエンドウ蛋白質加水分解物を終濃度１%となるように溶解させ試料とした。この乳酸菌飲料のマウスマクロファージ細胞に対する抗炎症効果を検証した結果、マウスマクロファージ細胞が分泌するIL-6を低下させることが確認された。 (Example 8) Preparation of lactic acid bacteria beverage and anti-inflammatory effect In order to prepare a lactic acid bacteria beverage containing pea protein hydrolyzate, the above-mentioned pea protein hydrolysis was applied to the commercially available lactic acid bacteria beverage "Yakult" (manufactured by Yakult Honsha Co., Ltd.). The sample was dissolved to a final concentration of 1% and used as a sample. As a result of examining the anti-inflammatory effect of this lactic acid bacteria beverage on mouse macrophage cells, it was confirmed that IL-6 secreted by mouse macrophage cells was reduced.
Claims (7)
- エンドウ蛋白質加水分解物を有効成分とする、経口性抗炎症機能剤。 An oral anti-inflammatory functional agent comprising pea protein hydrolyzate as an active ingredient.
- 加水分解物中のジ・トリペプチド含量が40重量%以上である、請求項１記載の抗炎症機能剤。 The anti-inflammatory functional agent according to claim 1, wherein the di-tripeptide content in the hydrolyzate is 40% by weight or more.
- エンドウ蛋白質加水分解物の、経口性抗炎症機能剤への使用。 Use of pea protein hydrolyzate as an oral anti-inflammatory function agent.
- ジ・トリペプチド含量が40重量%以上である、エンドウ蛋白質加水分解物の、経口性抗炎症機能剤への使用。 Use of a pea protein hydrolyzate having a di-tripeptide content of 40% by weight or more as an oral anti-inflammatory functional agent.
- エンドウ蛋白質加水分解物を有効成分とする、経口性抗炎症機能剤の製造方法。 A method for producing an oral anti-inflammatory functional agent comprising a pea protein hydrolyzate as an active ingredient.
- ジ・トリペプチド含量が40重量%以上である、エンドウ蛋白質加水分解物を有効成分とする、経口性抗炎症機能剤の製造方法。 A method for producing an oral anti-inflammatory functional agent comprising a pea protein hydrolyzate having a di-tripeptide content of 40% by weight or more as an active ingredient.
- 請求項１記載の抗炎症機能剤を含有した飼料。 A feed containing the anti-inflammatory function agent according to claim 1.
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| JP2021106624A (en) * | 2019-06-20 | 2021-07-29 | ユニ・チャーム株式会社 | Pet food |
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| NDIAYE,F. ET AL.: "Anti-oxidant, anti- inflammatory and immunomodulating properties of an enzymatic protein hydrolysate from yellow field pea seeds.", EUROPEAN JOURNAL OF NUTRITION, vol. 51, February 2012 (2012-02-01), pages 29 - 37 * |
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| JP2021106624A (en) * | 2019-06-20 | 2021-07-29 | ユニ・チャーム株式会社 | Pet food |
| JP7204816B2 (en) | 2019-06-20 | 2023-01-16 | ユニ・チャーム株式会社 | pet food |
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