JP2014024804A - Lipid metabolism-improving agent containing decomposition product of ovalbumin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a lipid metabolism-improving agent containing a component which is an active body for effectively exhibiting an improvement effect of lipid metabolism that albumin has.SOLUTION: A decomposition product is obtained by digesting ovalbumin with pepsin, and heat treatment of the digested ovalbumin. A lipid metabolism-improving agent and an inhibiting agent of fatty acid synthetase (FAS) contain the decomposition product of ovalbumin as an active ingredient. A drink and food, a pharmaceutical product, or an animal feed contains the lipid metabolism-improving agent or the inhibiting agent of FAS.

Description

  The present invention relates to an ovalbumin degradation product having an action of improving lipid metabolism, a lipid metabolism improving agent containing the ovalbumin degradation product, and a food, beverage, medicine or feed.

  In recent years, it has been reported that the incidence of so-called lifestyle-related diseases such as obesity, hyperlipidemia (hypertriglyceridemia and hypercholesterolemia), hypertension, diabetes, etc. is increased due to uneven diet and lack of exercise. It has become a big social problem.

  In the onset of the above lifestyle-related diseases, an increase in the amount of body fat and visceral fat and an increase in blood triglycerides and cholesterol are observed, both of which are related to abnormal lipid metabolism. To date, there are methods of using drugs such as fibrate drugs, nicotinic acid drugs, HMG-CoA reductase inhibitors, etc. as means for improving lipid metabolism, but they are not suitable for the purpose of improving a condition that has not reached the therapeutic range. Moreover, side effects such as diarrhea, nausea, loss of appetite, and angioedema may be caused by administration of the drug, and its use is limited under the control of a doctor. Therefore, it is desired to improve lipid metabolism by using a food material that can be easily consumed on a daily basis and has no side effects and is highly safe, and such a food material is being sought.

  On the other hand, egg white is known as one of food materials having an effect of improving lipid metabolism, and so far, a blood cholesterol lowering material composed of dried egg white that has been subjected to dry heat treatment at 100 to 150 ° C. (Patent Document 1), lactic acid fermentation Serum cholesterol lowering materials containing egg white (Patent Document 2), body fat / visceral fat accumulation inhibitor containing egg white as an active ingredient, and liver / blood triglyceride elevation inhibitor (Patent Document 3) have been reported. However, the components in egg white involved in improving lipid metabolism have not been elucidated.

JP2000-16943 WO2006 / 93013 JP2011-225496

  An object of the present invention is to elucidate a component serving as an active body in order to effectively exhibit the lipid metabolism improving action of egg white and provide it as a lipid metabolism improving agent.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have digested ovalbumin (ovalbumin) with pepsin and heat-treated ovalbumin degradation products obtained from blood neutral fat and cholesterol concentrations. It has been found that it has an action to reduce the amount of fat in the abdominal cavity, an action to reduce the amount of fat in the abdominal cavity, and an inhibitory activity on fatty acid synthase (FAS). The above ovalbumin degradation product contains a peptide consisting of Trp-Thr-Ser-Ser-Asn, which shows a direct inhibitory activity against purified fatty acid synthase (FAS) It was confirmed to reduce the rate of fatty acid synthase. The present invention has been completed based on such findings.

That is, the present invention includes the following inventions.
(1) An ovalbumin degradation product obtained by digesting ovalbumin with pepsin and heat-treating it.
(2) The ovalbumin degradation product according to (1), which contains a peptide consisting of Trp-Thr-Ser-Ser-Asn.
(3) A lipid metabolism improving agent comprising the ovalbumin degradation product according to (1) or a peptide comprising Trp-Thr-Ser-Ser-Asn as an active ingredient.
(4) A fatty acid synthase (FAS) inhibitor containing the ovalbumin degradation product according to (1) or a peptide comprising Trp-Thr-Ser-Ser-Asn as an active ingredient.
(5) A food or drink comprising the ovalbumin degradation product according to (1) or a peptide comprising Trp-Thr-Ser-Ser-Asn.
(6) The food or drink according to (5), wherein the food or drink is a health food, a functional food, a nutritional supplement, or a food for specified health use.
(7) A pharmaceutical product comprising the ovalbumin degradation product according to (1) or a peptide comprising Trp-Thr-Ser-Ser-Asn.
(8) A feed comprising the ovalbumin degradation product according to (1) or a peptide comprising Trp-Thr-Ser-Ser-Asn.

  According to the present invention, a lipid metabolism improving agent and a fatty acid synthase (FAS) inhibitor containing ovalbumin degradation products obtained by digesting ovalbumin (ovalbumin) with pepsin and heat-treated are provided. The lipid metabolism improving agent and fatty acid synthase (FAS) inhibitor of the present invention can be used by being contained in compositions such as foods and drinks, pharmaceuticals, and feeds. The above-mentioned ovalbumin degradation product has a lipid lipid (blood triglyceride and blood cholesterol) concentration lowering activity, fatty acid synthase (FAS) inhibitory activity, and intraperitoneal fat mass reducing activity to improve lipid metabolism. Therefore, it is effective for the prevention and treatment of diseases such as obesity, hyperlipidemia (hypercholesterolemia and hypertriglyceridemia), and hypertension that exhibit abnormal lipid metabolism. In addition, the ovalbumin degradation product, which is an active ingredient of the lipid metabolism improving agent and the fatty acid synthase (FAS) inhibitor of the present invention, and the peptides contained therein are derived from natural food materials, so humans and animals that have consumed them There are no side effects such as inhibiting the growth of the plant, and it is safe.

The scheme of the preparation method of the ovalbumin degradation product (PHOVA) of this invention is shown. The feeding and rearing schedule of the test rat is shown. Intraperitoneal fat weight (FIG. 3A), liver FAS activity (FIG. 3B), blood neutral fat (FIG. 3C), and blood total cholesterol (FIG. 3C) in ovalbumin degradation product (PHOVA) and casein rats Fig. 3D) is shown (there is statistical significance compared to the Casein control group: * p <0.05, *** p <0.005). Casein + normal fat (7%) diet, ovalbumin degradation product (PHOVA) + normal fat (7%) diet, casein + high fat (27%) diet, ovalbumin degradation product ( PHOVA) + total fat content of high fat (27%) diet (with statistical significance between different English letters: p <0.05). The MS / MS spectrum (FIG. 5A) of the FAS inhibition fraction obtained by fractionating ovalbumin degradation product (PHOVA) by reverse phase HPLC and the amino acid analysis results (FIG. 5B, C) are shown. Shows fatty acid synthesis rate of HepG2 hepatocytes cultured in medium supplemented with WTSSN peptide (no addition (DMSO): control, cerulenin: positive control, statistically different from control: ** p <0.01, *** p <0.005). 2 shows purified fatty acid synthase (FAS) activity in a reaction system to which a WTSSN peptide was added (no addition (DMSO): control).

The present invention is ovalbumin is the major protein of egg white (ovalbumin) was digested with pepsin, ovalbumin degradation product was heat-treated (P epsin and H eat-processed Ova lbumin: PHOVA), and containing the ovalbumin degradation product The present invention relates to a lipid metabolism improving agent or a fatty acid synthase (FAS) inhibitor.

  In the present invention, the type of ovalbumin may be any ovalbumin from any poultry, but chicken ovalbumin is preferred. Moreover, preparation of ovalbumin can be performed by a well-known method, for example, it may carry out as follows. After breaking the egg and stirring the egg white separated from the yolk, impurities are removed and diluted by adding a neutral buffer such as phosphate physiological saline and mixed uniformly, and the precipitate is removed by centrifugation or filtration. . Next, after adding a saturated ammonium sulfate solution to the supernatant liquid, stirring and allowing to stand, the resulting precipitate is removed, and the supernatant liquid is obtained as an ovalbumin-containing liquid. This ovalbumin-containing solution may be appropriately subjected to sterilization, freezing, concentration, dilution, spray drying, freeze drying, and the like. Commercially available ovalbumin may be used.

  The digestion of ovalbumin with pepsin is preferably carried out by adding water to the dried ovalbumin and adjusting the pH to 1.5 to 2.0, and then adding pepsin to react.

  The amount of water added to ovalbumin is preferably 13 to 14 times the amount of dried ovalbumin. The pH can be adjusted using a buffer solution or a pH adjusting agent.

  Any pepsin may be used as long as it can efficiently degrade ovalbumin. For example, commercially available pepsin derived from porcine gastric mucosa (manufactured by Sigma) can be used. The amount of pepsin added may be, for example, about 1 g of pepsin per liter of the above ovalbumin water solution.

  The decomposition reaction of ovalbumin with pepsin is carried out at 35 to 45 ° C., preferably 37 to 40 ° C., for 1 to 30 hours, preferably 10 to 24 hours.

  After completion of the reaction, the reaction solution is heat-treated at 90 to 95 ° C., preferably 95 ° C. for 5 to 30 minutes, preferably 10 to 20 minutes, and then centrifuged to remove the thermal coagulation fraction, and the supernatant is used in the present invention. Of ovalbumin (hereinafter sometimes referred to as “PHOVA” in the present specification). The obtained PHOVA may be used as it is in a liquid form, but is preferably concentrated under reduced pressure, freeze-dried, pulverized or coarsely processed into a dry powder form in view of storage stability and ease of handling. . The pulverization is performed according to a known method, for example, using a crusher, a mill, a blender or the like. Moreover, you may sterilize the obtained powder by techniques well-known to those skilled in the art, such as heat sterilization and high pressure sterilization, as needed.

The ovalbumin degradation product (PHOVA) thus obtained contains a peptide composed of Trp-Thr-Ser-Ser-Asn (SEQ ID NO: 1) having an activity of inhibiting fatty acid synthase (FAS). Therefore, the ovalbumin degradation product can be made into an isolated and purified product of the above-mentioned peptide by treating it by ultrafiltration or the like. Specifically, the ovalbumin degradation product is subjected to ultrafiltration with nitrogen gas pressure using a cut-off membrane (Millipore PLAC02510) to prepare a low molecular weight fraction (1 kDa or less). Chromatography (trifluoroacetic acid (TFA) / acetonitrile system, Cosmocil column C ₁₈ -AR-II (10 mm × 250 mm, Nacalai Tesque)) is used to fractionate the inhibition fraction. By repeating the fractionation of the peak fraction showing FAS inhibitory activity, the peptide can be isolated.

  The ovalbumin degradation product (PHOVA) of the present invention has an action of improving lipid metabolism, specifically, an action of reducing blood neutral fat concentration / cholesterol concentration, an action of reducing intraperitoneal fat mass, fatty acid synthase (FAS). Has inhibitory activity. In addition, a peptide consisting of Trp-Thr-Ser-Ser-Asn (SEQ ID NO: 1) contained in the ovalbumin degradation product (PHOVA) (hereinafter referred to as “WTSSN” or “WTSSN peptide” in this specification). ) Has the activity of directly inhibiting purified fatty acid synthase (FAS). Therefore, the PHOVA and WTSSN can be used as a lipid metabolism improving agent or a fatty acid synthase (FAS) inhibitor.

  The lipid metabolism-improving agent or fatty acid synthase (FAS) inhibitor of the present invention can be administered as it is, but other ingredients are appropriately blended within the range not impairing the effects of the present invention, food and drink, pharmaceuticals, It is preferable to provide it as various compositions such as feed.

  In the present invention, the food / beverage product is used to mean including a health food, a functional food, a dietary supplement, or a food for specified health use.

  The form of the food or drink may be any form suitable for edible use, for example, solid, liquid, granular, granular, powder, capsule, cream, or paste.

  Specific types of food and drink include breads such as bread and confectionery bread; noodles such as buckwheat, udon, harusame, Chinese noodles and instant noodles; rice cakes, chewing gum, candy, gummi, gum, caramel, chocolate, tablets Confectionery such as confectionery, snack confectionery, biscuits, etc .; confectionery such as jelly, jam, cream; frozen confectionery such as ice cream, ice sherbet, shaved ice; dairy products such as processed milk, fermented milk, yogurt, butter, cheese; Processed fishery and livestock products such as hamburger, ham and sausage; fats and oils such as margarine, mayonnaise, shortening, whipped cream and dressing; seasonings such as soy sauce, sauce, vinegar and mirin; soft drinks, carbonated drinks, Beverages such as nutritional drinks, fruit drinks and milk drinks (concentrated concentrates and powders for adjustment of these drinks) Including) including but are not limited to.

  The food / beverage products of the present invention may be appropriately blended with additives usually used depending on the type. Any additive that is acceptable for food hygiene can be used as the additive. For example, sweeteners such as sugar, fructose, isomerized liquid sugar, glucose, aspartame, stevia; citric acid, malic acid, tartaric acid Acidulants such as dextrin, starch, etc .; binders, diluents, fragrances, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspensions Agents, preservatives and the like.

  The amount of the fat metabolism improving agent or fatty acid synthase (FAS) inhibitor in the food and drink of the present invention may be any amount that can exert its lipid improving action and FAS inhibitory action, but the general intake of the target food and drink What is necessary is just to set suitably in consideration of quantity, the form of food / beverage products, efficacy and an effect, taste property, palatability, cost, etc. For example, in the case of a solid food, the PHOVA content is adjusted to 10% to 100% by weight, preferably 20% to 80% by weight.

  Since the food and drink of the present invention has an action to improve lipid metabolism, normal people as well as those who are prone to develop diseases such as hyperlipidemia that show abnormal lipid metabolism due to lifestyle, constitution, or genetic predisposition Even so, it can be taken on a daily basis for the purpose of preventing or ameliorating those diseases.

  Further, when the lipid metabolism improving agent or fatty acid synthase (FAS) inhibitor of the present invention is provided as a pharmaceutical product, the PHOVA or WTSSN peptide is pharmaceutically acceptable and appropriately selected according to the dosage form, and a carrier or carrier And additives (eg, excipients, diluents, binders, lubricants, disintegrants or disintegrants, solubilizers, stabilizers, preservatives, preservatives, extenders, dispersants, lubricants) Agents, wetting agents, buffering agents, fragrances, etc.) and various preparations that can be systemically or locally administered by various known methods orally or parenterally. The pharmaceutical of the present invention includes drugs used for animals, that is, veterinary medicine.

  The pharmaceutical of the present invention can be administered orally or parenterally, but is preferably administered orally. When the pharmaceutical product of the present invention is orally administered, it is formulated into tablets (including sugar-coated tablets), capsules, granules, powders, pills, liquids for internal use, suspensions, emulsions, syrups, etc. You may make it the dried product re-dissolved in the case. In addition, when the pharmaceutical product of the present invention is administered parenterally, it is formulated into an injection (eg, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), infusion, suppository, etc. In the case of pharmaceutical preparations, they are provided in the form of unit dosage ampoules or multi-dose containers. In formulating, one or more active ingredients other than the active ingredients of the present invention may be further blended.

  The pharmaceutical agent of the present invention can be used as a pharmaceutical agent for the prevention and / or treatment of diseases exhibiting abnormal lipid metabolism. Here, “lipid metabolism abnormality” means a condition in which the lipid metabolic pathway is abnormal and the blood concentration cannot be maintained within an appropriate range (in many cases, the blood concentration exceeds the appropriate range). To do.

  Accordingly, examples of the disease to be prevented and / or treated by the pharmaceutical of the present invention include, for example, hyperlipidemia (hypertriglyceridemia, hypercholesterolemia, hypoHDLemia, postprandial hyperlipidemia, etc.), obesity (Eg visceral fat accumulation, subcutaneous fat accumulation), fatty liver, hypertension, arteriosclerosis, diabetes mellitus, metabolic syndrome (pathology presenting two or more of hyperglycemia, dyslipidaemia and hypertension with visceral fat type obesity as common factors) ), Ischemic heart disease (angina pectoris, myocardial infarction), cerebrovascular disorder (cerebral infarction, cerebral hemorrhage) and the like, but are not limited thereto. The pharmaceutical agent of the present invention functions as a prophylactic agent that suppresses the onset of the above diseases and / or a therapeutic agent that improves the normal state.

  In addition, since fatty acid synthase (FAS) is highly expressed in many cancers such as esophageal cancer, stomach cancer, colon cancer, breast cancer, and lung cancer, the pharmaceutical of the present invention is based on its FAS inhibitory activity. It can be used as a preventive and / or therapeutic drug.

  The pharmaceutical of the present invention is safely administered orally or parenterally to mammals such as humans, mice, rats, rabbits, dogs, cats and the like when used as preventive and / or therapeutic drugs for the above-mentioned respective diseases. be able to. The dosage of the pharmaceutical agent of the present invention can be appropriately determined according to the type of disease, the age, sex, body weight, symptom level, method of administration, etc. of the administration subject. For example, when orally administered to a hyperlipidemic patient, the dose is converted into WTSSN peptide mass, and is divided into once to several times a day in the range of 100 mg to 1000 mg per day for an adult (body weight 60 kg).

  The lipid metabolism improving agent or fatty acid synthase (FAS) inhibitor may be added to the feed as an additive for feed. In the feed of the present invention, in addition to the above-described lipid metabolism improving agent or fatty acid synthase (FAS) inhibitor, the raw materials used in normal compounded feeds depend on the breeding environment such as the type of animal, the development stage, and the region. You may mix | blend suitably. Such raw materials include, for example, cereals or processed cereals (corn, milo, barley, etc.), potatoes (bran, rice bran, corn gluten feed, etc.), vegetable oils (soybean oil lees, sesame oil lees, cottonseed oil lees, etc.), Animal raw materials (fat dry milk, fish powder, meat and bone powder, etc.), minerals (calcium carbonate, calcium phosphate, salt, anhydrous silicic acid, etc.), vitamins, amino acids, yeasts such as brewer's yeast, fine powders of inorganic substances (crystals) Cellulose, talc, silica, etc.).

  The feed of the present invention is used for feeds such as excipients, extenders, binders, thickeners, emulsifiers, colorants, fragrances, food additives, seasonings and the like that are commonly used in blended feeds. Additives and other components (antibiotics, bactericides, anthelmintics, preservatives, etc.) may be added as desired.

  The form of the feed of the present invention is not particularly limited, and examples thereof include powders, granules, pastes, pellets, capsules (hard capsules, soft capsules), tablets and the like.

  The animal to be fed with the feed of the present invention is not particularly limited. For example, livestock such as cattle, horses, pigs, sheep, goats, deer, rabbits; chickens (both broilers and egg-laying chickens). ), Poultry such as turkey, duck, mallard, duck, pheasant, quail or goose; laboratory animals such as mice, rats and guinea pigs; pets such as dogs and cats.

  The compounding quantity of PHOVA in the feed of this invention is not specifically limited, For example, it is 5-50 weight% of the whole feed, Preferably it is the range of 10-30 weight%. However, the blending amount may be appropriately adjusted depending on the type of animal, weight, breeding conditions, feeding method, and the like.

  The feed of the present invention can reduce blood neutral fat and cholesterol in animals to be fed. Therefore, the feed of the present invention improves the lipid metabolism function of the fed animal and can suppress the decrease in productivity due to excessive accumulation of peritoneal fat. In the product (meat, egg, milk), For example, various functions such as a low cholesterol content can be imparted.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but these examples do not limit the present invention.

(Example 1) Preparation of ovalbumin degradation product (PHOVA) 75 g of ovalbumin (OVA; Wako) was suspended in 1000 ml of distilled water, and the pH was adjusted to 2.0 with HCl. 1 g of pepsin (Sigma) was added to this solution, and the mixture was stirred at 37 ° C. for 3 hours. Then, the pH was readjusted with 6N-HCl, and further reacted at 37 ° C. with stirring for 3 hours. Add 0.165g of defoamer to 1000ml of the reaction solution, adjust the pH to 5 with Na ₂ CO ₃ , heat-treat at 95 ° C for 10 minutes, and centrifuge (3000 rpm, 10 minutes). The insoluble heat coagulation fraction was removed. Next, the supernatant was concentrated under reduced pressure, freeze-dried and powdered to obtain an ovalbumin degradation product (PHOVA) of the present invention (FIG. 1).

(Example 2) Evaluation of ovalbumin degradation product (PHOVA) As shown in the feeding and breeding schedule shown in Fig. 2, SD rats (35) preliminarily reared with a basic diet for rats based on the AIN-93 formulation (35 (Age, male) is divided into Casein group (control) and PHOVA group (seven animals in each group), and the test feeds with the composition shown in Table 1 below are given freely. Dismantled.

  The disassembled rats were measured for the weight of intraperitoneal fat (weighing and weighing fat accumulated around the kidney in the peritoneal cavity), liver FAS activity, and blood neutral fat (triglyceride) and total cholesterol concentrations. The liver FAS activity was measured according to the method of Nepokroeff et al. Et al. (Nepokroeff CM, Lakshmanan MR, Porter JW. Fatty-acid synthase from rat liver. Methods Enzymol. 1975; 35: 37-44.). The neutral fat concentration in blood and the total cholesterol concentration in blood were measured by an enzyme method. Neutral fat in the blood is a commercially available measurement kit (Fuji Dry Chem Slide Triglyceride Kit: Fuji Film Co., Ltd.), and total cholesterol in the blood is a commercially available measurement kit (Fuji Dry Chem Slide Cholesterol Kit: Fuji Film Co., Ltd.) Used and measured according to the instructions for each kit.

  As a result, by feeding ovalbumin hydrolyzate (PHOVA), the weight of intraperitoneal fat was significantly reduced by 40%, liver FAS activity by 38%, blood neutral fat concentration by 23%, and blood total cholesterol concentration by 16%. (FIGS. 3A-D). All rats grew normally and no abnormalities were observed. There were no interval differences in the weights of major organs and skeletal muscles.

(Example 3) Effect of PHOVA on liver total lipid content of rats fed with high fat diet Feeding / breeding schedule, rat strain / sex / age, etc. are the same as in Example 2. After preliminary breeding, rats are divided into Casein + normal fat (7%) diet, PHOVA + normal fat (7%) diet, Casein + high fat (27%) diet, PHOVA + high fat (27%) diet (6 per group) Head), each of the test feeds having the compositions shown in Table 2 below was freely ingested, and the rats were disassembled on the 10th day of feeding the test feeds. The liver lipid was quantified by the Folch method. Homogenize 1 g of liver piece in acetic acid buffer using polytron homogenizer, add chloroform: methanol (2: 1) solution to homogenate, perform extraction operation, collect solvent fraction in pear-shaped flask, rotary evaporator After removing the solvent by drying under reduced pressure, the lipid weight was measured.

  As shown in FIG. 4, hepatic lipid accumulation increased with a high-fat diet, but it was confirmed that PHOVA suppressed it 43%. Therefore, it can be said that PHOVA is effective in preventing the occurrence of fatty liver and improving symptoms.

(Example 4) Identification of amino acid sequence of peptide contained in ovalbumin degradation product (PHOVA) Ovalbumin degradation product (PHOVA) prepared in Example 1 was subjected to nitrogen gas pressurization using a cut-off membrane (Millipore PLAC02510). Ultrafiltration was performed to prepare a low molecular weight fraction (molecular weight of 1 kDa or less), and 5 mg was dissolved in 1 ml of distilled water and used. Separation and purification of FAS-inhibiting peptides were performed by reversed-phase high performance liquid chromatography (JASCO LC-2000Plus system; column, Cosmosil C ₁₈ -AR-II (10 mm × 250 mm) Nacalai Tesque). Linear gradient elution using liquid A (0.1% TFA) and liquid B (0.1% TFA / 80% acetonitrile) as mobile phases (flow rate 4.0 ml / min, 35 minutes: liquid A 100% → 53.4%, liquid B 0 → 46.6%), the absorbance of the eluate at 220 nm was measured, and the peptides were fractionated. First, since FAS inhibitory activity was observed in the peak group fraction eluted at 13 to 18 minutes, the fractionation was repeated to obtain a freeze-dried powder. Next, in order to further fractionate this fraction, it was subjected to linear gradient elution (flow rate 2.0 ml / min, 57 min: solution A 91.9% → 90.7%, solution B 8.1 → 9.3%), and 36-37 min. The peak eluted as one peak was collected and subjected to mass spectrometry.

  The peptide mass was measured for accurate mass by Q-TOF LC / MS to search for peptide candidates derived from ovalbumin. Next, the MS / MS spectrum was measured by QQQ LC / MS (manufactured by Agilent Technologies), and the sequence was identified from the fragment pattern (FIG. 5A). As a result, the peptide was found to be tryptophan, threonine, serine, serine, asparagine (Trp-Thr-Ser-Ser-Asn: WTSSN, molecular weight 593.245), which is present in the 267th to 271st positions in the ovalbumin molecule (Fig. 5B, C).

(Example 5) Evaluation of WTSSN peptide contained in ovalbumin degradation product (PHOVA)
(1) Effect of WTSSN on fatty acid synthesis rate in HepG2 cultured hepatocytes
WTSSN (300 μg / mL) and cerulenin (15 μg / mL), which is a typical fatty acid synthase inhibitor as a positive control, were dissolved in DMSO (1%) and added to the culture solution of HepG2 cultured hepatocytes. Only DMSO was added to the non-addition group (control). Synthetic WTSSN was added to the medium (300 μg / mL) and cultured for 3 hours, and then [ ¹⁴ C] acetate was added and further cultured for 1.5 hours to recover the cells. Lipids were extracted from the collected cells, and [ ¹⁴ C] uptake was measured with a liquid scintillation counter, which was used as the fatty acid synthesis rate. As a result, compared with the non-added group, the fatty acid synthesis rate decreased by 30% in the WTSSN added group (FIG. 6).

(2) Direct inhibitory effect of WTSSN on purified chicken FAS
Fatty acid synthase (FAS) was isolated and purified from chicken liver according to the method of Arslanian and Wakil (Arslanian MJ & Wakil SJ, Methods in Enzymology, Vol 35, 59-65, 1975). Using this FAS, Zhao et al. (Zhao YX, Liang WJ, Fan HJ, Ma QY, Tian WX, Dai HF, Jiang HZ, Li N, Ma XF.Carbohydr Res. 346 (11): 1302-1306. 2011) FAS activity and WTSSN FAS inhibitory activity were evaluated. Since FAS quantitatively consumes NADPH as a reducing agent during fatty acid synthesis, FAS activity can be evaluated from the rate of decrease (consumption) of NADPH absorbance (340 nm). Specifically, the absorbance at 340 nm of a reaction system (1 mL) containing 100 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM dithiothreitol, 3 μM acetyl-CoA, 35 μM NADPH, and 5 μg purified chicken FAS was spectroscopically analyzed. Monitoring with a photometer (Shimadzu UV-1800), first, the FAS-independent decrease in absorbance was measured (blank reaction). Next, the enzyme reaction was started by adding malonyl-CoA (final concentration 10 μM), the absorbance was measured, the blank reaction was subtracted, and the molecular extinction coefficient of NADPH at 340 nm (6.22 × 10 ³ M ^-1 cm ^-1 ) The NADPH consumption per unit time was calculated and divided by the amount of added FAS protein, which was defined as the FAS specific activity. To the above reaction system, WTSSN dissolved in dimethyl sulfoxide (DMSO) was added (1.5 mg) to carry out an enzyme reaction, and direct inhibitory activity was evaluated based on the FAS specific activity determined as described above. As a result, compared with the non-added group, the FAS activity decreased by 42% in the WTSSN-added group (FIG. 7).

  The present invention can be used in the field of manufacturing foods and drinks, pharmaceuticals, feeds and the like.

Claims (8)

  An ovalbumin degradation product obtained by digesting ovalbumin with pepsin and heat-treating it.   The ovalbumin degradation product according to claim 1, comprising a peptide consisting of Trp-Thr-Ser-Ser-Asn.   A lipid metabolism improving agent comprising the ovalbumin degradation product of claim 1 or a peptide comprising Trp-Thr-Ser-Ser-Asn as an active ingredient.   A fatty acid synthase (FAS) inhibitor comprising the ovalbumin degradation product according to claim 1 or a peptide comprising Trp-Thr-Ser-Ser-Asn as an active ingredient.   A food or drink comprising the ovalbumin degradation product according to claim 1 or a peptide comprising Trp-Thr-Ser-Ser-Asn.   The food or drink according to claim 5, wherein the food or drink is a health food, a functional food, a nutritional supplement, or a food for specified health use.   A pharmaceutical product comprising the ovalbumin degradation product according to claim 1 or a peptide comprising Trp-Thr-Ser-Ser-Asn.   A feed comprising the ovalbumin degradation product according to claim 1 or a peptide comprising Trp-Thr-Ser-Ser-Asn.

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