JP5380649B2 - Milk component hydrolyzate - Google Patents

Description

  The present invention relates to a milk component hydrolyzate.

  Advanced Glycation End-products (AGEs) are irreversible highs caused by a series of reactions (Maillard reaction) starting from a non-enzymatic reaction between a carbonyl group of a reducing sugar such as glucose and an amino group of a protein. It is a molecular cross-linking substance. These reactions proceed slowly over a long period of time in vivo. For example, hemoglobin A1C (HbA1c) listed as one of the clinical test items for diabetes is a glycated product of hemoglobin, which is a protein of erythrocytes, and is a kind of AGEs.

  AGEs are causative substances for complications due to diabetes such as diabetic retinopathy and diabetic nephropathy. AGEs are also known to be associated with the development of diabetic angiopathy by binding to a specific receptor (RAGE) present in vascular endothelial cells. Considering the report that there are currently 16.3 million potential patients with diabetes in Japan, especially 1 out of 4.5 middle-aged and elderly people, the removal of AGEs in the body It is very important in preventing the onset and progression of diabetes complications. Furthermore, AGEs are also involved in the development of various debilitating diseases such as atherosclerosis, Alzheimer's disease, and rheumatoid arthritis.

  AGEs are generated not only from glucose but also from various sugars such as glucose autoxidation and degradation products. Among these, AGE-2, which is an AGE derived from glyceraldehyde, has a high binding ability to the AGE receptor (RAGE), and through this RAGE, diabetic vascular complications such as diabetic retinopathy or diabetic nephropathy. It is known to be strongly involved in the onset and progression of (Non-patent Documents 1 and 2). Thus, for the purpose of prevention and treatment of diabetes, AGEs, particularly AGE-2, has attracted attention for its role, and research continues.

  On the other hand, AGEs are also present in foods. In the first place, AGEs are substances that have attracted attention in browning due to non-enzymatic saccharification reaction (Maillard reaction) published in food chemistry research. It has been found that AGEs are hardly included in cooking methods such as boiling and steaming, and abundant in cooking methods such as baking, frying and frying. It is also known that AGEs are contained in a lot of foodstuffs such as cola, miso, and soy sauce. In this way, human beings always ingest a large amount of AGEs from food (especially processed foods) in their daily lives. Although it is thought that there is almost no harm in taking AGEs as a food ingredient, it is necessary to be careful when the kidneys are worsened, and after harmless food AGEs are absorbed into the body, There is a possibility of being converted to AGEs (for example, AGE-2).

  In this way, AGEs are generated both in vivo and ingested from outside the living body. For this reason, various drugs have been proposed in order to suppress the accumulation of excessive AGEs in vivo. For example, aminoguanidine, pyridoxamine derivatives and the like are conventionally known as AGEs production inhibitors. Further, Patent Document 1 discloses 3-methyl-1-phenyl-2-pyrazolin-5-one.

  Substances that destroy established AGE cross-links have also been studied, and for example, N-phenacylthiazolium bromide (PTB) is known. Recently, seven types of compounds such as 1,4-benzene-bis [4-methyleneaminophenoxyisobutyric acid] have been reported (for example, Patent Document 2). However, these AGE decomposition effects are not always sufficient.

  In recent studies, vitamin B6 has been found to have an AGE inhibitory action, and its use as an AGE inhibitor is expected.

  By the way, AGEs are known to accumulate with aging, and AGEs are known to act as a binding substance between collagens, and are thus considered to be involved in skin aging. It has been reported that aging of various body tissues accompanying aging can be suppressed by administering aminoguanidine (Non-patent Document 3), and aminoguanidine is widely used as an anti-aging substance in Europe and America. ing. However, aminoguanidine has been shown to be toxic in phase III clinical trials in the United States, with significant restrictions on its use and dosage.

Whey obtained by removing casein, which is the main component of milk protein, and milk fat from milk is attracting attention in the field of food and cosmetics due to its excellent nutritional value, antioxidant power, immunity enhancement, etc. It is a material. Since it is a natural material, safety is also expected in these fields.
JP 2004-300153 A JP-T-2004-529126 Yamagishi S. et al., Biochem. Biophys. Res. Commun., 2002, 290, 973-978 Okamoto T. et al., FASEB J., 2002, 16, 1928-1930 Li YM et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 3902-3907 Tessier et al., Biochem. J., 2003, 369, 705-719

  An object of this invention is to provide the substance which can be couple | bonded with AGEs with high safety | security to a biological body. Another object of the present invention is to provide foods and skin care products that can inhibit the absorption of AGEs into the body.

  The present invention provides a glyceraldehyde-derived advanced glycation end product (AGE-2) binding agent comprising a milk component hydrolyzate obtained by hydrolyzing whey with endoprotease, exoprotease, and endopeptidase.

  The present invention further provides a food product comprising a dairy hydrolyzate obtained by hydrolyzing whey with endoprotease, exoprotease, and endopeptidase.

  The present invention also provides a cosmetic comprising a milk component hydrolyzate obtained by hydrolyzing whey with endoprotease, exoprotease, and endopeptidase.

  In one embodiment, the dairy hydrolyzate has skin permeability.

  According to the present invention, a substance capable of binding to AGEs, particularly AGE-2 which is considered to be highly toxic to cells is provided. The milk component hydrolyzate of the present invention may be useful for reducing symptoms (for example, vascular disorder) due to an increase in AGEs associated with diabetic patients and aging. Furthermore, according to the present invention, a substance that can penetrate the skin and bind to AGEs is also provided.

(Milk component hydrolyzate)
The dairy hydrolyzate of the present invention can be prepared by treating whey with endoproteases, exopeptidases, and endopeptidases. For this reason, it is also referred to as “whey hydrolyzate”. Whey is a milk-derived component in which casein, which is the main component of milk protein, is removed from raw milk (eg, milk). Whey obtained as a by-product generated when producing a dairy product (eg, cheese, casein, etc.) from cow's milk or skim milk can be suitably used for preparing the milk component hydrolyzate of the present invention.

  As long as a milk component hydrolyzate capable of binding to AGE-2 and preferably capable of permeating through the skin membrane can be prepared, the type and mode of action of the enzyme for treating whey are not limited. As a starting material for preparing the dairy hydrolyzate of the present invention, mammalian secretions such as milk (e.g. milk) or skim milk can also be used, in this case prior to treatment with enzymes. It is preferable to remove casein. The processing conditions (including temperature and time) of the proteolytic enzyme for milk-derived protein in whey can be appropriately determined in consideration of protein denaturation and the enzyme's working temperature.

  As endoprotease, bovine gastric mucosa-derived pepsin; EC3.4.23.1 is preferable, as exopeptidase, Aeromonas Proteolytica-derived aminopeptidase; EC3.4.11.10 is preferable, and as endopeptidase, bovine pancreas-derived chymotrypsin type II; EC3.4 .21.1 is preferred. The processing temperature of the endoprotease, exopeptidase, and endopeptidase enzyme is preferably 20-60 ° C, more preferably 40-60 ° C, and even more preferably about 50 ° C. The endoprotease, exopeptidase, and endopeptidase may act simultaneously or separately. When it is made to act simultaneously, processing time becomes like this. Preferably it is 0.5 to 5 hours, More preferably, it is 1 to 3.5 hours. The ratio of the combination to act is Unit / 1 kg protein, preferably 1-1000: 1-10: 1-100, more preferably 100-1000: 1-2: 5-20, and even more Preferably, it is about 1000: 1: 10.

  The milk component hydrolyzate of the present invention can penetrate the skin. Such a milk component hydrolyzate can permeate a commercially available artificial skin membrane (for example, available from Toray Industries, Inc.). The artificial skin membrane may be a membrane capable of cutting off a molecular weight of about 2000.

  The milk component hydrolyzate of the present invention can bind to AGE-2. The binding to AGE-2 can be measured by reducing the fluorescence intensity of AGE-2 when a test substance is added to AGE-2 using the fact that AGE-2 emits fluorescence. For example, as described in Example 2 below, determination can be made by examining the binding and dissociation with AGE-2 using a quartz crystal microbalance (QCM) device.

  AGE-2 can be synthesized by any method such as a culture method or a chemical synthesis method. In the culture method, for example, human serum albumin (HSA) is incubated with D, L-glucose for several weeks or with D, L-glyceraldehyde or D, L-glycolaldehyde for several days. The chemical synthesis method is performed, for example, according to the method of Tessier et al. (Non-patent Document 4). Specifically, acetyl-lysine and glyceraldehyde are mixed in a phosphate buffer solution (pH 7.4), diethylenetriaminepentaacetic acid and 25% (v / v) methanol are added, and then at 37 ° C. for several days. It is obtained by reacting. The obtained AGE-2 is preferably immobilized on an appropriate solid phase (for example, beads).

  In order to prepare the milk component hydrolyzate of the present invention, it is preferable to cause a proteolytic enzyme to act on whey in substantially the same manner as described in Example 1 below.

  The dairy component hydrolyzate of the present invention may be in the form of an aqueous solution when included in a product as described below, or in the form of a powder obtained by removing the solvent (for example, by lyophilization). Good.

  The dairy component hydrolyzate of the present invention has binding properties to AGEs, particularly AGE-2. Therefore, when ingested as a food, the dairy component hydrolyzate of the present invention adsorbs food-based AGEs and inhibits absorption from the intestinal tract, thereby converting AGE-2 from food-based AGEs into the body. Reduce the amount. Further, when introduced into the body, it is useful as an anti-AGE-2 agent or as a prophylactic or therapeutic agent for diseases involving AGE-2, such as diabetes and its complications, or Alzheimer's disease.

  It is known that blood AGEs levels increase in patients with diabetes or with aging. It has been found that increase in blood AGEs (particularly glucose-derived AGE: AGE-1) also increases monocyte chemotactic activator (MCP-1), which is a vascular disorder marker in blood. The milk component hydrolyzate of the present invention can suppress the increase in MCP-1 that may be caused by such an increase in the blood AGEs concentration.

  The milk component hydrolyzate of the present invention can also be used as an absorption inhibitor of food AGEs, and is useful for suppressing excessive intake of AGEs in food.

  Furthermore, the milk component hydrolyzate of the present invention can be used as a material having skin permeability and suppressing the accumulation of subcutaneous AGEs, and is used as a cosmetic.

(Food)
The milk component hydrolyzate of the present invention can be used for food. Such foods may further include excipients, extenders, binders, thickeners, emulsifiers, colorants, flavors, food additives, seasonings and the like. Such foods can be formed into forms such as granules, tablets, and liquids depending on the application. Moreover, a milk component hydrolyzate can also be added to a food or a drink to make a food for health maintenance. Examples of such foods include home-use diabetic foods, liquid foods, foods for sick people (combination foods for adjusting diabetic foods, etc.), foods for specified health use, diet foods, and foods based on carbohydrates. However, it is not limited to these. Specific food forms include, for example, cooked rice products, wheat products, vegetable products, milk drinks, soft drinks, and the like, but are not limited thereto. Addition or processing to foods can be performed by methods commonly used by those skilled in the art. Addition to non-human animals such as livestock or pet feed is also possible.

  The amount of the milk component hydrolyzate of the present invention that can be contained in the food depends on the form, type, and mixed components of the food, but is preferably 0.1 to 30% by mass, more preferably 0.5 to 20% by mass. More preferably, it may be 1 to 10% by mass.

  The dairy component hydrolyzate of the present invention can be added to, blended with, or contained in a food product by procedures commonly used by those skilled in the art. For example, it can be added, blended, or contained in a food by dissolving in advance in a solvent such as water or alcohol and then mixing with other blending components, or by stirring and mixing with other blending components as powder.

(Cosmetics)
The milk component hydrolyzate of the present invention can also be contained in cosmetics. “Cosmetics” as used herein refers to things applied to the body by rubbing, spraying, etc. to keep the skin or hair healthy or to cleanse, beautify, enhance appeal and change the appearance of the body. Say. Cosmetics are classified into basic cosmetics, makeup cosmetics, hair cosmetics, and the like according to their uses. In the present specification, “cosmetics” includes those classified as quasi-drugs as defined in the Pharmaceutical Affairs Law, such as medicinal cosmetics. Examples of cosmetics include cold creams, vanishing creams, mixed creams that are oily, massage creams, emollient creams, hizenic creams, creams for the eyes and fingers, lip balms, shaving creams, and after shaving creams. Creams such as moisturizing lotions, astringent lotions, acidic lotions, alkaline lotions, carmine lotions, after shave lotions, etc .; emulsions such as emulsions containing polymer systems; essences and beauty oils; body Scrubs; packs and peelable packs; foundation creams, burnishing type foundations, fountains, water fountains, kneading trousers, oily foundations including sticks, oil-in-water or water-in-oil emulsions Foundation, powdery foundation, liquid foundation, lipstick, blusher, brushing powder, eyeliner, eye shadow, mascara, eyebrow, functional lipstick, lip liner pencil, lip gloss, lip balm, nail enamel, base coat, top coat, light removal Make-up products including liquids, nail creams, cuticle removers, etc .; shampoos, conditioning shampoos, rinses, hair treatments, shampoos with integrated rinses, hair restorers, hair nourishing agents, hair mousses and hair foams, hair sprays, hair mists, hair gels, set lotions, Hair liquid, scalp treatment, hair cream, hair oil, hair coating lotion, hair gloss spray, hair blow, pomade, Products for hair, including cosmetics, hair waxes, hair dyes, hair bleaches, etc .; and bath and facial soaps, body cleansers, baths, bubble baths, deodorants, antiperspirants, slimming, fragrances, Body care products including but not limited to perfumes, perfumes, eau de toilettes, eau de cologne and the like.

  In addition to the milk component hydrolyzate of the present invention, the components that can be included in the cosmetic of the present invention include the following: fats and oils, waxes, hydrocarbons, higher fatty acids, higher alcohols, esters, Oil-based raw materials including silicones; aqueous raw materials including purified water and alcohols; natural moisturizing factors including polyhydric alcohols, sugars, biopolymers, sodium pyrrolidonecarboxylate, amino acids, urea, sodium lactate; Natural surfactants including extracts, lactic acid bacteria cultures, casein, yeast extract, royal jelly, silk extract, seaweed extract, sodium alginate, etc .; semi-synthetic surfactants including carboxymethyl cellulose, etc .; methacrylic acid copolymer Synthetic surfactants including: carboxylates, sulfonates, phosphate esters, quaternary ammonium Hydrophobic surfactants including trimethylglycine, amine oxide, polyoxyethylene, etc .; nonionic systems including polyether modification, anionic systems including sulfonic acid modification, cationic systems including ammonium modification, sulfone Amphoteric silicone surfactants including betaine modification; polypeptide derivative surfactants including surfactin and lipopeptides; natural animal and plant surfactants such as glycolipids, phospholipids and bile acids; sophorolipid Microbial production-type natural surfactants including: powder raw materials for shaping and spreading; UV protection, exfoliation and dissolution of stratum corneum, deodorization, deodorization, nutritional supplementation, antiperspirant, whitening, cell activation, blood circulation Pharmaceutical raw materials for the purpose of promotion, anti-inflammatory, astringent, sebum control; antiseborrheic agent; and hormonal agent.

  The above components can be added to, blended with, or contained in the cosmetic so as not to impair the effectiveness of the dairy component hydrolyzate of the present invention. The content of the dairy component hydrolyzate of the present invention depends on the type and ingredients of the cosmetic, but is preferably 0.01 to 10% by mass, more preferably 0.05 to 5% by mass, and still more preferably 0.00. It may be 1-2% by mass. This content is not greatly different depending on the form or application (for foundation, for washing, etc.) such as cream, liquid, emulsion, mousse or solid.

  The dairy component hydrolyzate of the present invention can be added to, blended with, or contained in cosmetics according to procedures commonly used by those skilled in the art. For example, it can be added, blended or contained in the cosmetic by dissolving in advance in a solvent such as water or alcohol and then mixing with other blending components, or by stirring and mixing with other blending components in powder form.

Example 1
The remaining whey after producing casein sodium from raw bovine milk was obtained from Tatsua Japan Co., Ltd. Endoprotease (bovine stomach mucosa-derived pepsin; EC3.4.23.1; Sigma) 1000 Unit / 1 kg protein, exopeptidase (Aeromonas Proteolytica-derived aminopeptidase; EC3.4.11.10; Sigma) 1 Unit / 1 kg protein, and whey Endopeptidase (bovine pancreas-derived chymotrypsin type II; EC3.4.21.1; Sigma) 10 Unit / 1 kg protein was added and treated at 50 ° C. for 3.5 hours. The hydrolyzate obtained by such enzyme treatment was freeze-dried to obtain a whey hydrolyzate.

  The lyophilized whey hydrolyzate was dissolved in pure water so as to be 10 mg / mL, and this was designated as solution A. A transverse membrane permeable cell having phases on both sides sandwiching an artificial skin membrane (Toray Industries, Inc.) was produced. Liquid A was put in one phase of the membrane permeation cell, and pure water was put in the other phase, and the whole cell was brought to a constant temperature by circulating water at 25 ° C. Both phases were stirred for 10 minutes at a speed of 120 rpm. After stirring for 10 minutes, the solution was taken out from the phase containing pure water, and this was designated as solution B.

The contents of solution A (solution before starting stirring) and solution B in which lyophilized whey hydrolyzate was dissolved in pure water were subjected to the conditions described below according to the method used in the transdermal absorption evaluation test. Measured by high performance liquid chromatography (HPLC) at:
Column ODS (made by Shiseido) 4.6 x 150 mm
Eluent A: 0.02% (v / v) TFA (solvent H ₂ O)
B: 0.016% (v / v) TFA (solvent ACN)
A: B = 95: 5 (v / v) used Flow rate 0.75 ml / min Temperature 40 ° C.
Detector UV 220 nm.

  As a result, there was a substance that passed through the artificial skin membrane and moved from the A liquid to the B liquid.

(Example 2)
Using the intermolecular interaction quantitative quartz crystal balance (QCM) apparatus “AFFINIXQ” (model number: QCM2000; Initium Co., Ltd.), the A solution and the B solution of Example 1 above (dissociation constant) with AGE-2 I investigated.

  AGE-2 was prepared as follows.

  First, 180 mg of DL-glyceraldehyde and 39 mg of diethylenetriamine-pentaacetic acid as a chelating agent were weighed and placed in a 50 mL falcon tube. Next, 20 mL of 0.2 M phosphate buffer (pH 7.4) was added to the falcon tube, and DL-glyceraldehyde and diethylenetriamine-pentaacetic acid were dissolved with a vortex mixer. Furthermore, 500 mg of human serum albumin (HSA) (manufactured by Sigma) was added to the falcon tube and dissolved with a vortex mixer. Subsequently, the obtained solution was made into a sterile solution by passing through a filter having a pore size of 0.22 μm in a clean bench. The lid of the 50 mL falcon tube was sealed with parafilm and incubated at 37 ° C. for 1 week to react DL-glyceraldehyde with HSA. After the incubation, the solution was applied to a PD-10 column (manufactured by GE Healthcare Bioscience) to remove unreacted DL-glyceraldehyde, and the result was confirmed by HPLC.

  The production of AGE-2 was confirmed by ELISA using a monoclonal antibody. The anti-AGE-2 monoclonal antibody was consigned to Toyobo Co., Ltd. and produced from mice using AGE-2 as an antigen. This anti-AGE-2 monoclonal antibody is biotinylated with a biotinylation reagent EZ-Link (registered trademark) Sulfo-NHS-Biotinylation Kit (PIERCE, product code 21420), and this biotinylated anti-AGE-2 monoclonal antibody is labeled with streptavidin peroxidase. (Nacalai Tesque, product code 02517-61). ELISA POD Substrate A. B. T.A. Using the S kit (Nacalai Tesque, product code 14351-80), the well prepared by immobilizing the HSA antibody (RayBiotech, Inc) was used to bind the AGE-2 prepared above and peroxidase-labeled streptavidin. The generation of AGE-2 was confirmed by adding AGE-2 monoclonal antibody and detecting chemiluminescence.

  1 μL of 100 μg / mL AGE-2 is dripped onto a dedicated sensor chip of the intermolecular interaction quantitative quartz crystal balance (QCM) apparatus “AFFINIXQ” (model number: QCM2000; Initium Co., Ltd.), then air-dried sufficiently, and then ultrapure water The chip was washed with. The chip on which AGE-2 was fixed was mounted on the above apparatus and inserted into a test container containing 8 mL of ultrapure water. 8 μL of a test substance solution in which the lyophilized product of the solution A or solution B was 1 mg / mL with ultrapure water was taken and added to a test container. After confirming that the binding between AGE-2 on the chip and the test substance has become stable on the display of the apparatus, 8 μL of the test substance solution is further added. This operation was repeated 2 to 4 times to prepare an equilibrium curve (adsorption curve) indicating the interaction between AGE-2 and the test substance. The results were analyzed with dedicated measurement analysis software built in the instrument, and the dissociation constant was calculated.

About each of A liquid and B liquid, from the analysis by software, dissociation constant _Kd = 1.60 * 10 < ^-4 > M in A liquid, and dissociation constant _Kd = 5.87 * 10 < ^-4 > M in B liquid. Therefore, both the substance dissolved in the liquid A which was the solution before stirring and the substance dissolved in the liquid B obtained by permeation through the artificial skin membrane had binding properties to AGE-2.

  Thus, it was found that the whey hydrolyzate and the substance that permeated the artificial skin membrane have binding properties for AGE-2.

(Example 3)
An artificial intestinal fluid (pH 8.3) having the following composition was prepared:
NaCl 60mmol / L
NaHCO ₃ 50 mmol / L
Taurocholic acid 5mmol / L
Oleic acid 10mmol / L
NaH ₂ PO ₄ 10 mmol / L

  AGE-2 was added to this artificial intestinal fluid so that the amount of protein would be 5 g / L (about 0.1 mmol / L). AGE-2 was prepared in the same manner as in Example 2 above.

  100 mg of the lyophilized whey hydrolyzate produced in Example 1 was added to 100 mL of the artificial intestinal fluid to which AGE-2 was added, and the mixture was mixed in a 37 ° C. constant temperature bath with shaking at 20 strokes / minute. After 3 hours, 1 mL of this solution was collected, centrifuged at 3000 rpm, and the supernatant was collected. In this supernatant, as in Example 2 above, residual AGE-2 (unbound to whey hydrolyzate) was quantified by ELISA using HSA antibody and AGE-2 monoclonal antibody.

  The amount of whey hydrolyzate adsorbed was determined by drawing a calibration curve using synthetic AGE-2 as a reference substance. As a result, adsorption of 0.74 g of AGE-2 was observed as the amount of protein per 1 g of whey hydrolyzate.

  This suggested that exogenous AGE-2 can be captured in the digestive tract.

(Reference Example 1)
It was shown that administration of glucose-derived AGE (AGE-1) increases AGE-2 and monocyte chemotactic activator (MCP-1, which is a vascular injury marker) in the blood.

  AGE-1 was synthesized as follows. DL-glucose (Nacalai Tesque, Inc.) 100 mM, diethylenetriaminepentaacetic acid (Wako Pure Chemical Industries, Ltd.) 5 mM, bovine serum albumin (BSA; Sigma Corporation) 0.37 mM, incubated at 37 ° C. for 1 week (60 swings / min. And the resulting crude AGE-1 product was dialyzed against pure water for 3 days to remove impurities. AGE-1 purified by dialysis was lyophilized and used as a sample for administration.

  A 5-week-old male Sprague-Dawley rat (Kudo Co., Ltd.) was acclimated for 1 week, and each group was administered to an AGE-1 5 mg / kg administration group, an AGE-1 10 mg / kg administration group, and a placebo administration group. Five animals were sorted by random sampling. Rats are kept in a breeding device controlled at a temperature of 23 ± 2 ° C and a humidity of 55 ± 10% (however, it is allowed to change the temperature or humidity temporarily due to the effect of cleaning the breeding room, etc.) It was raised in a breeding cage and fed with food for raising rats (CE-2; Claire Japan) and tap water. Using the rat sonde, for the AGE-1 administration group, the dose was calculated to be 5 mg / kg and 10 mg / kg for each individual based on the body weight measured at the nearest time point and forced into the stomach for 30 days. Orally administered. For the placebo group as a control, the same calculated amount and the same amount of pure water were also forcibly orally administered into the stomach.

  30 days after administration, the concentrations of AGE-2 and MCP-1 in the blood of the rats in each administration group were measured. The blood AGE-2 concentration was measured using a competition method ELISA kit prepared by requesting Mizuho Media Co., Ltd. together with the AGE-2 monoclonal antibody obtained as described above. The blood MCP-1 concentration was measured using a rat serum kit sold by Cosmo Bio.

  Administration of AGE-1 for 30 days increased blood AGE-2 concentration in a dose-dependent manner. The blood MCP-1 concentration also increased in a dose-dependent manner, with a statistically significant increase (p <0.01) in the 10 mg / kg group.

Example 4
The rats were treated in the same manner as in Reference Example 1 except that a group in which the freeze-dried whey hydrolyzate produced in Example 1 was administered together with AGE-1 was added and the administration period was 14 days, After treatment, the blood MCP-1 concentration of the rat was measured. More specifically, the rat treatment group includes an AGE-1 10 mg / kg administration group, an AGE-1 10 mg / kg + 100 mg administration group of freeze-dried whey hydrolyzate prepared in Example 1, and a placebo administration group as a control ( 5 animals in each group).

  FIG. 1 is a graph showing changes in blood MCP-1 concentration by AGE-1 administration single administration treatment and AGE-1 and whey hydrolyzate coadministration treatment. The vertical axis indicates the ratio of the blood MCP-1 concentration after treatment to the blood MCP-1 concentration before treatment in%. As a result, the blood MCP-1 concentration was statistically significantly increased in the AGE-1 10 mg / kg administration group as seen in Reference Example 1 (p <0.01). In the lyophilized whey hydrolyzate 100 mg administration group produced in Example 1, the increase in blood MCP-1 concentration observed in the case of AGE-1 alone administration was suppressed (the concentration of MCP-1 in blood is AGE- 1 was statistically significantly lower than the blood MCP-1 concentration of single administration).

(Example 5)
The manufacture example of the cosmetics of this invention is shown. A cosmetic cream containing the following ingredients was prepared by methods commonly used by those skilled in the art: water, glycerin, vegetable squalane, cetyl ethylhexanoate, dimethicone, cetanol, glyceryl stearate, polyglycerin, PEG-60 glyceryl isostearate. The lyophilized whey hydrolyzate prepared in Example 1 above, cetyl palmitate, hydroxyethylethylenediaminesanacetic acid-trisodium salt (HEDTA-3Na), inulin laurylcarbamate, and methylparaben.

(Example 6)
An example of producing cookies using the lyophilized whey hydrolyzate produced in Example 1 above is shown:
Soft flour 70 grams Lyophilized whey hydrolyzate prepared in Example 1 above 10 grams Almond powder 20 grams Baking powder 1/4 teaspoon
40 grams of sugar
Unsalted butter 60 grams 1 egg Caramel chocolate chip 30 grams A few fragrance essences The above ingredients were prepared and cookies were produced according to the usual method. The freeze-dried whey hydrolyzate produced in Example 1 was added together with the powders.

(Example 7)
A production example of a lactic acid bacteria beverage using the freeze-dried whey hydrolyzate produced in Example 1 will be shown:
Plain yogurt 200 grams Freeze-dried whey hydrolyzate prepared in Example 1 30 grams Water 200 cc
Sugar 180 grams Lemon juice 2 tablespoon dry yeast 1/3 teaspoon The above ingredients were prepared and a lactic acid bacteria beverage was prepared according to the usual method. The freeze-dried whey hydrolyzate produced in Example 1 was added in small portions when plain yogurt was added to sugar water, and then yeast was fermented.

(Example 8)
An example of production of tofu using the freeze-dried whey hydrolyzate produced in Example 1 above is shown:
Soybean 300 grams Lyophilized whey hydrolyzate prepared in Example 1 15 grams Coagulant A little of the above ingredients were prepared and tofu was produced according to the usual method. The freeze-dried whey hydrolyzate produced in Example 1 was dissolved in water, and the boiled soybean juice was cooled to about 50 ° C. and then added.

  According to the present invention, a substance capable of binding to AGE-2, which is considered to be highly toxic to cells, is provided. The milk component hydrolyzate of the present invention is useful for inhibiting the absorption of (exogenous) AGE-2 derived from food in the digestive tract. The milk component hydrolyzate of the present invention also accumulates in the body with aging, and is useful for suppressing the accumulation of AGE-2, which is considered to cause aging. The dairy component hydrolyzate of the present invention may also be useful for reducing symptoms (eg, vascular disorders) due to increased AGEs with diabetics and aging. The milk component hydrolyzate of the present invention is useful as a material for cosmetics or foods.

It is a graph which shows transition of the MCP-1 density | concentration in blood by the AGE-1 administration single administration processing and the simultaneous administration processing of AGE-1 and a whey hydrolyzate.

Claims (2)

Whey EC. 3.4.23.1 enzyme , exoprotease , and EC. A glyceraldehyde-derived terminal saccharification product binder comprising a milk component hydrolyzate obtained by hydrolysis with an enzyme represented by 3.4.21.1 ,
The EC. 3.4.23.1, the exoprotease , and the EC. The ratio of the enzyme combination represented by 3.4.21.1 is Unit / 1 kg protein, and is 100 to 1000: 1 to 2: 5 to 20, respectively.
Glyceraldehyde-derived terminal saccharification product binder.   A blood MCP-1 concentration increase inhibitor comprising the binder according to claim 1.

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