NZ623006B2

NZ623006B2 - Sensation-improving agent

Info

Publication number: NZ623006B2
Application number: NZ623006A
Authority: NZ
Inventors: Ken Katoh; Toshiya Kobayashi; Takahiro Moriya; Yutaro Obara; Yuko Ono; Noriko Ueda; Hiroshi Ueno
Original assignee: Megmilk Snow Brand Co Ltd
Priority date: 2011-10-04
Filing date: 2012-10-02
Publication date: 2016-11-01

Abstract

Disclosed is a sense-improving agent that is safe and has the effect of improving dulling of peripheral senses by routine consumption or application to the skin. Also disclosed is to provide a food and beverage product, feed, and cosmetic product for improving senses that have the effect of improving peripheral senses by oral ingestion or application to the skin. A sense-improving agent is provided that uses a milk-derived protein and/or milk-derived protein decomposition product which is one of actoperoxidase, lactoferrin, cystatin and angiogenin as the active ingredient. The milk-derived protein and/or milk-derived protein decomposition product is capable of improving dulling of the senses, particularly the peripheral senses, and can be used in a food and beverage product, feed, or cosmetic for improving the senses. g peripheral senses by oral ingestion or application to the skin. A sense-improving agent is provided that uses a milk-derived protein and/or milk-derived protein decomposition product which is one of actoperoxidase, lactoferrin, cystatin and angiogenin as the active ingredient. The milk-derived protein and/or milk-derived protein decomposition product is capable of improving dulling of the senses, particularly the peripheral senses, and can be used in a food and beverage product, feed, or cosmetic for improving the senses.

Description

SENSATION~IMPROVING AGENT TECHNICAL FIELD The present ion relates to a sensation—improving agent that contains a milk—derived protein and/or a hydrolysate therefrom as an active ingredient, the sensation-improving agent has an effect of improving dulling of peripheral nerves.

And the t ion relates to a sensation—improving food, beverage, feed, or cosmetics that includes the ion—improving agent.

BACKGROUND ART In recent years, increases in age—related diseases such as osteoporosis and dementia have become a serious social issue associated with aging. ‘Various drugs have been developed to prevent or cure these age-related diseases. However, side effects of such drugs always need to be taken into consideration. Recently, attempts have been made to prevent or cure age~related diseases through a reconsideration of dietary habits or ingestion of a specific food ingredient. For example, ingestion of a basic protein in bovine milk is known to prevent or cure orosis . Furthermore, a preventive and therapeutic agent against Alzheimer’s defects of memory, containing omyelin, a relatively abundant phospholipid in bovine milk, as an active ingredient is known.

An e of the age—related symptoms includes g of peripheral sensations, which is caused by not only aging, but also diseases such.as diabetes. The g of peripheral sensations may lead to troubles, for example, a higher risk of ing burns caused by failure to feel hot rightly on touching a hot object, or a risk of delaying the discovery of an injury caused by a dull sensation of pain. In recent years, studies that reduce dulling of peripheral sensations caused by aging or diseases have been conducted in order to prevent such risks. For example, it has been reported that exogenous ceramide and the enzymes sphingomyelinase and phosphatidylcholine-specific olipase C, the enzymes increase biosynthesis of endogenous ceramide, promote morphological differentiation.of P-lZ cells being neural.model cells through 3T3 cells being an established fibroblast cell line, that is, a neurotrophic factor secreted by 3T3 cells (Non Patent Document 1). The promotion of morphological differentiation of the neural.model cells indicates the effect of improving dulling of peripheral sensations. However, use of the above de and enzymes, which are not food ingredients, requires examination of their safety. In such situations, there is a need for a safer agent that can improve dulled peripheral sensations through daily ingestion or application to the skin.

Components in milk are known to have many physiological activities. For example, milk—derived sphingomyelin and phospholipids are known to have an effect of improving dulling ofperipheralsensations(PatentDocumentJ.andPatentDocument 2). However, the effect of milk—derived proteins on improvements in dulling of peripheral sensations is not yet known. Examples of the milk-derived proteins include lactoperoxidase, lactoferrin, cystatin, and angiogenin.

Lactoperoxidase, t in milk, is a heme iron—containing glycoprotein, while details on its structure are yet to be known. Lactoperoxidase has been found to have an effect of ting in vivo production of lipid peroxides and is used as an anti—aging agent that ts loss of sight andInotor skills, and‘decline in immune functions and.the like, verfunctionimprovementagent. heglycoprotein is known to be used as a low—cariogenic nutrient composition due to its low cariogenicity. However, it is yet to be known that lactoperoxidase and an enzymatic hydrolysate therefrom produced with a protease have an effect of improving dulling of peripheral , and are of use as a sensation—improving agent. {0006] Lactoferrin and its hydrolysate are known to have an effect of preventing adhesion of pathogens to cells and an antiviral action. Moreover, xture with an epidermal growth factor is reported to increase a skin cell ting effect of the epidermal growth.factor alone. Also, lactoferrir1is generally known to have an effect of ating stress associated with pain and emotional stress. However, it is yet to be known that lactoferrin and an enzymatic hydrolysate therefrom produced with a protease have an effect of improving dulling of peripheral , and are of use as a sensation—improving agent.

Cystatin is a cysteine protease inhibitor that inhibits the proteolytic activity of a cysteine protease having a SH group in the active center, and is found in animal tissues, cells, blood ine. Also, cystatin's effect of inhibiting virus growth is found to be a beneficial effect. However, it is yet to be known that cystatin and an enzymatic hydrolysate therefrom produced with a protease have an effect of reducing dulling of peripheral nerves, and are of use as a sensation—improving agent. {0008] Angiogenin is one of enesis factors. Human angiogenin is known to be a protein having a molecular weight of 14, 400, and is present in blood and milk. Bovine angiogenin is isolated from bovine milk and is subjected to amino acid sequencing, and the results have already been reported. A production of angiogenin from bovine milk by subjecting milk to cation ge chromatography to apply the milk on the cation exchange column, eluting the adsorbate with an alkali metal salt solution of a weak organic acid, and subjecting the resulting eluate to cation exchange chromatography again and gel filtration chromatography to collect angiogenin is disclosed. Moreover, angiogenin is found to specifically inhibit melanin production in melanoma B-l6 cells, and is ed to be used also as a possible ing agent.

However, it is yet to be known that enin and.an tic hydrolysate therefronlproduced with a protease have an effect of improving dulling of peripheral nerves, and are of use as a sensation—improving agent.

RELATED ART DOCUMENT PATENT DOCUMENT Patent Document 1: Japanese Patent Application pen Publication No. 2009—126787 Patent Document 2: Japanese Patent Application Laid—Open Publication No. 2009—126788 NON PATENT DOCUMENT Non Patent Document 1: Annual Report of Cosmetology, vol. 10, (2002) Disclosure of The Invention SUMMARY OF THE INVENTION [0010a] According to a first aspect of the present invention there is provided the use of at least one milk-derived protein and/or at least one hydrolysate rom in the manufacture of a medicament for improving peripheral sensation in a subject with es- or ageing–related dulling of peripheral sensation; wherein the at least one erived n is selected from lactoperoxidase, lactoferrin, cystatin and angiogenin.

According to second aspect of the present invention there is provided a food, beverage, feed or cosmetic comprising the medicament according to the first aspect.

The present invention provides a safety sensationimproving agent that can improve dulled peripheral sensations through daily ingestion or application to the skin. r object of the present invention is to provide a sensation-improving food, beverage, feed, or cosmetics that can improve dulled eral sensations through oral ingestion or application to the skin.

[0012] The present inventors, who have diligently pursued a safe component highly effective for the improvement of dulled sensations, found that oral ingestion or direct application to the skin of any milk-derived protein and/or any hydrolysate rom can improve the dulling of sensations, particularly peripheral sensations. Use of such a milk-derived protein and/or a hydrolysate therefrom as an active ingredient has completed a sensation-improving agent. The present ors also found that the sensation-improving agent can be added with a food, beverage or a feed to form a sensation-improving food, beverage, feed, or cosmetics, and have completed the present invention. Throughout the specification, lactoperoxidase, lactoferrin, cystatin, and angiogenin, which are present in milk, are referred to as "milk—derived ns". These proteins in use for the sensation—improving agent according to the present invention may not necessarily be derived from milk. For example, they may be synthesized artificially or be purified from blood.

The present invention relates to the following aspects: (1) A sensation—improving agent containing a milk—derived n and/or a ysate rom as an active ingredient; (2) The ion—improving agent according to Aspect (1), wherein the milk—derived protein is at least one selected from lactoperoxidase, errin, cystatin, and angiogenin; (3) The sensation—improving agent according to Aspect (1) or (2), wherein the hydrolysate from the milk—derived protein is produced through hydrolisis of the milk-derived protein with a protease; (4) The sensation—improving agent according to Aspect (3), wherein the protease is at least one selected from the group consisting of pepsin, trypsin, chymotrypsin, and pancreatin; (5) A sensation—improving food, beverage, feed, or cosmetics containing the component according to any one of Aspects (l) to (4); (6) A method for improving a ion in a mammal, ing taking the mammal a milk-derived protein and/or a hydrolysate therefrom, or applying the milk—derived protein and/or the hydrolysate therefrom to the skin of the mammal; and (7) The method ing to Aspect (6), wherein the mammal is a human, and the erived protein and/or the hydrolysate therefrom is fed at a dose of 10 mg or more per day for an adult human.

EFFECT OF INVENTION

[0014] The sensation—improving agent according to the present invention can provide an effect of improving dulling of peripheral sensations.

DESCRIPTION OF EMBODIMENTS . [0015] The present ion is characterized by’a milk—derived proteinand/oreahydrolysatetherefrmnasenlactiveingredient. es of the milk—derived protein.include lactoperoxidase, lactoferrin, cystatin, and angiogenin. These milk—derived proteins may be prepared from milk of mammals such as human, cattle, buffalo, goat, and sheep, or be produced by a genetic engineering procedure. Hydrolysates of these milk—derived proteins can be prepared fronlthe milk-derived proteins by the action of a protease.

[0016] Lactoperoxidase is prepared from milk of mammals. The examples of the source of milk include mammals such as human, cattle, buffalo, goat, and sheep. Lactoperoxidase, which is a known and commercially ble substance, can be industrially produced using known methods, for example, a method for purifying lactoperoxidase with a sulfonated carrier (Japanese Patent Application Laid—Open Publication No. 3—109400). Lactoperoxidase producedkn/a c engineering ure can also be used in the present invention. A hydrolysate from lactoperoxidase is a peptide mixture prepared by limited proteolysis of the above-described lactoperoxidase with a protease such as trypsin, pancreatin, chymotrypsin, pepsin, , rein, cathepsin, lysin, or V8 protease so as to have a molecular weight of 10,000 or less.

The lower limit of the molecular weight is preferably 500 or more.

Lactoferrin is prepared from milk of mammals. The examples of the source of milk include mammals such as human, cattle, buffalo, goat, and sheep. Lactoferrine, which is a knownandcommerciallyavailablesubstance,canbeindustrially produced using known methods, for example, a method for purifying lactoferrin with a ated carrier (Japanese Patent Application Laid—Open Publication No. 3—109400).

Lactoferrin produced by a genetic ering procedure can also be used in the present invention. A hydrolysate from lactoferrin is a peptide mixture prepared by limited proteolysis of the above-described errin with a protease such as trypsin, atin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, or V8 protease so as to have a molecular weight of 10,000 or less. The lower limit of the molecular weight is preferably 500 or more.

Cystatin from any source can be used, including one derived from milk of s such as human, cattle, buffalo, goat, and sheep. For example, the gene sequence of in derived from human milk and bovine milk has already been determined; hence cystatin can be produced by recombinant production, and cystatin produced by a genetic engineering procedure can also be used in the present invention. atively, cystatin, which is relatively abundant in bovine colostrum, may be collected from the milk. Cystatin can also be collected from a cell culture medium, and such cell culture medium-derived cystatin may be used. For example, milk—derived cystatin can be produced in accordance with a known method (Japanese Patent Application Laid—Open Publication No. 2000—28158?) from milk such as raw milk, milk powder, skim milk, and tituted milk through ents such as heat treatment, salting treatment, l treatment, various chromatographic treatments such as ion exchange chromatography and gel filtration chromatography, and iltration treatment. A hydrolysate from cystatin can be a peptide mixture prepared by limited proteolysis of the cystatin with a protease such as trypsin, pancreatin, chymotrypsin, pepsin, , kallikrein, cathepsin, thermolysin, or V8 protease so as to have a molecular weight of 8,000 or less. The lower limit of the molecular weight is preferably 500 or more.

For sources of angiogenin, colostrum within 1 to 7 days after parturition, ularly preferably within 1 to 5 days after parturition obtained from mammals such as human, cattle, buffalo, goat, and.sheep is suitable because such colostrunlhas a high angiogenin content, although milk during the original lactationperiodcanalsobeusedasarawnmterialinthepresent invention. Angiogenin can be rially produced using known methods, for example, a method for purifying angiogenin by a combination of cation exchange tography and gel filtration chromatography (Japanese Patent Application pen Publication No. 2-296000). A hydrolysate from angiogeniniseapeptidendxturepreparedbylimitedproteolysis of the aboveedescribed angiogenin with ses such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, or V8 protease so as to have a molecular weight of 10,000 or less. The lower limit of the molecular weight is preferably 500 or more.

The sensation—improving agent ing to the present invention may be used as the above—described milk-derived protein,particularlylactoperoxidase,lactoferrin,cystatin, and angiogenin, or their ysates prepared from the milk—derived protein by the action of a protease, may be:mixed with other raw materials, such as saccharides, lipids, proteins, vitamins, minerals, and flavors, commonly used for pharmaceutical products, food and beverage, and feeds, or may beformulatedintopowders,granules,tablets,capsules,drinks and any other preparation in accordance with conventional methods. The sensation—improving agent according to the present invention can be used as application agent in any conventionalapplicationform,:mxﬂ1asemulsion,cream,lotion, or pack. These application agents may be produced through conventional methods by appropriately adding a milk—derived protein and/or a hydrolysate therefronlas an active ingredient in the t invention in the course of production, and can also be used as cosmetics. Also, other components that have a sensation—improving effect, for example, ceramide, sphingomyelinase, and.sphingomyelin.can.be'usedijicombinatior1 withthenﬁlk—derivedproteinand/orthehydrolysatetherefrom.

In the test described below for the effective amount of the ion—improving agent ing to the present invention, the milk—derived protein and/or the hydrolysate therefrom was orally ingested in a mouse at a dose of 10 mg or more, preferably mg or more per kg of body weight of the mouse to improve the peripheral sensations in the mouse. The dulling of sensations, particularly peripheral sensations can be ed to be improved by ingesting the milk-derived n and/or the ysate therefrom lly at a dose of 10 mg or more, preferably 20 mg or more per day for an adult human. It is desirable to ensure the ingestion at this necessary dose. If applied as an application agent to the skin, the density of the applied erived protein and/or ysate therefrom is 0.001 to 40% by weight, more preferably 0.1 to 10% by weight based on the total weight of the application agent.

The sensation-improving food, beverage according to the present invention may be produced by adding the erived protein and/or the hydrolysate therefrom with a conventional food, beverage, for example, yoghurt, amilk beverage, a wafer, and a dessert. Depending on the form of these sensation-improving food, beverage, a milk—derived protein and/or a hydrolysate therefrom is preferably combined in an amount of 0. 5 to 2000 mg per 100 g of the food, beverage in order to take a human the milk-derived protein and/or the hydrolysate therefrom at a dose of 10 mg or more per day for an adult human.

The sensation—improving feed according to the present invention may be produced by adding the milk-derived protein and/or the hydrolysate therefrom with a conventional feed, for example, a feed for domestic animals and a pet food. For example, if these feeds contain the milk—derived n and/or the hydrolysate therefrom, a milk-derived protein and/or a ysate therefrom is preferably added in an amount of 0.5 to 2000 mg per 100 g of the feed in order to feed a mammal the milk—derivedproteinand/orthehydrolysatetherefrmnataadose of 10 mg or more.

Thenulk—derivedproteinand/orthehydrolysatetherefrom may be combined by any method in the present invention. For example, for addition in solution, a milk—derived protein and/or a hydrolysate therefrom is suspended or dissolved in deionized water, and the mixture is stirred followed by formulation of the mixture into the form of a pharmaceutical t, a food, beverage, and a feed. The milk—derived protein and/or the hydrolysate therefronlin zed.water is stirred under such conditions that the milk-derived protein and/or a hydrolysate therefrom may be homogeneously mixed, and can be mixed with an ultradisperser or a TK homomixer. The solution can be concentrated with an RO ne or —dried, if necessary, to be readily used for a pharmaceutical product, a food, beverage, and a feed. The formulation process in the presentinventioncanincludesterilizationtreatmentcommonly used in the manufacture of pharmaceutical products, food, beverage, and feeds, and for the formulation in the form of powder, dry—heat sterilization. Accordingly, a pharmaceutical product, a food, beverage, and a feed that contain.a milk-derived protein and/or a hydrolysate therefrom according to the present invention can be ed in various forms such as liquid, gel, powder, or granule form.

The present invention will be described below in detail by way of examples and test examples, which are illustrative only and not intended to be limiting the present invention in any way.

Example 1 A column (diameter 5 cm.and height 30 cm) filled with 400 g of a<cation exchange resin, sulfonated earl (from Fuji ngCo.,Ltd.)wasthoroughlywashedwithdeionizaiwater, and 40 L of unsterilized skim milk (pH 6.7) was then passed h the column at a flow rate of 25 . The column was then thoroughly'washedwwith zed water, and the adsorbate was eluted with a 0.02 M carbonate buffer solution containing 2.0 M sodium chloride (pH 7.0). Eluted fractions containing lactoperoxidase were allowed to apply on an S-Sepharose FF column (from GE Healthcare Ltd.) , and the column was thoroughly washed with deionized.water, and was equilibrated.with a lOIMM phosphate buffer solution (pH 7.0). The adsorbate was then eluted by a linear gradient of O — 2.0 M sodium chloride to collect a fraction containing lactoperoxidase. The fraction was treated by gel filtration chromatography on HiLoad 16/60 Superdex 75 pg (from GE Healthcare Ltd.) to yield 3.0 g of eroxidase. The resulting lactoperoxidase had a purity of 94%, and can be used as a ion-improving agent (Example Product 1) without further purification.

Example 2 Lactoperoxidase (1 g) prepared in Example 1 was dissolved in 200 ml of water, and a pancreatin (from Sigma Co.) was added to the solution into a final tration of 0.01% by weight.

The solution was treated with the enzyme at 37°C for 5 hours.

The e was heat—treated at 90°C for 5 minutes to deactivate the enzyme, and was freeze—dried to yield 0. 8 g of a hydrolysate from lactoperoxidase. The resulting hydrolysate from lactoperoxidase had.a molecular weight of 10,000 or less, and can be used as a sensation—improving agent (Example Product 2) without further purification.

Example 3 {0026] Lactoperoxidase (1gﬂ preparede1Example1.was dissolved in 200 ml of water, and a trypsin (from Sigma Co.) was added to the solution into a final concentration of 0.01% by .

The solution was treated with the enzyme at 37°C for 5 hours.

The mixture was heat-treated at 90°C for 5 minutes to deactivate the enzyme, and was freeze—dried to yield 0. 9 g of a ysate from lactoperoxidase. The resulting hydrolysate from lactoperoxidase had a molecular weight of 10,000 or less, and can.be used as a sensation—improving agent (Example t 3) t r purification.

Example 4 A column (diameter 5 cm and height 30 cm) filled with 400 g of a cation exchange resin sulfonated Chitopearl (from Fuji SpinningCo.,Ltd.)wasthoroughlywashedwithdeionizedwater, and 40 L of unsterilized skim milk (pH 6.7) was then passed through the column at a flow rate of min. The column was then thoroughlyiwashed.with.deionized‘water, and the adsorbate was eluted with a 0.02 M carbonate buffer solution containing 2.0 M sodium de (pH 7.0). Eluted fractions containing lactoferrin were allowed to apply on an S—Sepharose FF column (from GE Healthcare Ltd.) , and the column was thoroughly washed with deionized water, and was equilibrated with a 10 mM phosphate buffer solution (pH 7.0). The adsorbate was then eluted by a linear gradient of 0 — 2.0 M sodium chloride to collect a fraction containing lactoferrin. The fraction was treated by gel filtration chromatography on HiLoad 16/60 Superdex 75 pg (from GE Healthcare Ltd.) to yield 8.0 g of lactoferrin. The resulting lactoferrin had a purity of 96%, and can be used as a sensation—improving agent (Example Product 4) without further purification.

Example 5 Lactoferrin (l g) prepared in Example 4 was dissolved in 200 ml of water, and a pancreatin (from Sigma Co.) was added to the solution into a final tration of 0.01% by weight.

The solution was treated with the protease at 37°C for 5 hours.

The e was heat-treated at 90°C for 5 minutes to deactivate the enzyme, and was freeze-dried to yield 0. 8 g of a hydrolysate from lactoferrin. The resulting hydrolysate from lactoferrin had a molecular weight of 10,000 or less, and can be used as a sensation-improving agent (Example Product 5) without r purification.

Example 6 Lactoferrin (l g) prepared in e 4 was dissolved in 200 ml of water, and a trypsin (from Sigma Co.) was added to the solution into a final concentration of 0. 01% by weight. The on was treated with the protease at 37°C for 5 hours. The mixture was heat—treated at 90°C for 5 minutes to deactivate the enzyme, and was freeze-dried to yield 0. 9 g of a hydrolysate from lactoferrin. The resulting hydrolysate from lactoferrin had a molecular weight of 10,000 or less, and can be used as a sensation—improving agent (Example Product 6) without further purification.

Example 7 A column filled with 3, 000 g of S—Sepharose was thoroughly washed with.deionized water, and 10,0001;of ilk.was then passed through the column. The column was thoroughly washed with deionized water, and the adsorbate was then eluted by a linearconcentrationgradientwithCL].tol.0blsodiumchloride.

Theresultingfractionswereheat—treatadat90°CforlOnﬁnutes and were then centrifuged to remove precipitates. The eluted fraction containing bovine milk~derived basic cystatin was again fractionated by Mono S ion exchange chromatography. This fraction was treated tially by Mono Q ion exchange chromatography and Superose 12 gel filtration chromatography in an FPLC system, and subsequently yapatite chromatography and C4 e phase chromatography in an HPLC systenlto yield 58 mg of in (Fraction A). The resulting cystatin can be used as a sensation—improving agent (Example Product 7) without further purification.

Example 8 A.5% whey n solution (10,000 L) was heat-treated at 90°C for 10 minutes, and was then centrifuged to remove precipitates. A column was filled with a carrier prepared by binding carboxymethylated papain to Tresyl-Toyopearl (from TOSOH CORPORATION), and was then equilibrated with a 0.5 M sodium chloride solution. The above-described whey protein solution was passed.through the column. The column.was washed with a 0.5 M sodium chloride solution and then a 0.5 M sodium chloride solution containing 0.1% Tween.20. Cysteine protease was then eluted with a 20 mM acetic acid—0.5 M sodium chloride on. Eluted fractions were immediately neutralized with aJ.M sodium hydroxide solution. The lized solution was, fractionated by Mono S anion ge chromatography, hydroxyapatite chromatography, and then C4 reverse phase chromatography in an HPLC system to yield 48 mg of milk—derived ystatin(FractionB). Theresultingcystatincanbetmed as a sensation-improving agent (Example Product 8) without further purification.

Example 9 Fraction A (25 mg) prepared in Example 7 was suspended in 100ml of water, and pancreatin was added to the suspension into a final tration of 1% by weight. The suspension was treated with the enzyme at 37°C for 5 hours. The suspension was heat—treated at 90°C for 5 minutes to deactivate the enzyme, and was freeze—dried to yield 23 mg of a ysate from cystatin (Fraction C). The fraction B (25 mg) prepared in Example 8 was d in a similar manner to yield 24 mg of a hydrolysate from cystatin (Fraction D). The resulting hydrolysate from cystatin had a molecular weight of 8,000 or less, and can be used as a sensation—improving agent (Example t 9) without r purification.

Example 10 A column (diameter 5 cm and height 30 cm) filled with 400 g of a cation exchange resin sulfonated Chitopearl (from Fuji SpinningCo.,Ltd.)wasthoroughlywashedwithdeionizedwater, and 40 L of unsterilized skim milk (pH 6.7) was then passed through the column at a flow rate of 25 ml/min. The column was then thoroughly washediyith deionized water, and the adsorbate was eluted with a 0.02 M carbonate buffer solution containing 2.0 M sodium chloride (pH 7.0). Eluted fractions containing angiogenin were allowed to apply on an S—Sepharose FF column (from GE Healthcare Ltd.) , and the column was thoroughly washed with deionized water, and was equilibrated with a 10 mM phosphate buffer solution (pH 7.0). The adsorbate was then eluted by a linear gradient of 0 — 2.0 M sodium chloride to t a fraction containing angiogenin. The fraction was treated by gel filtration chromatography on HiLoad 16/60 Superdeg 75 pg (from GE Healthcare Ltd.) to yield 1.8 g of a fraction abundantly containing angiogenin. The angiogenin content in the resulting fraction abundantly containing enin is 10%, and the on can be used as a sensation—improving agent (Example Product 10). e 11 A column filled with 3,000 g of a cation exchange resin, sulfonated ChitOpearl (from Fuji Spinning Co., Ltd.) was thoroughly washed with deionized water, and 100 L of unsterilized skim milk (pH 6.7) was then passed through the column. This column.was then thoroughly washednwith deionized water, and the adsorbate was eluted by a linear concentration gradient of 0.1 to 2.0 M sodium.chloride. The eluted fraction containing angiogenin was onated by S-Sepharose cation exchange chromatography (from GE Healthcare Ltd.), the resulting fraction containing angiogenin was heat—treated at 90°C for 10 minutes, and was centrifuged to remove precipitates .

This fraction containing angiogenin was sequentially d by Mono S cation exchange tography, Superose 12 gel filtrationchromatography,hydroxyapatitechromatography,and C4 reverse phase chromatography to yield 55 mg of angiogenin.

The ing angiogenin had a purity of 99%, and can be used as a sensation—improving agent (Example Product 11).

Example 12 Angiogenin.(5 mg) prepared in Example 11 was ved in ml of water, and pancreatin (from Sigma Co.) was added to thesolutionintoaifinalconcentrationof0.01%byweight. The solution was treated with the enzyme at 37°C for 5 hours. The solution was heat—treated at 90°C for 5 minutes to deactivate the enzyme, and was freeze-dried to yield 4 . 0 mg of a hydrolysate from angiogenin. The resulting hydrolysate from angiogenin had a molecular weight of 10,000 or less, and can be used as a sensation—improving agent (Example Product 12) without further cation.

Example 13 Angiogenin (5 mg) prepared in Example 11 was dissolved in 10 ml of water, and trypsin (from Sigma Co.) was added to the solution into a final concentration of 0.01% by weight. The solution was d with the enzyme at 37°C for 5 hours. The solution was heat-treated at 90°C for 5 minutes to deactivate the enzyme, and was freeze—dried to yield 4 .2 mg of a hydrolysate from angiogenin. The resulting hydrolysate from angiogenin had a molecular weight of 10,000 or less, and can be used as a ion—improving agent (Example Product 13) without further purification.

Test e 1 ication of promotion of cell differentiation) 3T3 cells being a fibroblast cell line that is known to bepresentintheskinwereincubatedfortwodaysinthepresence of Example Products 1, 3, 4, 5, and 7, Fraction C in e Product 9, and Example Products 11 and 13, each in a concentration of 0.03 to 1%. As a control, 3T3 cells were incubated for two days in the absence of any e Product (control). PC-12cellsbeingneuralmodelcellswereincubated with those culture supernatants, and morphological differentiation of the PC-12 cells was observed when a neurotrophic factor was secreted by 3T3 cells.

The results showed that all of the culture supernatants containing an Example Product differentiated PC~12 cells clearly. This experiment was repeated several times, and the proportion of differentiation was observed with an optical microscope. When any Example Product was added, differentiation was observed in 95% or more of the cells. In contrast, the control culture atant failed to entiate PC—12 cells, and observation with an optical microscope in the experiment that was repeated several times revealed no differentiation. This shows that a milk—derived n and/or a hydrolysate therefronlpromotes the secretion of a neurotrophic factor from 3T3 cells, and es the differentiation of PC—l2 cells being neural model cells.

Test example 2 (Verification of sensation—improving effect in animal experiments) A sensation—improving effect by thermal stimulation was evaluated in the hot plate that is a behavioral study to thermal stimulation develOped by Woolfe, MacDonald, et al. ks old hairless mice R—l) were divided into 13 groups with 6 mice in each group. Example Products 2, 6, and 8, Fraction D in Example Product 9, Example Products 10 and 12 were each orally administered to a mouse through a sonde in a dose of 10 mg or 20 mg once daily per kg body weight in a mouse, or a vehicle only was orally administered to a mouse through a tube once daily (control; 0 mg), and.these mice were bred for 4 weeks. At the end of the administration, mice were placed on a hot plate at 54°C, and the time until the mice exhibited escape behavior, such as pulling their paws away from the hot plate, standing up, and g was measured. The maximal strength of thermal stimulation was set to 30 s, the value at this maximal strength was assigned to 30 seconds. The results are shown in Table l.

[Table 1] escape behavior positive response time control 0 mg 29.2 i 0.14 seconds Example Product 2 10 mg 22.2 i 0.17 seconds mg 20.1 i 0.25 seconds Example t 6 10 mg 22.8 d: 0.18 seconds mg 20.5 :t 0.22 s Example Product 8 10 mg 23.1 :I: 0.27 seconds mg 21.0 :I: 0.13 Seconds Example Product 9 10 mg 23.3 :I: 0.10 seconds (Fraction D) 20 mg 21.1 :I: 0.21 s Example Product 10 10 mg 26.2 :I: 0.17 seconds mg 24.1 :I: 0.16 s Example Product 12 10 mg 23.9 :I: 0.21 seconds mg 21.8 :I: 0.20 seconds [0 0 4 1] Table 1 demonstrates that ingestion of Example Products 2, 6, and 8, Fraction D in Example Product 9, and Example Products and 12 each show a tendency toward a shorter escape behavior positive response time at a dose of 10 mg, and signifficantly shortened the time at a dose of 20 mg. This indicates that ingestion of Example Products 2, 6, and 8, Fraction D in Example Product 9, and Example Products 10 and 12 can prevent or improve thedullingofsensations,particularlyperipheralsensations.

Test example 3 (VerificatLMIOfsensation—improvingeffectknroralingestion) Healthy y subjects (average age 75i3) who experienced dulled sensations in the hand were divided into 9 groups with 10 subjects in each group. These groups consisted of Group A with ingestion of no Example Product, Group B with ingestion of Example Product 1 at a dose of 10 mg, Group Clﬂith ingestion of e Product 1 at a dose of 20 mg, Group D‘Nith ingestion of Example Product 4 at a dose of 10 mg, Group vaith ingestion of Example Product 4 at a dose of 20 mg, Group F with ion of Example Product 7 at a dose of 10 mg, Group vaith ingestion of Example t 7 at a dose of 20 mg, Group Hxvith ingestion of Example Product 11 at a dose of 10 mg, and Group I with ingestion of Example Product 11 at a dose of 20 mg, and such ingestion was continued for 6 weeks. As determined with an algesiometer (from Intercross) which is an instrument for determining superficial sensations in accordance with the manufacturer's directions for use before and after the 6-week ion, pain sensations in the palHIOf the hand and the sole of the foot were graded in four ranks from normal to declines I to III on the basis of pain sensations in the medial side of the arm. The results are shown in Tables 2 and 3. er, after the 6-week ingestion, a questionnaire survey was conducted to each subject on the improvement of his/her sensation in the hand. The results are shown in Tables 4 and rement) The pain sensation was evaluated using five pins that have different thicknesses in combination with five positions of a fulcrum“ The thinnest pin 1 was .along the medial side of the arm, and the subject was asked about the degree of normal pain sensation. The pin 1 was then rolled along the palm and the sole of the foot while the position of the fulcrum.for the holder was sequentially changed to determine the position of the fulcrum at which the same degree of pain sensation as the first pain sensation was .

(Evaluation) The algesiometer was designed.to cause pain sensations in the same degree in rolling the pin 1 (fulcrum: 50 g) along the medial side of the arm and in rolling the pin 2 (fulcrum: 50 g) along the palm, and was used in accordance with the manufacturer's directions for use to evaluate the pain sensation as described below. The evaluation of the pain was scored and the scores were ed.

Normal (score 0): The pain sensation in the same degree was caused in rolling the pin 2 (50g) Decline I (score 1): The pain sensation.in.the same degree was caused in rolling the pin 1 (50g) e II (score 2) : The pain sensation in the same degree was caused in rolling the pin 1 (60g) Decline III (score 3): The pain sensation in the same degree was caused in rolling the pin 1 (70g) [Table 2] Measurement of sensation in the hand (Before ingestion) Normal Decline I Decline II e III Average value Group A 0 2 3 5 2.3 Group B 0 1 5 4 2.3 Group C 0 1 5 4 2.3 Group D 0 1 4 5 2.4 Group E 0 1 5 4 2.3 Group F O 2 3 5 2.3 Group G 0 1 5 4 2.3 Group H 0 1 5 4 2.3 Group I 0 1 4 5 2.4 Measurement of sensation in the hand (After 6-week ingestion) Normal Decline I Decline II Decline III Average value Group A 0 2 4 4 2.2 Group B 0 3 5 2 1.9 Group C 2 4 3 1 1.3 Group D 1 2 4 3 1.9 Group E 2 2 5 1 1.5 Group F 0 3 5 2 1.9 Group G 2 4 3 1 1.3 Group H 1 2 4 3 1.9 Group I 2 2 5 1 1.5 [Table 3} Measurement of sensation in the sole of the foot (Before ingestion) Normal Decline I e II Decline III Average value Group A 0 2 3 5 2.3 Group B 0 1 4 5 2.4 Group C 0 1 3 6 2.5 Group D 0 1 6 3 2.2 Group E 0 1 4 5 2.4 Group F 0 2 3 5 2.3 Group G 0 1 4 5 2.4 Group H 0 1 3 6 2.5 Group I 0 1 6 3 2.2 Measurement of sensation in the sole of the foot (After 6-week ingestion) Normal Decline I Decline II Decline III Average value Group A 0 2 3 5 2.3 Group B 1 4 1 4 1.8 Group C 2 3 3 2 1.5 Group D O 3 6 1 1.8 Group E 1 3 5 1 1.6 Group F 1 4 1 4 1.8 Group G 2 3 3 2 1.5 Group H 0 3 6 1 1.8 Group I 1 3 5 1 1.6 [Table 4] Sensation in the hand Deteriorated Unchanged Recovered Group A 2 7 1 Group B 1 5 4 Group C 0 2 8 Group D 0 3 7 Group E 0 2 8 Group F 1 5 4 Group G 0 2 8 Group H 0 3 7 Group I 0 2 8 [Table 5] Sensation in the sole of the foot Deteriorated Unchanged Recovered Group A 2 7 1 Group B Group C Group D Group E Group F Group G Group H Group I OOOOOOOO ‘INO) mmmmmmmph Tables 2 to 5 demonstrate that ingestion of Example Products 1, 4, 7, and Example Product 11 each Show a tendency toward.improved sensations in the hand.and the sole of the foot at a dose of 10 mg, and signifficantly ed the sensations at a dose of 20 mg. The dulled sensations, particularly peripheral sensations can be expected to be improved by ingesting the milk—derived protein and/or the hydrolysate therefrom typically at a dose of 10 mg or more, preferably 20 mg or more per day for an adult human.

Example 14 (Preparation.of sensation—improving'cosmeticrproduct (cream)) A ysate from lactoperoxidase prepared in Example 3 (Example Product 3) was used to produce a sensation-improving ic product (cream) by mixing with raw materials in the proportion shown in Table 6.

[0049] [Table 6] Line 6: Hydrolysate from eroxidase Glyceryl monostearate (self-emulsiﬁable) 10.0 Puriﬁed lanolin 6.0 Liquid parafﬁn 5.0 Jojoba oil 5.0 Paraben 03 Decomposed product from lactoperoxidase 1.0 (Example Product 3) Flavoring agent riate amount Sterile ion exchanged water q.s to 100.0 Test example 4 (Test for sensation—improving effect by application to the skin) Healthy elderly subjects ge age 75i3) who experienced dulled sensations in the hand were divided into 2 groups, Group A and Group B with 15 subjects in each group. A cosmetic product (cream) prepared as in e Product 14 exceptthatanysensation—improvingagentwasnotcontainedwas applied to subjects in Group A, and a sensation—improving cosmetic product (cream) in Example t 14 to subjects in Group B once daily over their hands and feet. The application was continued for 6 weeks. As determinedxnith an ometer (from Intercross) which is an instrument for determining superficial sensations in accordance with the manufacturer's directions for use before and after the 6-week application, pain sensations in the palm.of the hand and the sole of the foot were graded in four ranks from normal to declines I to III on the basis of pain sensations in the medial side of the arm. The results are shown in Tables 7 and 8 . Moreover, after the 6—week application, a questionnaire survey was ted to each subject on the improvement of his/her sensation in the hand.

The results are shown in Tables 9 and 10. The measurement was carried out as in Test example 3.

[Table 7] Measurement of sensation in the hand (Before application) Normal Decline I Decline II Decline III Average value Group A 0 4 6 5 2.1 Group B 0 5 4 6 2.1 Measurement of sensation in the hand (After 6-week application) Normal Decline I Decline II Decline III Average value Group A 1 3 7 4 1.9 Group B 3 6 3 3 1.4 [Table 8] Measurement of ion in the sole of the foot (Before application) Normal Decline I Decline II Decline III Average value Group A 0 5 5 5 2.0 Group B 0 5 6 4 1.9 Measurement of sensation in the sole of the foot (After 6-week ation) Normal e I Decline Il Decline III Average value Group A 1 3 5 6 2.1 Group B 2 6 6 1 1.4 [Table 9] Sensation in the hand Deteriorated Unchanged Recovered Group A 2 12 1 Group B 1 6 8 [Table 10] Sensation in the sole of the foot Deteriorated Unchanged Recovered Group A 1 13 1 Group B 0 9 6

[0055] Tables 7 to 10 demonstrate that application of the sensation—improving cosmetic t (cream) in Example Product 14 shows a tendency toward improved sensations in the hand and the sole of the foot. This indicates that the dulled peripheral sensations can be expected to be improved by ng the cream containing a sensation—improving agent according to the present invention.

Example 15 (Preparation of sensation—improving liquid nutrient composition) A hydrolysate (5 g) from errin in Example Product was dissolved in 4995g of zed water, the solution was stirred with a TK homomixer (TKROBO MICS; from Tokusyukika) at 6000 rpm for 30 minutes to prepare a solution of the hydrolysate from lactoferrin having a content of the hydrolysate from errin of 100 mg/lOOg. To 5.0 kg of the solution of the hydrolysate from lactoferrin were added 4.0 kg of casein, 5.0 kg of soy protein, 1.0 kg of fish oil, 3.0 kg of perilla oil, 18.0 kg of dextrin, 6.0 kg of a mineral mixture, 1.95 kg of a vitanu11mixture, an.2.0 kg of emulsifier, 4.0 kg of stabilizer, _and 0.05 kg of a flavoring agent. The e was then placed into a 200 ml retort pouch. The retort pouch was sterilized with a retort sterilizer (Class—1 pressure vessel, TYPE: RCS-4CRTGN, from HISAKA WORKS, LTD.) at 121°C for 20 minutes to prepare 50 kg of a ion—improving liquid nutrient compositionaccordingto1ﬂuapresentinvention. Theresulting sensation—improving liquid nutrient composition had no precipitate, nor any abnormality of taste. The sensation—improving liquid.nutrient composition had.a content of the hydrolysate from lactoferrin of 10 mg/lOOg. e 16 (Preparation of sensation—improving gel food) Cystatin (2 g) in Example Product 8 was dissolved in 708 g of deionized water, and the solution was stirred with an ultra—disperser‘(ULTRA—TURRAXT-ZS; fromIHGXJapanJ at9500 rpm for 30 minutes. To the solution were added 40 g of sorbitol, 2 g of an acidulant, 2 g of a ing agent, 5 g of pectin, g of a whey n concentrate, l g of calcium lactate, and 235ggofdeionizedwater. Thendxturewasstirredandwasplaced into a 200 ml cheer pack. The cheer pack was sterilized at 85°C for 20 minutes, and was then sealed to prepare 5 bags (each 200 g) of a ion—improving gel food according to the present invention. The resulting sensation—improving gel food had no precipitate, nor any abnormality of taste. The sensation—improving gel food had a cystatin content of 200 mg/lOOg.

Example 17 (Preparation of sensation—improving ge) An acidulant (2 g) was dissolved in 706 g of deionized water, and 4 g of angiogenin in Example Product 11 was then dissolved in the solution. The mixture was stirred with an ultra—disperser (ULTRA—TURRAXT—ZS; frontIKleapan) at 9500 rpnt for 30 minutes. To the mixture were added 100g of maltitol, g of a reduced starch syrup, 2 g of a flavoring agent, and 166 g of deionized water, and xture was filled into a 100 ml glass bottle. The bottle was sterilized at 95°C for 15 seconds and was sealed to e 10 bottles (each 100 ml) of a sensation-improving beverage. The resulting sensation—improving beverage had no precipitate, nor any ality'of'taste. The sensation-improvirm;beverage had an angiogenin content of 400 mg/lOOg.

Example 18 (Preparation of sensation-improving feed) A hydrolysate from lactoperoxidase (2 kg) in Example t 2 was dissolved in 98 kg of zed water, and the solution was stirred with a TK homomixer (MARK II Model 160; from Tokusyukika) at 3600 rpm for 40 minutes to prepare a solutionofaahydrolysatefromlactoperoxidasehavingaicontent of the hydrolysate from lactoperoxidase of 2 g/100 g. To 10 kg of the solution of the hydrolysate from eroxidase were added 12 kg of soybean cake, 14 kg of skim milk powder, 4 kg of soybean oil, 2 kg of corn oil, 23.2 kg of palm oil, 14 kg of corn starch, 9 kg of wheat flour, 2 kg of bran, 5 kg of a vitamin mixture, 2.8 kg of cellulose, and 2 kg of a mineral mixture, and.the mixture was sterilized at 120°C for 41ninutes arelOOkgofeasensation-improvingcaninefeedaccording totjuapresent invention. The sensation—improving canine feed had a content of the hydrolysate from lactoperoxidase of 200 mg/lOOg; Example 19' (Preparation of sensation—improving agent (tablet)) The raw materials were mixed in the proportion shown in Table 11, and the mixture was shaped into 1—g tablets in accordance with a conventional method to prepare a sensation—improving agent according to the present invention.

The ion—improving agent.hadealactoperoxidase content of 100 mg/g.

[Table 11] Hydrous crystalline glucose 83.5 % (% by weight) Lactoperoxidase (Example Product 1) 10.0 % Mineral mixture 5.0 % Sugar ester 1.0 % Flavoring agent 0.5 % Example 20 ration of sensation-improving cosmetic product {lotion)) The raw materials were mixed in the proportion shown in Table 12 to prepare a sensation—improving ic product (lotion).

[Table 12] Sorbitol 3.0 Sodium DL-pyrrolidone carboxylate 2.0 Carboxymethyl cellulose 0.3 Paraben 0.1 errin (Example Product 4) 1.5 Flavoring agent Appropriate amount Sterile ion exchanged water q.s to 100.0 [0063a] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. [0063b] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or ion or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common l knowledge in the field of endeavour to which this specification relates.

Interwoven\NRPortbl\DCC\MM\10191829_1.docx-30/05/2016 THE

Claims

CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS

1. Use of at least one milk-derived protein and/or at least one hydrolysate therefrom in the manufacture of a medicament for improving peripheral sensation in a subject with diabetes- or ageing–related dulling of peripheral sensation; wherein the at least one milk-derived n is selected from cystatin and angiogenin.

2. Use according to claim 1, wherein the at least one erived protein further comprises lactoperoxidase and/or lactoferrin.

3. Use according to claim 1 or 2, wherein the at least one ysate is produced through hydrolysis of the at least one milk-derived protein with at least one protease.

4. Use according to claim 3, wherein the at least one protease is selected from the group consisting of pepsin, trypsin, chymotrypsin, and pancreatin.

5. Use according to any one of claims 1 to 4, wherein the medicament is for administration orally or for application to the skin of a mammal.

6. Use according to claim 5, n the mammal is a human, and the ment is for administration such that the at least one milk-derived protein and/or the at least one hydrolysate therefrom is administered at a dose of 10 mg or more per day for an adult human.

7. Use ing to any one of claims 1 to 6 wherein the medicament is in the form of a food, a beverage, a feed or a cosmetic.