CA2373599A1 - Legume products, compositions thereof and uses therefor - Google Patents

Abstract

The present invention features legume products having trypsin inhibitory activity and reduced microbial content, methods of decontaminating such legume products, compositions containing such legume products, and the topical application of such legume products or compositions to skin, nails, and hair.

Description

LEGUME PRODUCTS, COMPOSITIONS THEREOF AND USES THEREFOR
FIELD OF THE INVENTION
The present invention relates to legume products, topical compositions containing such legume products, and the manufacture and use thereof.
BACKGROUND OF THE INVENTION
Legume fruits contain high levels of proteins, to lipids and carbohydrates. Consequently) legume fruits, such as soybeans, and compositions containing legume fruits are considered a great nutrient for human use. Legume fruits also contain compounds that inhibit protease activity. For example, two protein protease inhibitors were isolated from soybeans in the early 1940's, the Kunitz-type trypsin inhibitor (soybean trypsin inhibitor, STI) and the Bowman-Birk protease inhibitor (BBI). See, e.g., Birk, Int. J.
Pept. Protein Res. 25:113-131 (1985) and Kennedy, Am. J.
Clin. Neutr. 68:14065-1412S (1998).
STI inhibits the proteolytic activity of trypsin by the formation of a stable stoichiometric complex. See, e.g., Liu, K., Chemistry and Nutritional value of soybean components. In: Soybeans, chemistry, technology and utilization. pp. 32-35 (Aspen publishers, Inc., Gaithersburg, MD, 1999). STI consists of 181 amino acid residues with two disulfide bridges and is roughly spherically shaped. See, e.g., Song et al., J. Mol. Biol.
275:347-63 (1998).

J&J-1998 BBI is an 8 k-Da protein that :inhibits the proteases trypsi.n and chymotrypsin at separate reactive sites. See, e.g., Billings et al., Pro. Natl. Acad.
Sci. 89:3120-3124 (1992). STI and BBI are found only in the soybean seed, and not in any other part of the plant. See, e.g., Birk, Int. J. Pept. Protein Res.
25:113-131 (1985).
However, due to its natural origin, high levels of microorganisms are carried on the outside of legume 1o fruits, such as soybeans. Consequently, decontamination processes such as heat, organic/aqueous solvent extraction, and high shear purification may be used to reduce such microorganism concentrations to allow it to be safe for human use, e.g., skin care applications.
Applicants, however, have found that these processes, which frequently denature the active compounds in the soy, result irl a compromised biological efficacy (e. g., a reduction in protease inhibitory activity) which is important for cosmetic and therapeutic uses to the skin, 2o hair, and nails. Furthermore, such processes also can lead to instability of the soy product as well as to an undesirable odor and color generation. Therefore, there is a commercial need to develop a mf=ans to reduce the levels of microbials in soy products without compromising the biological benefits of such products.
The object of the present invention is to provide for a soy product (e.g., that can be used as an ingredient in a skin, hair, o-r nail care composition) that has reduced microbial content but maintains its 3o protease inhibitory activity. Another object of the J&J-1998 l invention is to provided for a skin, hair, or nail care composition containing such soy product optionally with other active agents.
The present invention relates to legume products s containing reduced microbial content that retains legume's beneficial biological activities, processes for obtaining such legume products, and uses thereof in cosmetic compositions.
SU1~IARY OF THE INVENTION
The present invention features legume products having trypsin inhibitory activity and reduced microbial content, methods of decontaminating such soy products, and compositions containing such soy products. In one preferred embodiment, the legume product is a soy product.
The present invention also relates to the topical application of legume products or compositions containing 2o such legume products for use in the maintenance of healthy skin, nails, and hair as well as the prevention or the treatment of skin, rails, and hair disorders, including, but not limited to: regulating firmness of the skirl, hair, or nails; cleansing the skin, hair or nails;
reducing and/or delaying hair or nail growth;
straightening and /or lightening of hair; treatment and/or prevention of acne; regulating the tone of skin, hair, or nails; regulating the texture of skin, hair, or nails; regulating wrinkles in skin; treatment of external 3o aggressions in,skin, hair, or nails; and beautifying the skin, hair, or nails.

J&J-1998 ' ~ Other features and advantages of the present invention will be apparent from the detailed description of the invention and from the claims DETAILED DESCRIPTION OF THE INVENTION
It is believed that one skilled in the art can, based upon the description herein, utilize the present invention to its fullest extent. The following specific embodiments are to be construed as merely illustrative, so and not limitative of the remainder of the disclosure in any way whatsoever.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordin<~ry skill in the art to which the invention belongs. Also, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference. As used herein, all percentages are by weight unless otherwise specified.
2o As used herein, "trypsin inhibitory activity" means the ability of the legume product at a concentration of 0.1% (w/w) to inhibit the activity of the protease trypsin, as measured by the assay set forth below in Example 2. In one embodiment, the :Legume products of the present invention have a trypsin inhibitory activity of at least about 150. In a further embodiment, the legume products of the present invention have a trypsin inhibitory activity of at least about 25%, such as at least about 500.

J&J-1998 As used herein, "thiol retention activity" means the ability of the legume product at a concentration of 1%(w/v) to inhibit smoke-induced loss of thiols, as measured by the assay set forth below in Example 3. In one embodiment, the legume products of the present invention have a thiol retention activity of at least about 750. In a further embodiment, the legume products of the present inver~tion have an th:iol retention activity of at least about 90%, such as at least about 95% .
As used herein, "microbial content" means the amount of bacteria, fungi, and yeasty present in the legume product. Examples of means to measure microbial content include, but are not limited to, the AOAC 986.23 z5 Method as set forth in "Official Methods of Analysis of AOAC International," edited by Patr:icia Cunniff, Sixteenth Edition, 5th Revision, 1999 (AOAC
International) or the USP Method as set forth in "Official Compendia of Standards, USP 24 USP/NF 19", 2o United States Pharmacopeial Convention, Inc., 2000 (Board of Trustees, United States Plzarmacopeial Convention, Inc.).
"Objectionable microbial content" means the amount of bacteria, fungi, and yeast present in the legume 25 product that are harmful to humans, including but not limited to coliform, E. Coli, Salmonella, thermophilic spores, Bacillus, Enterococcus, Staphylococcus, fecal streptococcus, and those listed in "Disinfection, sterilization, and preservation" 4th edition, Seymour S.
30 Block, pp. 887-888 (1991, Lea & Febiger, Malvern, PA).

J&J-1998 ' As used herein, "topical application" means directly laying on or spreading on outer skin using, e.g., by use of the hands or an applicator such as a wipe, puff, roller, or spray.
As used herein, "cosmetically-acceptable" means that the products) or compounds) which the term describes are suitable for use in contact with tissues (e. g., the skin) without undue toxicity, incompatibility, instability, irritation, allergic response, and the like.
As used herein, "topical carrier" means one or more compatible solid or liquid filler diiuents that are suitable for topical aaministration to a mammal.
Examples of topical carriers include, but are not i5 limited to, water, waxes, oils, emollients, emulsifiers, thickening agents, gelling agents, and mixtures thereof.
As used herein, "regulating the firmness°' means the enhancing of the firmness or elasticity, preventing the loss of firmness or elasticity, or preventing or treating 2o sagging, lax and loose skin, hair, or nails. The firmness or elasticity of the skin can be measured by use of a cutometer. See Hanabook of Non-Invasive Methods and the Skin, eds. J. Serup & ~. Jemec, Chapter 14.3 (1995).
The loss of skin elasticity or firmness may be a result 25 of a number of factors, including beat not limited to aging, external aggressions, or the result of an application of a cosmetic to the skin, hair, or nails.
As used herein, "regulating the tone" means the lightening and/or darkening of the appearance of the 3o skin, hair, or nails (e. g., lightening pigmented lesions, darkening skin sallowness, and/or evening the color of the skin) .
As used herein, "delaying or reducing nail growth"
means the delaying or reducing the growth rate of the nail.
As used herein, "delaying or reducing hair growth"
means the delaying or reducing the growth rate of the hair and/or width of hair shaft, including, but not limited to, the reducing the visibility or appearance of hair (e. g., hair on the arms, legs, and face).
As used herein, "cleansing" means the removal of dirt and/or oil from the skin, hair, or nail surface.
As used herein, "regulating the texture" means the smoothing of the surface of the skin, hair, or nail to remove either bumps or crevasses on the surface, including, but not limited to, smoothing or evening the appearance of the skin.
As used herein, "regulating wrinkles in skin"
means preventing, retarding, arresting, or reversing the process of wrinkle or fine line formation in skin, including, but not limited to, reducing the visibility or appearance of wrinkles.
As used herein, "treatment of external aggressions" means the reduction or prevention of the damage from external aggressions in skin, hair, or nails caused by physical contact, chemical contact, or temperature.
Examples of external aggression include, but are not limited to, damage to the skin, hair, and nails from: the use of chemicals such as cleansers (e. g., skin and hair cleansers containing surfactants) and make-up; physical contact such as damage caused by shaving and cutting; and the environment such as chemical damage from ozone, exhaust, pollution, chlorine and compounds containing chlorine, and cigarette pmoke, mechanical damage caused by the wind, and damage caused by cold or hot temperature. Effects of external aggressions on the skin, nails, and skin include, but are not limited to, oxidative and/or nitosative damage to and modifications on lipids, carbohydrates, peptides, proteins, nucleic acids, and vitamins. Effects o;f external aggressions also include, but are not limited to, loss of cell viability, loss or alteration of cell functions, and changes in'gene and/or protein expression.
As used herein, "safe and effective amount" means an amount of compound or composition (e. g., the legume product) sufficient to induce a positive modification in the condition to be regulated or treated, but low enough to avoid serious side effects. The safe and effective amount of the compound or composition will vary with the particular condition being treated, the age and physical condition of he end user, the severity of the condition being treated/preserved, the duration of the treatment, the nature of other treatments, the specific compound or product/composition employed, the particular cosmetically-acceptable carrier utilized, and like factors.
Legume Product What is meant by a "legume product" is a substance derived from a legume fruit. A legume is a plant from the family Leguminosae, which has a dehiscent fruit such as a J&J-1998 bean, pea, or lentil. Examples of legumes, include but are not limited to, beans such as soybeans, lentil beans, peas, and peanuts.
The legume product may contain the entire legume fruit (e.g., the legume fruit ground into a powder) or only a portion of the legume (e.g., an extract of the legume). The legume product may be in the form of a fluid (e.g., a mixture of the legume fruit and water) or a solid (e. g., legume fruits powders). When in the form of a fluid, the term "legume product" refers to the solid constituents of the fluid derived from the legume.
The compositions of the present invention comprise a safe and effective amount of the legume product (e. g., soy product). In one embodiment, the composition contains from about 0.001% to about 500, from about 1o to about 30%, of the legume product (e. g., soy product).
Soy Product What is meant by a "Soy Product" is a substance 2o derived from the soybean. The soy product may contain only a portion of the soybean (e.g., an extract of the soybean such as a lipid reduced soybean powder or filtered soymilk) or may contain the entire soybean (e. g., a ground powder of the legume). The soy product may be in the form of a fluid (e. g., soymilk) or a solid (e.g., a soybean powder or soymilk powder). When in the form of a fluid, the term "soy product" refers to the solid constituents of the fluid that are derived from the soybean.

J&J-1998 In one embodiment, the soy product is soybean powder. Soybean powder may be made :by grinding dry soybeans. In one embodiment, the soybean powder has a average particle size of less than about 10 micrometers such as less than about 1 micrometer. In one embodiment, the soybean powder has a moisture content of less than about 10o such as less than about 5%. In one embodiment, the soybean powder is lyophilized.
In one embodiment, the soy product is soymilk or 1o soymilk powder. Soymilk is a combination of solids derived from soybeans and water; the mixture of which has some or all of the insoluble constituents filtered off.
Soymilk powder is evaporated soymilk, which in one embodiment, is in a lyophilized or spray-dried form.
Procedures for manufacturing soymilk include, but are not limited to, the following three procedures. First, soymilk may be made by placing soybeans into water to allow them to absorb the water. The swelled beans are then ground and additional water is then added. The 2o mixture may then filtered to remove any insoluble residue. Second, soymilk may also be prepared from soybean powder. Soybean powder is thoroughly mixed with water (e.g., for at least one hour), which may then be followed by a filtration process to :remove insoluble residues. Third, soymilk can also :be reconstituted from soymilk powder by adding water. In one embodiment, soymilk comprises from between about to to about 50%, by weight (e.g., from about 5% to about 200, by weight) of solids from the soybean.

J&J-1998 Anti-microbial Treatment of Legume product As discussed above, the surfacE=_ of legume fruits often contain high levels of microorganisms. Thus, prior to use by humans, the legume ~?roduct needs to be treated to reduce or eliminate such microorganisms.
In one embodiment, the legume products of the present invention have a total microbial content of less than about 10,000 colony-forming units ("cfu") per gram.
In a further embodiment, the soy products of the present 1o invention have a microbial content of less than about 1,000 cfu per gram (such as less than about 100 cfu per gram) of the legume product.
In one embodiment, the legume products of the present invention have a total objectionable microbial content of less than 300 cfu per gr<~m such as less than 150 cfu per gram. In a further embodiment, the legume products of the present invention have an undetectable amount of any objectionable microbi<~ls for at least one gram (e. g., at least ten grams) of legume product.
2o In one embodiment, the legume product is exposed to gamma irradiation. In a further embodiment, the legume product is exposed to between about 2 to about 30 kGy of gamma irradiation, such as between about 5 and about 10 kGy of gamma irradiation. Applicants have unexpectedly found that such treatment reduces the microbial content of the legume product, while maintaining its biological activity (e. g., serine protease inhibitory activity).
Applicants have also found that treatment of legume products with gamma irradiatior~ maintains the cosmetic 3o elegance of the legume product, such as maintained its J&J-1998 ' natural colors and did not induce significant malodors.
Other anti-microbial processes that also maintain the protease inhibitory activity of the legume product that can be practiced alone or in combination with gamma irradiation, include, but are not limited to, exposure to x-rays, high energy electron or proton beams, ultraviolet radiation, hydrostatic pressure, and addition of chemical agents possessing antimicrobial activity, and combinations thereof. A complete list of methods for 1o microbial content reduction is set forth in "Disinfection, sterilization, and preservation" 4th edition, Seymour S. Block, pp. 887-888 (1991, Lea &
Febiger, Malvern, PA).
Applicants have found that processes using thermal i5 treatment may result in a substantial loss in protease inhibitory activity and, thus, should be used with caution. For example, applicants have found that heating soymilk to 100°C for only 10 minutes reduced the trypsin inhibitory activity of the soymilk from 860 20 (when maintained at 4°C) to 460. Applicants have found that heating soymilk can also result:. in a change of the color or odor of the soybean product.
Topical Compositions 25 The topical compositions useful in the present invention involve formulations suitable for topical application to skin. In one embodiment, the composition comprises the soy product and a cosmetically-acceptable topical carrier. In one embodiment, the cosmetically-3o acceptable topical carrier is from about 50o to abut J&J-1998 r 99.990, by weight, of the composition (e. g., from about 80% to about 950, by weight, of the composition.
The compositions may be made into a wide variety of product types that include but are not limited to lotions, creams, gels, sticks, sprays, shaving creams, ointments, cleansing liquid washes <~nd solid bars, shampoos, pastes, powders, mousses, shaving creams, wipes, patches, nail lacquers, wound dressing and adhesive bandages, hydrogels, films and make-up such as 1o foundations, mascaras, and lipsticks. These product types may comprise several types of cosmetically acceptable topical carriers including, but not limited to solutions, emulsions (e.g., microemulsions and nanoemulsions), gels, solids and liposomes. The following are non-limitative examples of such carriers.
Other carriers can be formulated by those of ordinary skill in the art.
The topical compositions useful in the present invention can be formulated as solutions. Solutions 2o typically include an aqueous solvent (e. g., from about 50% to about 99.99% or from about 90% to about 990 of a cosmetically acceptable aqueous solvent).
Topical compositions useful in the subject invention may be formulated as a solution comprising an emollient. Such compositions preferably contain from about 2o to about 50% of an emollient(s). As used herein, "emollients" refer to mater_Lals used for the prevention or relief of dryness, as well as for the protection of the skin. A wide variety of suitable 3o emollients are known and may be used herein. Sagarin, J&J-1998 Cosmetics, Science and Technology, 2nd Edition, X701. 1, pp. 32-43 (1972) and the International Cosmetic Ingredient Dictionary and Handbook, eds. Wenninger and McEwen, pp. 1656-61, 1626, and 1654--55 (The Cosmetic, s Toiletry, and Fragrance Assoc., Washington, D.C:, 7tn Edition, 1997) (hereinafter "ICI Handbook") contains numerous examples of suitable materials.
A lotion can be made from such a solution. Lotions typically comprise from about 1% to about 20o (e. g., 1o from about 5% to about 100) of an ernollient(s) and from about 50% to about 90% (e. g., from about 60% to about 80%) of water.
Another type of product that may be formulated from a solution is a cream. A cream typically comprises from i5 about 5% to about 500 (e. g., from about 10o to about 20%) of an emollients) and from about 45o to about 85%
(e. g., from about 50% to about 750) of water.
Yet another type of product that may be formulated from a solution is an ointment. An ointment may 2o comprise a simple base of animal or vegetable oils or semi-solid hydrocarbons. An ointment may comprise from about 2% to about 100 of an emollients) plus from about 0.1% to about 20 of a thickening agent(s). A more complete disclosure of thickening agents or viscosity 25 increasing agents useful herein can be found in Sagarin, Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 72-73 (1972) and the ICI Handbook pp. 1693-1697.
The topical compositions useful in the present invention formulated as emulsions. I:f the carrier is an 3o emulsion, from about 1% to about 10° (e. g., from about J&J-1998 2% to about 5o) of the carrier comprises an emulsifier(s). Emulsifiers may be nonionic, anionic or cationic. Suitable emulsifiers are disclosed in, for example, U.S. Patent No. 3,755,560, U.S. Patent No.
4,421,769, McCutcheon's Detergents and Emulsifiers, North American Edition, pp. 317-324 (1986), and the ICI
Handbook, pp.1673-1686.
Lotions and creams can be formulated as emulsions.
Typically such lotions comprise from 0.5% to about 50 of 1o an emulsifier(s). Such creams would typically comprise from about 1% to about 200 (e. g., from about 5% to about 10%) of an emollient(s); from about 20% to about 80o (e. g., from 30o to about 700) of water; and from about 1o to about 10% (e.g., from about 2'5 to about 5o) of an emulsifier(s).
Single emulsion skin care preparations, such as lotions and creams, of the oil-in-water type and water-in-oil type are well-known in the cosmetic art and are useful in the subject invention. Multiphase emulsion 2o compositions, such as the water-in-oil-in-water'type, as disclosed in U.S. Patent No. 4,254,?_05 and 4,960,764, are also useful in the subject invention. In general, such single or multiphase emulsions contain water, emollients, and emulsifiers as essential ingredients.
The topical compositions of this invention can also be formulated as a gel (e.g., an aqueous gel using a suitable gelling agent(s)). Suitab7_e gelling agents for aqueous gels include, but are not ls_mited to, natural gums, acrylic acid and acrylate polymers and copolymers, 3o and cellulose derivatives (e. g., hydroxymethyl cellulose J&J-1998 and hydroxypropyl cellulose). Suitable gelling agents for oils (such as mineral oil) include, but are not limited to, hydrogenated butylene/et=hylene/styrene copolymer and hydrogenated ethylene/propylene/styrene copolymer. Such gels typically comprises between about 0.1% and 50, by weight, of such gelling agents.
The topical compositions of the present invention can also be formulated into a solid formulation (e.g., a wax-based stick, soap bar composition, powder, or a wipe i0 containing powder).
Liposomal formulations are also useful compositions of the subject invention. Examples of liposomes are unilamellar, multilamellar, and paucilamellar liposomes, which may or may not contain phospholipids. Such compositions can be prepared by first combining hesperetin with a phospholipid, such as dipalmitoylphosphatidyl choline, cholesterol and water according to the method described in Mezei &
Gulasekharam, "Liposomes--A Selective Drug Delivery 2o System for the Topical Route of Adm~_nistration; Gel Dosage Form", Journal of Pharmaceutics and Pharmacology, Vol. 34 (1982), pp. 473-474, or a modification thereof.
Epidermal lipids of suitable composition for forming liposomes may be substituted for the phospholipid. The liposome preparation may then incorporated into one of the above carriers (e. g., a gel or an oil-in-water emulsion) in order to produce the liposomal formulation.
Other compositions and pharmaceutical uses of topically applied liposomes are described in Mezei, M., "Liposomes 3o as a Skin Drug Delivery System", Topics in J&J-1998 Pharmaceutical Sciences (D. D. Breimer and P. Speiser, eds.,), Elsevier Science Publishers B. V., New York, N.Y., 1985, pp. 345-358, PCT Patent Application No.
W096/31194 and U.S. Patent No. 5,260,065.
The topical compositions useful in the subject invention may contain, in addition i~o the aforementioned components, a wide variety of additional oil-soluble materials and/or water-soluble materials conventionally used in compositions for use on skin, hair, and nails at 1o their art-established levels.
Additional Cosmetically Active Agents In one embodiment, the topical composition further comprises another cosmetically active agent in addition i5 to the legume product. What is meant by a "cosmetically active agent" is a compound that has a cosmetic or therapeutic effect on the skin, hair, or nails, e.g., lightening agents, darkening agents such as self-tanning agents, anti-acne agents, shine control agents, anti-2o microbial agents, anti-inflammatory agents, anti-mycotic agents, anti-parasite agents, external analgesics, sunscreens, photoprotectors, antioxidants, keratolytic agents, detergents/surfactants, moisturizers, nutrients, vitamins, energy enhancers, anti-perspiration agents, 25 astringents, deodorants, hair removers, firming agents, anti-callous agents, and agents for hair, nail, and/or skin conditioning.
In one embodiment, the agent is selected from, but not limited to, the group consisting of hydroxy acids, 3o benzoyl peroxide, sulfur resorcinol, ascorbic acid, D-J&J-1998 panthenol, hydroquinone, octyl methoxycinnimate, titanium dioxide, octyl salicylate, homosalate, avobenzone, polyphenolics, carotenoids, free radical scavengers, spin traps, retinoids such as retinol and retinyl palmitate, ceramides, polyunsaturated fatty acids, essential fatty acids, enzymes, enzyme inhibitors, minerals, hormones such as estrogens, steroids such as hydrocortisone, 2-dimethylaminoethanol, copper salts such as copper chloride, peptides 1o containing copper such as Cu:Gly-His-Lys, coenzyme Q10, peptides such as those disclosed in PCT Patent Application W000/15188, lipoic acid, amino acids such a proline and tyrosine, vitamins, lactobionic acid, acetyl-coenzyme A, niacin, riboflavun, thiamin, ribose, electron transporters such as NADH and FADH2, and other botanical extracts such as aloe vera, and derivatives and mixtures thereof. The cosmetically active agent will typically be present in the composition of the invention in an amount of from about. 0.0010 to about 200 2o by weight of the composition, e.g., about 0.010 to about 10% such as about 0.10 to about 50.
Examples of vitamins include, but are not limited to, vitamin A, vitamin Bs such as vir~amin B3, vitamin B5, and vitamin B12, vitamin C, vitamin 1~, and vitamin E and derivatives thereof.
Examples of hydroxy acids include, but are not limited, to glycolic acid, lactic acid, malic acid, salicylic acid, citric acid, and tartaric acid. See, e.g., European Patent Application No. 273,202.

J&J-1998 Examples of antioxidants include, but are not limited to, water-soluble antioxidants such as sulfhydryl compounds and their derivatives (e. g., sodium metabisulfite and N-acetyl-cysteine), lipoic acid and dihydrolipoic acid, resveratrol, lactoferrin, and ascorbic acid and ascorbic acid der_Lvatives (e. g., ascorbyl palmitate and ascorbyl pol~rpeptide). Oil-soluble antioxidants suitable for use in the compositions of this invention include, but are not limited to, butylated hydroxytoluene, retinoids (e. g., retinol and retinyl palmitate), tocopherols (e. g., tocopherol acetate), tocotrienols, and ubiquinone.
Natural extracts containing antioxidants suitable for use in the compositions of this invention, include, but i5 not limited to, extracts containing flavonoids and isoflavonoids and their derivatives (e.g., genistein and diadzein), extracts cor_taining resveratrol and the like.
Examples of such natural extracts include grape'seed, green tea, pine bark, and propolis. Other examples of 2o antioxidants may be found on pages l_612-23 of the ICI
Handbook.
Other Materials Various other materials may also be present in the 25 compositions useful in the subject z.nvention. These include humectants, proteins and po~.ypeptides, preservatives and an alkaline agent. Examples of such agents are disclosed in the ICI Handbook, pp.1550-1667.
The compositions of the present invention may also 30 comprise chelating agents (e. g., EDi'A) and preservatives J&J-1998 (e.g., parabens). Examples of suitable preservatives and chelating agents are listed in pp. 1626 and 1654-55 of the ICI Handbook. In addition, the topical compositions useful herein can contain conventional cosmetic adjuvants, such as dyes, opacifiers (e. g., titanium dioxide), pigments, and fragrances.
Mineral Water The legume product (e. g., soymilk) and compositions of the present invention may be prepared using a mineral water. In one embodiment, the mineral water has a mineralization of at least about 200 mg/L (e. g., from about 300 mg/L to about 1000 mg/L). In one embodiment, the mineral water comprises at least; about 10 mg/L of i5 calcium and/or at least about 5 mg/I~ of magnesium.
The composition and formulations containing such compositions of the present invention may be prepared using methodology that is well known by an artisan o.f ordinary skill.
Example 1: Gamma Irradiation of Legume Product Applicants have found that soymilk powder prior to any antimicrobial processing such as gamma irradiation has high levels microbial content, ranging from up to 50,000 cfu per gram. Such products were also found to have detectable levels of objectionable microbial content, such as fecal streptococci, at levels up to 20,000 cfu per gram.

J&J-1998 Applicants have exposed various amounts (e. g., from about 1 g to about 200 kg) of soymilk powder to gamma irradiation varying from 1 kGy to 16 kGy. The dose or gamma irradiation needed for a reduction a total microbial content to less than about 100 cfu per gram was found to be about 10 kGy. The dose for one log reduction for fecal streptococci is determined to be about 3 kGy and a dose of about 5 k<~y was found to consistently reduce this microbial content within a 10 gram sample of soymilk powder to undetectable levels.
However, the amount of gamma irradiation used on the legume product will ultimately be determined by the microbial content and size of the soy product to be so treated.
Example 2: Trypsin Inhibitory Activity of Legume Product The inhibition of trypsin-induced cleavage of a fluorescent casein peptide was measured using the EnzChekT''t protease assay kit, following manufacturer's instructions (EnzChekTM Protease Assay Kits Product Information, Revised 3/15/99; Molecular Probes, Eugene OR). In summary, various soy preparations were first diluted in 1X digestion buffer (provided in kit) and incubated at different concentrations with 1000 units of trypsin (Sigma, St. Lcuis, MO) dissolved in 1X digestion buffer. A pure serine protease inhibitor (soybean trypsin inhibitor, from Sigma, St. Louis, MO) was used as a positive control at 0.1, 0.010, and 0.001% w/v. Then, 1.0 mg/ml stock solution of BODIPY ~'L casein was prepared by adding 0.2 mL of deionized water to the vials supplied J&J-1998 with this substrate (provided in kit), then made to a final working concentration of 10 microgram/ml in digestion buffer. Following incubation of the trypsin, with or without the test material, with the BODIPY
fluorescent casein substrate at room temperature for one hour, fluorescence was measured (excitation 485 nm /emission 530 nm) on a SpectraMax° Gemini microtiter plate reader (Molecular Devices Corporation, Sunnyvale, CA) using Softmax~ Pro 3.0 software (Molecular Devices 1o Corporation). Each experiment was performed in three replicates and was repeated twice.
This assay was performed on soy products processed seven different ways. Example A was soybeans ground into powder (Sunlight Foods Corporat:.ion, Taipei County, Taiwan, R.O.C.). Example B was soybean powder of Example A exposed to about 8-15 kGy of gamma irradiation. Example C was soybean ~>owder in which the oil in the soybean powder was removed by extraction (Soyafluff~ 200W from Central Soya Company, Inc., Fort Weyne, IN). Example D was soymilk powder made with dehulled soybeans and water that wa~> subsequently filtered and heated and spray dried (Devansoy Farms, Carroll, Iowa) and exposed to between about 7 - 9 kGy of gamma irradiation. Example E was soymilk powder obtained by mixing soy beans and water, heating the mixture overnight, and adding 1,3-butylene glycol to the mixture (Flavosterone SB from Ichimaru Pharcos Co., Ltd, Gifu Japan). Example F was soymilk powder obtained by mixing soy beans and water, heating tile mixaure overnight, and 3o subsequently adding ethanol to the mixture (Flavosterone J&J-1998 SE from Ichimaru Pharcos Co., Ltd, Gifu Japan). Example G was an extract of soy proteins (Vegeseryl HGP LS 8572 from Laboratories Serobiologiques S.A., Pulnoy, France).
These soy products were compared to Soy Trypsin Inhibitor (STI) (Sigma).
The percent inhibition of trypsin cleavage of the substrate by the different soy prep<~rations was calculated using Microsoft ExcelTM and is reported in Table 1.
1o Table 1 Tested Product Concentration o Inhibition of Trypsin STI 0.01 43.0 STI 0.1 76.1 Example A 0.01 32.8 Example A 0.1 67.1 Example B 0.01 31.5 Example B 0.1 67.2 Example C 0.01 22.7 Example C 0.1 36.2 Example D 0.01 8.92 Example D 0.1 17.4 Example E 0.01 7.83 Example E 0.1 10.8 Example F 0.01 4.87 Example F 0.1 5.99 Example G 0.1 ~ 6.85 As shown in Table 1, STI can inhibit trypsin-induced cleavage in a dose response manner. Example A, which is soybean powder, also significantly inhibited trypsin activity. Further gamma irradiation of the soybean powder (i.e., Example B), awhile reducing the microbial content of the soybean powder, unexpectedly did not significantly impact the trypsin inhibition activity of a J&J-1998 the soybean powder. The heat and/or extraction processing of Examples C-G, however, did significantly reduce the trypsin inhibitory activ~_ty of the soybean powder .
Example 3: Thiol Retention Activity of Legume Product The ability of soy powder to prevent smoke-induced loss of thiols was evaluated in normal human dermal fibroblasts (Clonetics, San Diego, CA). Thiols, chiefly 1o glutathione, are part of the endogenous cellular antioxidant defense system. Glutath:ione serves as a redox buffer, thereby, maintaining the balance between oxidants and antioxidants. Glutathione is also the preferred substrate for several enzymes such as the s5 glutathione peroxidases (decomposing peroxides) and the glutathione-S-transferases (a major group of detoxification enzymes}. See, A. Meister, Cancer Res.
54:1969s-1975s (1994).
Cutaneous antioxidants (both enzymatic and non-2o enzymatic), including glutathione, are depleted after UV
or ozone exposure. See, M. J. Connor and L. A. Wheeler, Photochem. Photobiol. 46:239-246 (198'7) and R. M. Tyrrell and M. Pidoux, Photochem. Photobiol. 47:405-412 (1988).
In cell culture models, low intracel7_ular glutathione 25 (GSH) levels lead to a higher UV radiation sensitivity.
Topical application of cysteine derivati~,res on rat skin has been shown to protect against UV radiation-induced photodamage; this benefit was correlated with an increase in GSH synthesis. See, L. T. van den Broeke and G. M. J.
3o Beijersbergen van Henegouwen, J. Photochem. Photobiol. B

J&J-1998 Biol. 27:61-65 (1995); K. Hanada, et al., J. Invest.
Dermatol. 108:727-730 (1997); and D. P. T. Steenvoorden, et al., Photochem Photobiol. 67:651-656 (1998).
Consequently, glucothione is a major endogenous antioxidant, highly responsive against environmental challenges, able to regulate the tone and the wrinkling of skin, as well as treat external aggression.
In this experiment, normal human neonatal dermal fibroblasts seeded in 24-well format Transwell inserts (Corning Costar, Cambridge, MA) were incubated with media containing various concentrations of soymilk powder (gamma irradiated at about 5 kGy) for 24 hours prior to exposure with either placebo (mock) or cigarette smoke (1 cigarette, BASIC Full Flavor 100's cigarettes, Philip Morris, Richmond, VA) for 10 minutes.
Prior to smoke exposure, the medium above the inserts containing the soy product was removed, and the cells were washed 3 times with Dulbecco's Phosphate-Buffered Saline (Life Technologies, Gaitnersburg, MD) before 2o being smoke-exposed with only media below the inserts.
Immediately after exposure, the cells were incubated for another 24-hour period with the previous medium. The cells were washed again, 5 times with Dulbecco's Phosphate-Buffered Saline, and intracellular thiols were then measured by adding 60 ~M monobromobimane (Molecular Probes, Eugene, OR, USA) to the cells and incubating at 37°C for 30 minutes before the fluorescence reading. In the presence of thiols, the monobromobimane becomes fluorescent. This fluorescence was measured using a 3o CytoFluor~ Fluorescence Plate Reader (PerSeptive Biosystems, Framingham, MA, USA) set with the following filter combination: excitation at 360nm and emission at 460nm:
The results of this experiment are set-forth in Table 2.
Table 2 Soymilk Powder Thiols (Percent of concentration Thiols contained in (weight %) No Smoke Group;

Mean S.E.M.) No Smoke 0 100 6.71 Smoke ( 10 min 0 6 5 . 3 8 7 . 16 . ) 0.5 91.24 + 14.25 1 95.39 + 4.52 2 106.92 + 17.06 These results indicate that gamma irradiated soymilk powder surprisingly afforded a protection against smoke-induced loss of thiols (data represent the mean ~
Standard of the mean of replicates from 3 independent experiments).
It is understood that while the invention has been described in conjunction with the detailed description thereof, that the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the claims.

Claims (65)

1. A soy product having:
(a) a trypsin inhibitory activity of at least about 15%; and (b) a microbial content less than about 1000 cfu per gram.

2. A soy product of claim 1, said soy product having an objectionable microbial count less than about 150 cfu per gram.

3. A soy product of claim 1, said soy product having no detectable objectionable microbial content per gram.

4. A soy product having:
(a) a trypsin inhibitory activity of at least about 50%; and (b) a microbial content less than about 10,000 cfu per gram.

5. A soy product of claim 4, said soy product having a microbial content of less than about 1000 cfu per gram.

6. A soy product of claim 4 or 5, said soy product further having an objectionable microbial count less than about 150 cfu per gram.

7. A soy product of claim 4 or 5, said soy product having no detectable objectionable microbial content per gram.

8. A soy product of any one of claims 1 to 7, wherein said soy product has a thiol retention activity of at least about 75%.

9. A soy product of any one of claims 1 to 8, wherein said soy product has been exposed to gamma irradiation.

10. A soy product of any one of claims 1 to 8, wherein said soy product has been exposed to about 5 to about 16 kGy gamma irradiation.

11. A soy product of any one of claims 1 to 10, wherein said soy product is soymilk powder.

12. A soy product of any one of claims 1 to 10, wherein said soy product is soybean powder.

13. A method of decontaminating a soy product, said method comprising exposing said soy product to gamma irradiation.

14. A method of claim 13, wherein said soy product is exposed to about 6 to about 16 kGy gamma irradiation.

15. A method of claim 13 or 14, wherein said soy product is soymilk powder.

16. A method of claim 13 or 14, wherein said soy product is soybean powder.

17. A composition for topical application comprising:
(a) a soy product having a trypsin inhibitory activity of at least about 15% and a microbial content less than about 1000 cfu per gram; and (b) a cosmetically-acceptable topical carrier.

18. A composition of claim 17, said soy product having an objectionable microbial count less than about 150 cfu per gram.

19. A composition of claim 17, said soy product having a microbial content of less than about 100 cfu per gram.

20. A composition for topical application comprising:
(a) a soy product having a trypsin inhibitory activity of at least about 50% and a microbial content less than about 10,000 cfu per gram; and (b) a cosmetically-acceptable topical carrier.

21. A composition of claim 20, said soy product having a microbial content of less than about 1000 cfu per gram.

22. A composition of claim 20 or 21, said soy product having an objectionable microbial count less than about 150 cfu per gram.

23. A composition of claim 20 or 21, said soy product having no detectable objectionable microbial content per gram.

24. A composition of anyone of claims 17 to 23, wherein said soy product has been exposed to gamma irradiation.

25. A composition for topical application comprising:
(a) a soy product having been exposed to gamma irradiation; and (b) a cosmetically-acceptable topical carrier.

26. A composition of any one of claims 17 to 25, wherein said soy product has a thiol retention activity of at least about 75%.

27. A composition of any one of claims 17 to 26, wherein said soy product is soymilk powder.

28. A composition of any one of claims 17 to 26, wherein said,soy product is soybean powder.

29. A composition of any one of claims 17 to 28, wherein said composition further comprises a cosmetically active agent selected from the group consisting of vitamin A, vitamin B3, vitamin B5, vitamin B12, vitamin C, vitamin K, vitamin E, derivatives thereof and mixtures thereof.

30. A composition of any one of claims 17 to 29, wherein said composition further comprises 2-dimethylaminoethanol.

31. A composition of any one of claims 17 to 30, wherein said composition further comprises salicylic acid, lactic acid, glycolic acid or a mixture thereof.

32. A composition of anyone of claims 17 to 31, wherein said composition further comprises N-acetyl-cysteine.

33. A composition for treatment of external aggressions to the skin, nails, or hair, said composition comprising a safe and effective amount of a soy product and a cosmetically-acceptable topical carrier.

34. A composition of claim 33, wherein said composition is for the treatment of external aggressions to the skin.

35. A composition of claim 33 or 34, wherein said external aggression is a result of chemical contact.

36. A composition of claim 33 or 34, wherein said external aggression is a result of physical contact.

37. A composition of claim 33 or 34, wherein said external aggression is damage from use of cleansers.

38. A composition of claim 33 or 34, wherein said external aggression is damage from shaving.

39. A composition of anyone of claims 33 to 38, wherein said soy product has a thiol retention activity of at least about 75%.

40. A composition of any one of claims 33 to 39, wherein said soy product has been exposed to gamma irradiation.

41. Use topically of a safe and effective amount of a soy product of any one of claims 1 to 12 for maintaining healthy skin, nails or hair, or preventing or treating disorders of skin, nails or hair.

42. Use of a safe and effective amount of a composition of any one of claims 17 to 32 for maintaining healthy skin, nails or hair, or preventing or treating disorders of skin, nails or hair.

43. Use according to claim 41 or 42 for: regulating firmness of skin, nails or hair; cleansing skin, nails or hair; reducing and/or delaying growth of nails or hair;
straightening and/or lightening hair; treating and/or preventing acne; regulating tone of skin, nails or hair;
regulating texture of skin, nails or hair; regulating wrinkles in skin; treating external aggressions in skin, nails or hair; or beautifying skin, nails or hair.

44. Use according to claim 41 or 42 for treating external aggressions in skin.

45. Use according to claim 44, wherein said external aggression is a result of chemical contact.

46. Use according to claim 44, wherein said external aggression is a result of physical contact.

47. Use according to claim 44, wherein said external aggression is damage from use of cleansers.

48. Use according to claim 44, wherein said external aggression is damage from shaving.

49. A commercial package comprising of a soy product of any one of claims 1 to 12 together with instructions for its use in maintaining healthy skin, nails or hair, or preventing or treating disorders of skin, nails or hair.

50. A commercial package comprising a composition of any one of claims 17 to 32 together with instructions for its use in maintaining healthy skin, nails or hair, or preventing or treating disorders of skin, nails or hair.

51. A commercial package according to claim 49 or 50, wherein the instructions are for use in: regulating firmness of skin, nails or hair; cleansing skin, nails or hair;
reducing and/or delaying growth of nails or hair;
straightening and/or lightening hair; treating and/or preventing acne; regulating tone of skin, nails or hair;
regulating texture of skin, nails or hair; regulating wrinkles in skin; treating external aggressions in skin, nails or hair; or beautifying skin, nails or hair.

52. A commercial package according to claim 49 or 50, wherein the instructions are for use in treating external aggressions in skin.

53. A commercial package according to claim 52, wherein said external aggression is a result of chemical contact.

54. A commercial package according to claim 52, wherein said external aggression is a result of physical contact.

55. A commercial package according to claim 52, wherein said external aggression is damage from use of cleansers.

56. A commercial package according to claim 52, wherein said external aggression is damage from shaving.

57. A method for treating external aggressions to the skin, nails or hair comprising topically applying to the skin, nails or hair a soy product.

58. A method of claim 57, wherein said soy product has a thiol retention activity of at least about 75%.

59. A method of claim 57 or 58, wherein said soy product has been exposed to gamma irradiation.

60. A method for treating external aggressions to the skin, nails or hair comprising topically applying to the skin, nails or hair a soy product of any one of claims 1 to 12 .

61. A method for treating external aggressions to the skin, nails or hair comprising topically applying to the skin, nails or hair a composition of any one of claims l7 to 32.

62. A method of any one of claims 57 to 61, wherein said external aggression is a result of chemical contact.

63. A method of any one of claims 57 to 61, wherein said external aggression is a result of physical contact.

64. A method of any one of claims 57 to 61, wherein said external aggression is damage from use of cleansers.

65. A method of any one of claims 57 to 61, wherein said external aggression is damage from shaving.