METHOD FOR PROTECTING AN ANIMAL SKIN PRODUCT FROM METALOPROTEINASE ACTIVITY
FIELD DF THE INVENTION The present invention relates to leather processing and in particular to a method for protecting animal skins or leathers from enzymatic degradation when treating animal skins or hides with a metalloproteinase inhibitor. BACKGROUND OF THE INVENTION The deterioration of fresh hides and skins due to growth and microbial activity is a major problem in the leather and tanning industry and a major cause of economic loss. Harmful microorganisms such as bacteria and fungi can come from many sources, including the animal skin itself, the slaughterhouse and leather tanning and the processing environment. Fresh hides and skins have a high moisture content, a favorable pH, and large amounts of available nutrients, which allow the rapid growth of microorganisms, which results in the deterioration of the essential components of skins and leathers. Microorganisms that grow on or near the hides and skins can cause rotting of hides and skins by producing enzymes such as collagenase that can degrade the protein component of hides or skins.
Proteins constitute approximately 33% of the composition of fresh hides or skins (the rest being mainly water and fat) and much of this protein is collagen. (John Henry Sharphouse, Leather Technicians Handbook, 1995. Leather Producers' Association, Moulton Park, Northhampton, Great Britain). The finished leather is mainly collagen, which is reticulated during the tanning process. Since the finished leather is primarily collagen, the damage to the skins and collagenase skins produced by the microorganisms during the processing and storage stages of the animal skins and leathers is particularly harmful and adversely affects the quality of the finished leather. Different methods have been used to temporarily preserve skins and hides while in storage before processing or while being transported to other locations for processing. In particular, biocides or preservatives have been used in an effort to control harmful microorganisms. (see, for example, the following North American patents incorporated herein by reference: U.S. Patent No. 4,164,393, U.S. Patent No. 4,224,028, U.S. Patent No. 4,278,432, U.S. Patent No. 4,322.2 10, U.S. Patent No. 4,379,706, American patent No.
4,478,728, U.S. Patent No. 4,889,811, U.S. Patent No. 4,935,031, U.S. Patent No. 4,985,039, U.S. Patent No. 5,096,553, U.S. Patent No. 5,435,808, U.S. Patent No. 6,086,633, and U.S. Patent No. 6,45 1,062). While biocides or preservatives can work well in inhibiting or killing microorganisms, they are generally not effective in blocking collagenase or other enzymes responsible for degrading collagen. These enzymes can remain active and can cause damage even after the microorganisms that produced them have been exterminated. Additionally, the use of biocides to preserve skins and leathers can be costly and impractical since large quantities of biocides are required to kill enough microorganisms to completely prevent the production of collagenase. In addition, a biocide is not effective against endogenous enzymes, that is, against enzymes that were produced by the animal itself and that remain on the skin or hide after slaughter. On the other hand, in some cases, it may not be desirable to completely cleanse all of the microorganisms that come in contact with a skin or leather, since some microorganisms can produce enzymes that are useful in the decomposition of the foreign organic material adhered to the leather or the skin. skin. Collagenase belongs to the class of proteins
known as meta] opoteinases, which are proteases that require the presence of a metal ion in order to function. It has been previously reported that the activity of metalloproteinases can be blocked or inhibited by exposing metalloproteinases to a metal chelator. For example, derivatives of aminocarboxylic acid such as ethylenediaminetetraacetic acid (EDTA) and ethylene glycol] -bis (ß-aminoethylether) N, N, N ', N' -tetraacetic acid (EGTA) are metal chelators which are known to inhibit metalloproteinases such as collagenase. (David S. Auld 1995"Removal and Replacement of Metals with Metallopeptidases." In: Allan J. Barrett (Ed), Methods of Enzymology, 248: 228-242, Academic Press, New York). However, until now the use of target chelators] to inhibit metalloproteinases has been mainly in the fields of medicine and biotechnology. The usefulness and effectiveness of aminocarboxylic acid derivatives as collagenase inhibitors have not been known or appreciated in the leather and tanning industry and the use of protease inhibitors to protect skins and leathers has not been previously reported. Accordingly, there is a need for a composition and method for protecting skins and leathers of proteolytic enzymes such as metalloproteinases that come into contact with skins and leathers or that are present in skins and surfaces with which skins or leathers
they come into contact, particularly during the stages of the leather tanning process. In addition, there is a need for a composition and method for protecting skins and leathers of metalloproteinases that are produced by microorganism or that are endogenous enzymes that were present in the skin or hide of the animal at the time the animal was sacrificed. In addition, there is a need for a composition or method for protecting hides and skins of metalloproteinases using compounds and compositions that are not expensive and easily obtainable. In addition, there is a need for a composition and method for protecting skins and leathers from metalloproteinases by using compounds and compositions which have low toxicity, are environmentally favorable and are compatible with the tanning and leather processes. BRIEF DESCRIPTION OF THE INVENTION It is a feature of the present invention to provide a composition and method for protecting animal skins and hides from proteolytic enzymes such as metalloproteinases that are produced by microorganisms that come into contact with skins and hides and that are present in fluids and surfaces with which the skins and hides come into contact, for example during a tanning process of the skin, or which are proteolytic enzymes
endogenous that were present in the skin or leather when the animal was sacrificed. The present invention further provides a composition and method for protecting skins and leathers of metalloproteinases using compounds and compositions that are inexpensive and readily obtainable. The present invention further provides a composition and method for protecting skins and leathers from metalloproteinases using compounds and compositions that have a low toxicity, are environmentally favorable and / or are compatible with the tanning and leather processes. Additional advantages of the present invention will be set forth in part in the description that follows, and in part will be apparent from the description or may be learned by the practice of the present invention. The objects and advantages of the present invention will be realized and will be achieved by means of the elements particularly indicated in the appended claims. In order to achieve the objectives noted in the foregoing in accordance with the purpose of the present invention, as embodied and amply described herein, the present invention provides a method for preventing or inhibiting putrefaction, degradation, and / or deterioration of a skin or animal leather due to an action of a loproteinase goal. The method includes applying a solution that
it contains at least one metalloproteinase inhibitor to the skin or animal hide or to a fluid or solid surface which makes contact with the skin or animal hide. The present invention further provides a method for preventing or inhibiting putrefaction or degradation or deterioration of an animal skin or hide, the method comprising applying a solution containing at least one polyaminocarboxylic acid, or a salt thereof to the skin or leather. animal or to a fluid or solid surface that makes contact with the skin or animal hide. The present invention further provides a method for producing leather that includes the steps for separating a skin or animal hide from a slaughtered animal, curing the skin or animal hide, soaking the skin or cured animal hide, removing the flesh and hair from the skin or animal hide soaked and tanned animal skin or hide from which the flesh and hair have been removed to form the hide, where after the step to separate the skin or animal hide from the sacrificed animal and before the tanning stage of the skin or animal hide from which the flesh and hair have been removed to form the leather, a solution containing at least one metalloproteinase inhibitor is applied to the skin or animal hide or to a fluid or solid surface which makes contact with skin or animal hide in an amount sufficient to prevent or inhibit the degradation or deterioration of the skin or
animal leather due to an action of a metalloproteinase. The present invention further provides a composition containing at least one aqueous immersion or soaking solution wherein at least one skin or animal hide can be immersed or soaked therein, wherein the soaking or immersing solution contains minus a metalloproteinase inhibitor. The present invention further provides a composition containing an animal skin or leather having a solution containing at least one metalloproteinase inhibitor applied to at least one surface thereof. It will be understood that both the foregoing general description and the following detailed description are exemplary only and are not restrictive of the present invention, as claimed. All patents, patent applications and publications mentioned in the foregoing and throughout the present application are incorporated in their entirety by reference herein. DETAILED DESCRIPTION OF THE PRESENT INVENTION The present invention is directed to a method of treating an animal skin or hide to prevent putrefaction, degradation, or deterioration caused at least in part by proteolytic enzymes, such as metalloproteinases.
As used herein, the terms "skin", "animal skin", "leather", or "animal hide" are all used interchangeably to refer to skinned or separated skin or outer layer of an animal, particularly an animal whose skin is useful to turn it into leather. Examples of animals from which skin can be taken to make leather include, but are not limited to, cattle, pigs, deer, kangaroos, goats, camels, sheep, horses, alligators, crocodiles, snakes, birds, seals, eels and walruses. The term "skin or leather" is intended to refer to a skin or leather at any stage of processing after it is removed from a dead animal, which includes any intermediate stage in the processing or preservation of the leather. The method of the present invention can be carried out at any time after an animal dies or is sacrificed or its skin or hide is skinned or separated from the animal carcass. In typical leather processing, for example, an animal hide or hide is separated from a fallen or slaughtered animal, and then the animal skin or hide is cleaned, cured, soaked, treated for the removal of meat and hair, it is macerated and tanned to form leather. The skin or animal hide can be stored or transported to another location after skinning and before the start of leather processing. Many variations of
these processes. The method of the present invention can be carried out at the same time as any of these processes or it can be carried out as a separate step between any of these processes. For example, a skin or leather can be treated with a metalloproteinase inhibitor according to the method of the present invention at least once between the time that a skin or hide is separated from an animal and the time that a tanning process has been carried out. Typically, it is no longer necessary to treat a skin or leather after it has been tanned, since the tanning process typically causes the crosslinking of the collagen fibers, making them less susceptible to enzymatic attack. On the other hand, the method of the present invention is not limited to leather processing and can be combined with any other process to preserve a leather or leather. For example, the method of the present invention can be used if a skin or leather is to be dried without tanning. In one embodiment of the method of the present invention, a metalloproteinase inhibitor is applied to an animal skin or hide or to a fluid that contacts skin or leather or a surface that contacts skin or leather in order to prevent or inhibit putrefaction, degradation, or deterioration of skin or animal hide particularly, putrefaction, degradation, or deterioration,
due at least in part to the action of a metalloproteinase. As used herein, the term "proteolytic enzyme" refers to any enzyme of a bacteria, fungus, or animal source that cleaves or hydrolyzes the peptide bonds and breaks down a protein. The terms "metalloproteinase", "metalloprotease", and "metallopeptidase" are used interchangeably to refer to proteolytic enzymes that require a metal ion in order to function. As an example, collagenase is a metalloproteinase that hydrolyzes collagen peptide bonds and that requires a zinc ion (eg, Zn2 +) at its catalytic site. The metalloproteinase inhibitor used in the method of the present invention is a material, such as a compound, or a mixture of compounds, or a composition that is capable of preventing or inhibiting the action of at least one proteinase. As a non-limiting example, an inhibitor of a metalloproteinase can be a chelator, such as a chelator of a divalent metal ion, which can act to inhibit a metalloprotemase by binding to the metal ion required for metalloproteinase function. For example, a metal chelator can bind to the zinc ion at the catalytic site of a collagenase, thereby blocking, or preventing, or reducing the action of collagenase. The metalloproteinase inhibitor can be, but
it is not limited to an aminocarboxylic acid or a polyaminocarboxylic acid or a salt thereof. As used herein, the term "polyaminocarboxylic acid" refers to an aminocarboxylic compound that contains more than one amine. Preferred chelators are compounds that contain at least two groups that can bind to a metal ion, such as, for example, at least two carboxylic acid groups. Another way to affirm this is that the chelator is preferably at least bidentate. Non-limiting examples of metalloproteinase inhibitors include ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as, for example, disodium salt of ethylenediaminetetraacetic acid (Na2EDTA or disodium EDTA), thapsodium salt of eti lendiaminotetraacetic acid (Na3EDTA) or tpsodium EDTA), tetrasodium salt of ethylenediaminetetraacetic acid (Na4EDTA) or tetrasodium EDTA), dipotassium salt of ethylenediaminetetraacetic acid (K2EDTA), tripotassium salt of ethylenediaminetetraacetic acid (K3EDTA), ammonium salt of ethylenediaminetetraacetic acid (NH4EDTA) ), or diammonium salt of ethylenediaminetetraacetic acid (NH4) 2EDTA). Other non-limiting examples of acid include ethyl endiammatr racético (ED3A) or salts thereof. Other non-limiting examples of metalloproteinase inhibitors include ethylene glycol-bis (β-ami noethylether) N, N, N ', N' -tetraacetic acid (EGTA)
or salts thereof, such as, for example, tetrasodium salt, Na4EGTA. Examples of aminocarboxylic acids, polyaminocarboxylic acids and salts thereof, and method of their preparation, are disclosed, for example, in the following North American patents incorporated herein by reference: U.S. Patent No. 2,407,645, U.S. Patent No. 5,250,728, U.S. Pat. No. 5,449,822 and North American Patent No. 6,297,397. ESTA and EGTA are commercially available, and their salts are either commercially available or readily prepared by known methods using commercially available raw materials. These compounds are typically inexpensive and have an acceptable toxicity and an acceptable environmental cost. Other non-limiting examples of metalloproteinase inhibitors include S, S'-ethylenediamine disuccinic acid (EDDS), 1,2-di aminocyclohexan- N, N, N ', N' -tetraacetic acid (CDTA) and N- (2-) acid. h? drox? et? l) ethylenediamma-N, N'-triacetic (HEEDTA). Still other non-limiting examples of metalloproteinase inhibitors include glycine glycaeneacetic acid (MGDA), N, N-bis (carboxymethyl) glutamate (GLUDA), ortho phenanthrolma, 8-hydroxyquinoline, and phosphonic acid derivatives such as amino-tris methylene phosphonic acid , for example, sold by Buckman Laboratories under the trade name of "Phos 2", diethylene tpamma pentameti J enfosphonic acid, for example,
sold by Buckman Laboratories under the trade name "Busperse 254", 2-phosphono-1,2,4-butanetr? carboxylic acid, for example, sold by Buckman Laboratories under the trade name "Phos 9", hydroxyethylidene -diphosphonic, for example, sold by Buckman Laboratories under the trade name "Phos 6", or a mixture of 2-methylpentanediamine tetracis (methylen phosphonic acid) and 1, 2, diaminocyclohexanotetracis (methylene phosphonic acid), for example, sold by Buckman Laboratories under the trade name of "BPS 319". Still other examples of metalloproteinase inhibitors include citric acid and salts of citric acid, glycoprotein and salts of gluconic acid, cysteine, iodoacetic acid and sodium iodoacetate. Mixtures, in any combination, of any of the compounds named herein may also be used. For purposes of the present invention, more than one metalloproteinase inhibitor may be used at one time, different times or sequentially, or in any combination. As used herein, the phrase "preventing or inhibiting the putrefaction, degradation, or deterioration of an animal skin or hide due to an action of a metalloproteinase" refers to any reduction in the putrefaction, degradation, or deterioration of a skin. or leather
animal and does not intend to impose a requirement that all metalloproteinase activity and all putrefaction, degradation, or deterioration of an animal skin or hide due to an action of metalloproteinases be completely stopped, although preferably all or substantially all of the activity of the animal. Metalloproteinase stops. For example, preferably, at least 90%, at least 95%, at least 99% of all the push-tylenase metaJ activity and / or related effects is stopped. The metalloproteinase inhibitor can be applied to an animal skin or leather by any convenient method, such as, for example, by applying the metalloproteinase inhibitor directly to the skin or animal hide or by combining the metalloproteinase inhibitor with a liquid such as, for example, water, to form a solution and spray the solution on the skin or animal hide. If the metalloproteinase inhibitor is applied directly to the skin or animal hide, instead of a solution, it is not mixed with an extender or carrier material. The metalloprotemase inhibitor can be applied by immersion, spraying, propagation, roller, and / or coating drum techniques. Essentially any technique that can apply a substance or a substrate can be used in the present invention. The metalloproteinase inhibitor can be combined in one solution with other components while the other
components do not adversely affect the effectiveness or stability of the metaphorease inhibitor. In one or more modalities, it is not recommended to combine the opioidinase goal with an extender or carrier (eg, solid, such as a solid absorbent material, such as sawdust.) In one or more embodiments, the metalloproteinase inhibitor can be applied only as a solid, for example, powder or liquid without any solid carrier or extender, such as sawdust.In the liquid form, such as solutions, a solid extender or carrier is preferably not used, such as an extender or carrier is not soluble in the solution and / or is not dispersible, and / or floats, and / or interferes with the leather processing system.In addition, the metalloproiemase inhibitor can be combined with any liquid bath or process water used in the treatment of leather or leathers animals as long as it does not interfere with such treatment.As a non-limiting example, an aqueous solution containing the metalloproteinase inhibitor can be sprayed on an animal skin or leather. immediately after skinning (for example, within the hours of skinning) to provide immediate protection against putrefaction. The aqueous solution may also contain bactericides, fungicides, and / or insecticides and / or may contain agents for washing or cleansing the skin or animal hide. As another
non-limiting example, for short-term preservation of an animal skin or hide in preparation for storage or transportation, the skin or animal hide may be immersed or soaked in a cooling solution containing the inhibitor of meta J opoteinase. Adding a metalloproteinase inhibitor to a cooling solution can allow cooling to be done at a much higher temperature than usual, thereby reducing energy costs The reason cooling can be carried out at a higher temperature is that if the activity of the metalloprotemase is inhibited by the metalloproteinase inhibitor, it becomes less crucial to completely delay the bacterial growth by cooling to very low temperatures. As an example, the cooling solution may be at a temperature greater than that of freezing and lower than the ambient temperature (for example, less than 30 ° C). As another non-limiting example, the metalloproteinase inhibitor may be combined with an aqueous brine solution, such as a saturated or supersaturated solution of sodium chloride in water, in which the animal hides are immersed or soaked, or may be combined with a curing or pickling solution. As another non-limiting example, the metalloproteinase solution can be added to any aqueous solution used for depilation, wool removal,
fur, fleshy or removal of fur. As another non-limiting example, the metalloproteinase inhibitor can be added to a dipping or soaking solution which is used to soak, soften or hydrate an animal skin or leather. The amount of the metalloproteinase inhibitor applied to the animal skin or leather is not critical and can be any effective amount to prevent or inhibit the putrefaction, degradation, and / or deterioration of an animal skin or leather due to an action of a metal tyrosinease. The amount of the metal inhibitor oproteinasa can vary, for example, according to the method of application of the inhibitor of metalloproteinase, according to environmental conditions, according to the amount and condition of the metalloproteinase although to be present, and / or according to to the degree of prevention or inhibition desired. For example, the amount of the metalloproteinase inhibitor adi coned to an animal skin or hide may be from about 0.00001% to about 10% or more by weight based on the skin or animal hide. As another example, the amount of the metalloproteinase inhibitor added to an animal skin or leather may be from about 0.0001% to about 5% by weight based on the skin or animal hide. As another example, the amount of the anti-digoxin metal inhibitor added to an animal skin or hide may be about 0.001% at
about 2% by weight based on skin or animal hide. When the metalloproteinase inhibitor is combined with a fluid, the fluid must be agitated to spread the metalloproteinase inhibitor throughout the fluid, and when the animal skins or leathers are added to a fluid such as an immersion or soaking solution or bath , the animal skins or leathers are preferably drummed to spread the metalloproteinase through all the skins or animal hides. EJ metal oppressinase inhibitor can also be added to a fluid that makes contact with an animal skin or hide such as, for example, any immersion or soaking solution or bath in which skins, sheepskins, or animal hides are immersed or They are soaked during the processing or conservation of leather. The addition of the metalloproteinase inhibitor to a fluid not only provides a way to deliver an effective amount of a metalloproteinase inhibitor to a skin or animal hide, as discussed above, but also reduces the likelihood that an opioidinase metal can be spreading from one skin or animal hide to another or from a batch of fur or animal hides to another, such as might happen when more than one skin or animal hide share a solution or dip or roasting bath or when a solution or bath of immersion or soaking is refused to treat more than one lot
of skins or animal skins. As a non-limiting example, if a solution of immersion, soaking, salting, curing, or aqueous pickling to treat pellets or animal skins is refused, a metalloproteinase inhibitor may be added to the solution of immersion, soaking, salting, curing or aqueous solution between the times it is used, to deactivate any metalloprotemase that may remain in the solution. The amount of the metalloproteinase inhibitor added to a fluid is not critical and may be a concentration that is substantially lower than the concentration at which the metalloproteinase inhibitor becomes lethal to the microorganisms. For example, the amount of the metalloproteinase inhibitor added to the fluid may be from about 0.00001% to about 10% or more, preferably from about 0.0001% to about 5% and most preferably from about 0.001% to about 2% by weight of the fluid. Alternatively, the amount of the metalloproteinase inhibitor added to the fluid may be from about 0.00001% to about 10% or more, preferably from about 0.0001% to about 5% and most preferably from about 0.001% to about 2% by weight of the skins or animal skins that will be submerged or soaked in the fluid. The metalloproteinase inhibitor can also be
apply to any solid surface that comes into direct or indirect contact with a pile or animal hide to inactivate or inhibit metalloproteinases that can be produced, for example, by bacterial or fungal contaminants on the solid surface and to reduce the likelihood of a metalloproteinase It can be spread from the solid surface to the skin or animal hide or from one skin or animal hide to another. As used herein, the term "solid surface" refers to any surface in a slaughterhouse or leather processing area or tanning facility, such as walls, raceways, floors, platforms, structures, elevators, shovels, tables, hooks or cutting instruments in a slaughterhouse, leather processing facility, or leather storage facility, which the animal skin or hide may come into contact with during slaughter, storage or processing. The term "solid surface" is not intended to include particulate materials. It is proposed that the method of the present invention also include the treatment of solid surfaces that indirectly contact an animal skin or hide. For example, a surface in a leather processing facility can host microorganisms that produce metalloproteinases, and such metalloproteinases can be splashed on a contacting surface.
directly with an animal skin or leather. It can be easily observed that it would be desirable to treat such surfaces with a metalloproteinase inhibitor as well as those surfaces that come into direct contact with a skin or animal hide. As an example of the method for treating a solid surface, the metalloproteinase inhibitor can be applied directly to a surface that makes contact directly or indirectly with the skin or animal hide or can be combined with a fluid medium, such as, for example, water , to form a mixture that is applied to the surface. The amount of the metalloproteinase inhibitor added to the liquid that is applied to a surface is not critical. For example, the amount of the metalloproteinase inhibitor added to the liquid may be from about 0.00001% to about 10%, preferably from about 0.0001% to about 5% and most preferably from about 0.001% to about 2% by weight of the liquid. One of ordinary skill can easily determine the effective amount of the metalloproteinase inhibitor useful for a particular application by simply testing several concentrations before treatment of a complete substrate or system. The present invention also relates to a composition containing an immersion solution or a
useful soaking for at least one foot L or animal hide that is immersed or soaked in it, wherein the soaking or soaking solution contains at least one metalloproteinase inhibitor. The metalloproteinase inhibitor is as defined in the above and can be a chelator, such as a chelator of a divalent metal ion. As a non-limiting example, metalloproteinase inhibitor may be an aminocarboxylic acid or a polyaminocarboxylic acid or a salt thereof. As a non-limiting example, the metalloproteinase inhibitor can be ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as, for example, disodium salt of ethylenediaminetetraacetic acid (Na2EDTA) or disodium EDTA), trisodium salt of ethylenediaminetetraacetic acid (Na3EDTA) or trisodium EDTA), tetrasodium salt of ethylenediaminetetraacetic acid (Na4EDT?) Or tetrasodium EDTA), dipotassium salt of ethylenediamine notetraacetic acid (K2EDTA), tripotassium salt of ethylenediaminetetraacetic acid (K3EDTA), salt of ammonium of ethylenediaminetetraacetic acid (NH4EDTA), or diammon salt of ethylenediaminetetraacetic acid (NH4) 2EDTA). Other non-limiting examples of acid include ethanediaminetriacetic acid (ED3A) or salts thereof. Other non-limiting examples of metalloproteinase inhibitors include ethylene glycol-bis (β-ammoetherether) N, N, N ', N' -tetraacetic acid
(EGTA or salts thereof, such as, for example, tetrasodium salt, Na4EGTA Examples of arylocarboxylic acids, polyaminocarboxylic acids and salts thereof, and method of their preparation, are disclosed, for example, in the following North American patents. gray hair incorporated herein by reference: US Patent No. 2,407,645, US Patent No. 5,250,728, US Patent No. 5,449,822 and US Patent No. 6,297,397. ESTA and EGTA are commercially available, and their salts are either commercially available or readily available. prepared by known methods using commercially available raw materials These compounds are typically inexpensive and have acceptable toxicity and acceptable environmental cost Other non-limiting examples of metalloprotease inhibitors include S, S'-ethylenediamine (EDDS) disuccinic acid, 1, 2-d Laminocyclohexen-N, N, N ', N'-tetraacetic acid (CDTA) and N- (2-h? drox? et? l) ethylenediamine-N, N'-tpaacetic (HEEDTA). Still other non-limiting examples of metalloproteinase inhibitors include methylglycineadiacetic acid (MGDA), N, N-bis (carboxymethyl) glutamate (GLUDA), ortho fenantrolma, 8-hydroxyquinoline, and phosphoric acid derivatives such as amino-tris methylene phos Conical, for example, sold by Buckman Laboratories under the trade name "Phos 2", diethylene pentamethylene phosphonic acid amine, for example,
2S
sold by Buckman Laboratories under the tradename of "Busperse 254", 2-phosphono-1,2,4-butanetricarboxylic acid, by e-commerce, sold by Buckman Laboratories under the trade name "Phos 9", hydroxyethylidene acid. diphosphonic, for example, sold by Buckman Laboratories under the trade name "Phos 6", or mixture of 2-methylpentanodiamma tetracis (methylene phosphonic acid) and], 2, diaminocyclohexanotetracis (methylene phosphonic acid), for example, sold by Buckman Laboratories under the commercial name "BPS 319". Still other examples of metalloprot kinase inhibitors include citric acid and citric acid salts, gluconic acid, and salts of gluconic acid, cistern, iodoacetic acid and sodium iodoaceta. Mixtures of any of the compounds named herein can also be used. The amount of metalloproteinase inhibitors contained in the immersion or soaking solution is not typical and can be any amount effective to prevent or inhibit the putrefaction, degradation, and / or deterioration of the animal hides or skins in the composition. As an example, the amount of the metalloproteinase inhibitors in the immersion or removal solution may be from about 0.00001% to about 10%, preferably from about 0.0001% to about 5% and most preferably from about 0.001% to
about 2% by weight based on the weight of the skin or animal hide contained in the composition. Alternatively, the amount of the metalloproteinase inhibitor in the immersion or stirring solution may be about 0.00001% to about 30%, preferably from about 0.0001% to about 5% and most preferably from about 0.001% to about 2% by weight based on the immersion or soaking solution. The soaking or soaking solution can be any solution, such as, for example, an aqueous solution, which is used to treat animal skins or leathers, which include, but are not limited to, cleaning, cooling, curing, pickling, furring and / or removal of fur, and / or solutions to soften or hydrate an animal skin or hide. The present invention is further related to an animal skin or leather having at least one meta] oprotein inhibitor applied to at least one surface thereof. The metalloproteinase inhibitor is as defined in the foregoing, and may be a chelator, such as a chelator of an equivalent metal ion. As a non-limiting example, the metalloproteinase inhibitor can be an aminocarboxylic acid or a carboxylic acid or a salt thereof. As a non-limiting example, the metalloproteinase inhibitor can be ethylenediaminetetraacetic acid
(EDTA) or a salt thereof, such as, for example, disodium salt of ethylenediaminetetraacetic acid (Na2EDTA) or disodium EDTA), trisodium salt of ethylenediaminetetraacetic acid (Na3EDT?) Or trisodium EDTA), tetrasodium salt of ethylend aminotetraacetic acid (Na ^ EDTA) or tetrasodium EDTA), dipotassium salt of ethylenediaminetetraacetic acid (K2EDTA), tripotassium salt of ethylenediaminetetraacetic acid (K3EDTA), ammonium salt of ethylenediaminetetraacetic acid (NH4EDTA), or diammonium salt of ethylendiammotetraacetic acid (NH) 2EDTA). Other non-limiting examples of acid include ethylenediammatpaacetic (ED3A) or salts thereof. Other non-limiting examples of metalloproteinase inhibitors include ethylene glycol bis (β-ammoethylene ether) N, N, N ', N' -tetraacetic acid (EGTA or salts thereof, such as, for example, tetrasodium salt, Na4EGTA. Examples of aminocarboxylic acids, polyaminocarboxylic acids and salts thereof, and method of their preparation, are disclosed, for example, in the following North American patents incorporated herein by reference: U.S. Patent No. 2,407,645, U.S. Patent No. 5,250,728, U.S. Pat. No. 5,449,822 and US Patent No. 6,297,397, EJ ESTA and EGTA are commercially available, and their salts are either commercially available or readily prepared by US Pat.
known methods using commonly available raw materials. These compounds are typically inexpensive and have an acceptable toxicity and an acceptable environmental cost. Bulls non-limiting examples of metalloproteinase inhibitors include S, S'-ethylenediamine disuccinic acid (EDDS), 1,2-d? Am? Noc? Clohexen-N, N, N ', N' -tetraacetic acid (CDT? ) and N- (2-hydroxyethyl) ethylenedi amine-N, N'-tp acetic acid (HEEDTA). Still other non-limiting examples of metalloproteinase inhibitors include methylglycineadiacetic acid (MGDA), N, N-bis (carboxymethyl) glutamate (GLUDA), ortho-phenanthroline, 8-hydroxyinoline, and phosconic acid derivatives such as amino-tris methylene phosphonic acid, for example, sold by Buckman Laboratories under the trade name "Phos 2", diethylene triamine pentamethylene phosphonic acid, for example, sold by Buckman Laboratories ba or the trade name of "Busperse 254", 2-phosphono-1, 2, 4 acid -butanetricarboxylic, for example, sold by Buckman Laboratories under the trade name of "Phos 9", hydroxyethylidene diphosphonic acid for example, sold by Buckman Laboratories ba or the trade name of "Phos 6", or mixture of 2- methylpentanodiami tetracis (methylene phosphonic acid) and 1, 2, diaminocyclohexanotetracis (methylene phosphonic acid), for example, sold by Buckman Laboratories under the tradename "BPS 319". Still
Other examples of metalloproteinase inhibitors include citric acid and salts of citric acid, gluconic acid, and salts of gluconic acid, cysteine, iodoacetic acid, and sodium iodoacetate. Mixtures of any of the compounds named herein may also be used. The amount of the metalloproteinase inhibitor applied to the skin or animal hide is not critical and can be any amount effective to prevent or inhibit the putrefaction, degradation, and / or deterioration of the animal skin or hide. As an example, the amount of the metalloproteinase inhibitor applied to the animal skin or leather may be from about 0.00001% to about 10%, preferably from about 0.0001% to about 5% and most preferably from about 0.001% to about 2% in Weight based on the weight of the skin or animal hide. The metalloproteinase inhibitor can be rinsed immediately or before it is hardened or can be left on the animal skin. In general, any treatment time for any of the modalities discussed in the above can be used. The treatment time is variable and depends on the method chosen to apply the inhibitor to the hides or skins. For example, if it is incorporated into brine curing, the treatment time can be from one hour to one day or longer, and when it is used for the preservation of
Fresh leather, the inhibitor can be added to the mixing drum and placed in the drum for one hour or two hours or more. Essentially, the treatment time can vary from one minute to one hour, to six hours, to twenty-four hours, forty-eight hours, seventy-two hours, or longer, depending on the concentration of the inhibitor used and / or the amount of desired protection. The following examples are given to illustrate the nature of the invention. It will be understood, however, that the invention is not to be limited to the specific conditions or details set forth in these examples. EXAMPLES E ploses 1 and 2 Evaluation of collagenase activity: Inhibition of collagenase from pure bacterial cultures by disodium EDTA, trisodium EDTA, tetrasodium EDTA, tetrasodium DGTA and trisodium EDDS. Method Azocoll-modified nutrient agar (a substrate consisting of insoluble collagen particles impregnated with azo-bright dye) was used as the test medium to study the activity of collagenase. The nutrient agar was prepared and placed in an autoclave and then cooled to 55 ° C. Azocol was added to give a final concentration of 0.5%. The inhibitors were added to the dishes
petri, and the nutrient agar-azocoj medium was added and mixed carefully to give the desired concentration of the inhibitor. Using a sterile spatula, or a cork borer, the average potion of each plate about the size of a quarter of the United States was dried with a spoon. The azocol agar medium of fresh nutrient that does not contain 1 inhibitor was added to replace the spoon-removed portion of the plate. Two positive bacteria of collagenase, Pseudomonas s fl uorescens (Example 1) and Aeromonas sa lmoni cida (example 2), which were isolated from rotting hides were used as sources of collagenase. The bacteria developed overnight on the nutrient agar. A suspension of each culture was made in sterile water and used to inoculate the middle portion of each plate. The treated samples were incubated at 30 ° C and were valued after 24 hours, 2 days and 7 days for the activity of the collagenase and also for the growth of the bacteria. The test cultures secreted collagenase in the medium as they grew to degrade the collagen in the azocol medium, and the activity of the collagenase was determined by visual inspection of the impure J region around the bacteria. All% are in by weight of the active ingredient. The results are summarized in Tables 1 and 2: Table 1: Anticolagenase activities of chemicals using Pseudomonas s fl uorescens as a source of
collagenase (Example 1). Compound Conc Growth / Days Collagenase Activity / Days 1 1 Na2EDTA 038% 0 0 ++ 0 0 0 025% ++ 4 (- 1 + 0 0 0 02% 41 + +++ +++ 0 0 0 015% 14 + +++ +4 + 0 0 0 01% ++++ 1 f ++ 111- + 0 0 0 005% ++++ ++++ 0 0 + 0025% 4 +++ 1 + 4 + ++ +1 ++++) - (- ++ ++++ 0% 4 +++ 4 I ++ + M + ++++ + H- + ++++
Na3EDl A 038% + + + 0 0 0 025% ++ ++ 4 ++ 0 0 0 015% +41 + 1 + t - ++ 0 0 0 01% ++++) +++ ++ H 0 0 0 005% + 11 + +++ 1 ++ 41 0 + + 0025% + 1 t- + I +++ I ++ 4 ++++ I - +++ ++++ 0% ++++ ++ ++ ++! 1 ++++ ++++ ++++ Na, ED 1 A 05% 0 1 t - ++ 0 0 0 04% +! - + ++ 4 0 0 0 03% 11 + 11 + +4 ( - 0 0 0 02% +++ +++ +++ 0 0 0 01% +++ I ++ +++ - 0 0 0 0075% 1 t - ++ 1 +++ t - ++ 4 0 0 0 005% 1 t - ++ +++ 4 ++++ 0 0 0 0025% 1 +++ + -14 + ++ 1 + 1 +++ ++++ ++++ 0% t - ++ + t - +++ ++ 11 H ++ ++++ p 1 1 r- N, EGrA 05% ++++ 1 I ++ ++ 1 t 0 0 0 04% 44 ++ ++++ + 11 + 0 0 0 03% 1 (- ++ 41-1- + ++++ 0 0 0 02% ++++ I +++ +++ I 0 0 0 01% 1 (++ ++ - 14 ++++ 0 0 0 0075 ++++ ++++ 1 - +++ 0 0 0 005% ++++ ++++ ++++ 0 0 0 0025 t +++ + t - ++ 1 + 41 (- ++ + 4 +++ ++++ 0% ++++ ++++ 1 ilr ++++ 4 +++ ++++ Na3EDDS 18% ++++ d ++++ and H- + t '0d 0e o1 15% ++++ ++ H- 4 +++ 0 0 0 13% 44 ++ 1 t - + - > - 4 t - ++ 0 0 0 1% ++++ ++ ++) - +++ 0 0 0
0 9% ++++ +++ 4 -II - ++ 0 0 0 0 8% ++ - 1- + ++++ I - +++ 0 0 0 0 7% + 44- + +++ + ++++ 0 0 0 0 6% ++++ -I +++ 4 +++ 0 0 + 0.5% ++++ 4 +++ + 4-H- 0 0 ++++ 0.4% +++ + +4 - ++ 41 - ++ 0 ++ ++++ 0 3% ++++ 1 -I - ++ ++++ 0 +++ ++++ 0.2% ++++ ++ ++ I - +++ ++++ ++++ 0.1% ++ - H- ++++ ++ 4 + ++++ + -H- + ++++ 0% ++++ 1-41-4- ++++ -1 +++ ++++ ++++
Legend d, e, f Data were collected on days 2, 3 and 7, respectively. Growth: 0 = No Growth + = Growth Very Little + ^ = Average Growth +++ = Heavy Growth ++++ = Very Heavy Growth Collagenase activity 0 = No collagenase activity 4 = Very little collagenase activity ++ = Average collagenase activity? i- = Strong collagenase activity ++++ = Very strong collagenase activity
Table 2: Anticolagenase activity of the chemists using Ñeromonas sa l oni cida as a source of collagenase (Example 2).
Compound Conc. Growth / Days Collagenase Activity / Days 1 Na2EDTA 0.2% ++ + 4-1- ++++ 0 0 0 0.1% ++++ ++++ ++++ 0 0 0 0.05% ++++ ++++ 4 - +++ ++++ +++ - (- ++++ 0.025% ++++ ++++ ++++ ++++ ++++ ++++ 0 % ++++ ++++ -I - +++ ++++ ++++ ++++ Na3EDTA 0.2% (- +++++ ooo
0. 1% 4- ++++ ++++ 0 0 0 0.05% ++++ ++ - I- + 4-I-1- + ++++ +4 - ++ ++++ 0% + +++ ++++ + 4-4- + ++++ ++++ ++++
NaEDTA 0.5% 0 +++ a +++ 0 0a 0 0.25% 0 +++ '' +++ 0 0a 0 0.1% 0 +++ "+++ 0 0a 0 0% ++++ +++ + a ++++ ++++ ++++ a ++++
NÍHEGTA 0.5% ++++ +4 - ++ ++ 4- + 0 0 0 0.25% ++++ ++++ ++++ 0 0 0 0.1% ++++ ++++ ++ - I- + 0 0 0 0% ++++ ++++ + -H- + ++++ ++++ ++++
Na3EDDS 2% 4-1- +++ + 4-1- 0 0 0 1.8% +++ ++++ -II-1- + 0 0 0 1.5% ++++ ++++ + 4-4 -4- 0 0 0 1.3% ++++ ++++ + 4-4- + 0 0 +++ 1.2% 4-4 - ++ + 4-I- + 4-I-4- + 0 + ++++ 1% ++++ ++++ ++++ 0 ++ ++++ 0.7% ++++ ++++ ++++ 0 ++++ ++++ 0.5% ++++ ++++ ++++ 0 ++++ ++++ 0.2% ++++ ++ - 1- + ++++ ++++ ++++ ++++ 0 % ++++ ++++ ++++ ++ - H- ++++ ++++
Legend Growth: 0 = No Growth + = Growth Very Little ++ = Average Growth +++ = Heavy Growth
++++ = Very Heavy Growth Collagenase activity 0 = No activity of cogenase Lagenase + = Very little collagenase activity ++ = Average collagenase activity +++ = Strong collagenase activity ++++ = Collagenase activity very strong a The data was collected on the fourth day. As shown in Tables 1 and 2, the three salts of EDTA, Na4EGTA and Na3 EDDS were performed very well as collagenase inhibitors against the collagenase produced by the bacteria studied. In addition, the compounds were effective as co-janase inhibitors at concentrations that were far below that would be required to retain bacterial growth. EXAMPLE 3 Inhibition of co-magease from the growth of mixed bacteria on Cresco leathers by disodium EDTA, trisodium EDTA, tetrasodium EDTA and tetrasodium EGTA. Method Nutrient agar modified with 0.5% of
AZOCOLL as the test medium to study the activity of collagenase. The nutrient agar was prepared and placed in an autoclave and then cooled to 55 ° C. Azocol was added
to give a fine concentration of 0.5%. The inhibitors were added to the petri dishes, and the nutrient azocol agar medium was added and mixed thoroughly to give the desired concentrations of the inhibitor. The fresh leathers were obtained from a tannery and cut into pieces of approximately 2 cm squares a 2x2 leather sample was placed on top of the agar containing the inhibitors. The treated samples were incubated at 30 ° C and evaluated after 24 hours, 2 days, 3 or 4 days and 7 days for collagenase activity and also for the growth of the bacteria. Bacteria from the leathers secreted collagenase in the medium as they grew to degrade the collagen in the azocol medium and the activity of the collagenase was determined by vidual inspection of the clean area around the bacteria. The results are summarized in Table 3 Table 3: Anticolagenase activities of chemicals using collagenase from mixed bacteria on samples of fresh leather (Example 3) Compound Conc Growth / Days Collagenase Activity / Days
1 Na2ED IA 0 5% 0 +++ ++++ 0 0 0 0 0 4% 0 +++ ++ 1 (- 0 0 0 0 0 3% 0 +++ ++ 41 0 0 0 + 0 2 % 0 +++ +411 0 0 0 + 0 1% t- + 1 (- + I- 4 +++ 0 + +++ +++ 0 075 1 - +++ ++++ +++ 1 + +++ ++++ ++++ 0 05% ++++ I t - ++ +4 + 1- 0 1 1 11 11 ++++ ++++ 0 025% 4 +4 + + ++ 1- (-1 1 + 1 - +++ ++++ ++ (- + ++++ 0% ++++ +++ 4 +++ 4 ++++ ++++ + +++ ++++
Na3EDTA 05% + t ++++ ++ - M 0 0 0 + 04% ++++ +++ 4 4 +++ 0 0 0 + 03% 1-1 1- (++ +4 + 0 0 ++ ++ 02% ++++ ++++ +++ 4 0 0 ++ ++ 4
01% ++++ ++ 4 (1 ++ 4 0 + +++ +++
0075% +4 1 +! - +++ + 11+ 0 + +++ il i1 li ^ 005% + t - ++ ++++ 4 + -r + 1 + +++ ++++ ++ 4H - 0025% ++++ 4 Hl +41 t ++++ ++++ ++++ ++++
0% + (++ (++ (- ++++ ++++ ++++ ++++
NajEDT? 05% ++++ H (-1 + 111 0 0 ++ 04% ++ H- +++ - > - ++++ 0 + + +++
03% 4 t - ++ ++++ ++ 1 0 + + + -H- 02% + 4 ++ +++ (++++ 0 + ++ +++
01% ++++ ++++ +++ 4 0 + ++ +++
0075% ++++ ++++ t - +++ + ++ +++ 005% ++++ l + + + (4 (+++ 4 ++++ ++++ ++++
0025% +41 + +++ (++ I-1- ++++ ++++ ++++ +4 - ++
0% 1-414 1 +++ ++++ ++++ + H- + ++++ +++ 4
Nc, EG TA 05% +++ ++++ +++ 4 0 0 or ** 0 04% +4 (- ++ 41 ++++ 0 0 0 + 03% ++++ t ++ t - +41 + 0 0 + + 02% ++++ H- + 4 +++ - t 0 0 + + 01% ++++ +++ 4 t - +++ 0 0 + H- 0075 ++ ++ +++ 1 ++ t ~ 0 ++ ++++ 005% ++ (- (- ++++ + (- + - ++++ ++++ ++++ ++++
0025 ++++ ++ 41 ++++ ++++ ++++ ++++ +++ 4
0% ++++ I +++ ++++ ++++ + -H- + ++++ ++++
Legend ** Data collected on Day 3 Growth: 0 = No Growth + = Growth Very Little ++ = Growth Mean + 1 i- = Heavy Growth ++++ = Very Heavy Growth Collagenase activity 0 = No activity of the collagenase
+ = Very little collagenase activity ++ = Average collagenase activity +++ = Strong collagenase activity +++ 1- = Very strong collagenase activity The three salts of EDTA and Na4EGTA performed very well as inhibitors of collagenase against the collagenase produced by the mixed culture of bacteria in the leathers. In addition, the compounds were effective as collagenase inhibitors at concentrations that were far below that would be required to arrest bacterial growth. Other embodiments of the present invention will be apparent to those skilled in the art upon consideration of the present specification and practice of the present invention disclosed herein. It is proposed that the present specification and examples be considered as exemplary only with a true scope and spirit of the invention which is indicated by the following claims and equivalents thereof.